Mesure de la biomasse et de l'activité bactérienne dans l'eau de distribution Measurement of bacterial biomass and activity in tap water

Résumé

Afin d'étudier la reviviscence bactérienne dans les réseaux de distribution, des méthodes de mesures de la biomasse et de l'activité bactérienne ont été investiguées sur des eaux provenant d'un réseau de distribution. Trois méthodes d'estimation de la biomasse bactérienne ont été comparées : le comptage sur gélose, selon la norme française d'examen bactériologique des eaux de consommation, le dosage de l'ADN contenu dans les particules retenues sur une membrane de porosité de 0.2 µm et le comptage direct au microscope à épifluorescence après coloration des bactéries à l'acridine orange. Les comptages sur gélose, tout comme en milieu aquatique naturel, sous-estiment très largement le nombre de bactéries; ceci semble principalement lié à la présence de bactéries viables mais non cultivables. Le dosage de l'ADN et les comptages directs corrèlent assez bien avec en moyenne un contenu en ADN par bactérie de 4,1 x 10^-15 g d'ADN, mais la première méthode semble moins précise. Le comptage direct semble donc la méthode la plus adaptée à l'estimation du nombre total de bactéries dans ce type de milieu.

Afin d'estimer l'activité bactérienne, les protocoles expérimentaux de deux méthodes utilisées en écologie bactérienne ont été adaptés aux conditions particulières de l'eau de distribution : l'incorporation de thymidine tritiée dans l'ADN bactérien et l'incorporation de leucine tritiée dans les protéines. La comparaison des deux méthodes sur une série d'échantillons montre une bonne corrélation, avec un rapport molaire entre incorporation de leucine et de thymidine compatible avec les facteurs de conversion des deux méthodes cités dans la littérature et établis pour les milieux aquatiques naturels. Les deux méthodes sont utilisables pour mesurer l'activité bactérienne dans l'eau potable, néanmoins l'incorporation de thymidine est plus aisée à mettre en oeuvre, car elle ne nécessite de travailler qu'à une seule concentration en traceur radioactif.

Mots-clés:

• Biomasse bactérienne,
• comptage sur gélose,
• énumération microscopique,
• activité bactérienne,
• ³H-thymidine,
• ³H-leucine,
• eau de distribution

Abstract

Bacterial regrowth in distribution systems is an important problem for drinking water producers. It is linked to the more and more frequent utilization of low quality surface waters, containing high concentration of organic matter, as raw water, and also to the increase in size and complexity of the distribution networks with high residence time of the water between its production and utilization. At the present time chlorination of treated water, with sometimes rechlorination in the network, is the usual way to limit growth in distribution systems. This solution however presents disadvantages, the major one is the formation of unpleasant organochlorine compounds which are responsible for tastes and odours of water. An alternative strategy consists of developing treatment lines in which biodegradable dissolved organic carbon is removed. It allows through a reduction of the chlorine demand of the water to increase the stability of the chlorine residual of the water. In this context, it is important to get a good knowledge of the factors controlling bacterial development in distribution networks. Up to now, studies on this subject have met some methodological problems linked to the fact that classical bacteriological methods are inadequate to study this kind of systems.

In this paper, various methods have been investigated to estimate bacterial biomass and activity in tap water. For this study, the analyzed water samples have been collected in the distribution system of the Parisian suburbs.

Three methods have been tested for the determination of bacterial biomass : plate count, measurement of DNA associated with particles with a size higher than 0.2 µm and direct microscopic enumeration. Heterotrophic plate counts have been performed following the French standard and results are expressed in CFU (Colony Forming Units) per ml; the DNA collected after filtration of 500 ml to 1500 ml of water on a 0.2 µm pore size membrane was estimated using a fluorimetric method, as proposed by Mc COY and OLSON (1985); direct enumerations were performed by epifluorescence microscopy after acridine orange staining (AODC) following the procedure proposed by HOBBIE et al. (1977), the comparison between plate counts and AODC (fig. 1) shows the important underestimation of the bacterial numbers when estimated by the CFU (up to 3 orders of magnitude). Such discrepancy has already been observed in natural aquatic ecosystems and is usually explained by the presence of numerous dead cells enumerated by microscopy. Now, it seems that the difference between, plate counts and direct counts may rather be explained by the presence in water of « viable but non culturable » bacteria.

A comparison between DNA estimation and direct counts have also been performed. Figure 2 shows the results of this comparison. In spite of the dispersion, the correlation between both methods is significant and the correlation straight line indicates an average DNA content per bacteria of 4.1 x 10-15g DNA in good accordance with the values quoted in the literature. The dispersion of the data around this average can be explained by various ways : the variability of DNA per cell content for the different bacterial strains present in the water samples, the precision of the DNA method which is not higher than 20 % and possible contamination by other organisms than bacteria, as flagellates or ciliates, which are retained on the 0.2 µm pore size membrane.

On the basis of these tests, it seems that the direct count by epifluorescence microscopy is the most adapted method for studying the bacterial regrowth in distribution system.

The understanding of bacterial dynamics in a distribution system requires measurements of bacterial activity. Various methods have been developed in order to estimate bacterial activity in natural aquatic ecosystems. They are primarily based on the use of radioactive tracers. At the present time, the tritiated thymidine incorporation method, which measures the replication of bacterial DNA, is the most usually used one, but the incorporation of tritiated leucine into proteins, which measures increase in bacterial biomass, seems to be also an interesting method. These methods have been selected, on one hand, because of their specificity towards bacteria and, on the other hand, because of their high sensibility which is required for measurements of bacterial activity in the conditions of drinking water. Up to now, these methods have never been applied to drinking water. We have modified the experimental procedure of both methods : incubation time, radioactive tracers concentrations and volume of the sample have been tested and adapted in order to allow measurement in the conditions of drinking water samples. For thymidine incorporation, the volume of sample, incubated during 20 hours in the presence of 20 nM concentration of ³H-thymidine, was 100 ml. The incorporation was measured in the DNA, using the biochemical procedure proposed by WICKS and ROBARTS (1977), rather than in the total macromolecules. For leucine incorporation, we measured the incorporation rate at four leucine concentrations (2, 27, 52, 77 nM : 2 nM of ³H-leucine + non radioactive leucine) in 25 ml samples and the incubation lasted 3 to 4 hours. The incorporation rate was calculated as the reciprocal of the angular coefficient of the correlation straight fine obtained when the reciprocal of the fraction of leucine incorporated per hour was plotted against leucine concentration (fig. 3). Comparison of both methods on samples of drinking is presented at figure 4, a good linear correlation was found. The equation of the correlation straigth line is :

log [Inc.leu (pmol/l.h)] = 0,97 log [Inc.thy (pmol/l.h)] + 1.35(n = 69, r = 0.84)

The molar ratio between leucine and thymidine incorporation found in these samples (20 to 25) seems to be in good agreement with the usual conversion factors found for both methods in natural aquatic ecosystems. Bath methods seem to be available to bacterial activity estimations in drinking water, the triatiated thymidine incorporation method which requires working with only one concentration of radioactive tracer seems easier to use.

Keywords:

• Bacterial biomass,
• plate count,
• microscopic enumeration,
• bacterial activity,
• ³H-thymidine incorporation,
• ³H-leucine incorporation,
• drinking water