Mesure de la biomasse et de l'activité bactérienne dans l'eau de distribution

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.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

Comparaison de deux méthodes d'estimation du broutage des bactéries par les protozoaires en milieux aquatiques [Courte note]

L'objectif du présent travail est de comparer deux méthodes indépendantes permettant d'estimer, dans les milieux aquatiques, le flux de carbone transitant du compartiment bactérien vers les protozoaires. Les deux méthodes utilisées sont, d'une part, celle basée sur le suivi de la décroissance de radioactivité du matériel génétique bactérien après marquage à la thymidine tritiée (SERVAIS et al., 1985) et, d'autre part, celle de mesure du taux d'ingestion de bactéries fluorescentes (FLB) par les protozoaires. Elles ont été appliquées en parallèle sur des échantillons de la rivière Meuse (Belgique). L'emploi de la première méthode a montré des taux de broutage compris entre 0.002 h-1 et 0.016 h-1 qui représentent en moyenne 72 % des taux de mortalité totale. Une excellente corrélation entre les estimations de flux de broutage obtenues par les deux techniques a été trouvée, mais les valeurs estimées à partir de la méthode FLB sont systématiquement inférieures (d'environ 30% en moyenne) à celles obtenues par l'autre méthode. Une part de cette différence peut vraisemblablement s'expliquer par la non prise en compte par la méthode FLB du broutage par des organismes de taille supérieure à 100 µm.The goal of the present work was to compare two methods allowing to estimate, in aquatic ecosystems, the carbon flux due to grazing of bacteria by protozoa. The first method follows the decrease of labeling in the DNA of natural assemblages of bacteria previously labeled with tritiated thymidine (SERVAIS et al., 1985) and the second procedure is based on the estimation of bacterial ingestion rate by protozoa using fluorescently labeled bacteria (FLB). Both methods were applied in parallel on river Meuse (Belgium) samples. Using the first method, grazing rates in the range 0.002 h-1 to 0.016 h-1 were observed; they represented in average 72 % of the total bacterial mortality rates. A very good correlation between both estimates of the grazing fluxes was found but the data obtained by the FLB method were systematically lower (around 30% in average) than those estimated with the other method. A part of this difference is probably due to he fact that the FLB method does not take into account grazing by organism higher than 100 µm

Estimation de la biomasse bactérienne dans les effluents urbains par mesure de l'activité exoprotéolytique potentielle

Des études récentes ont montré que, lors du rejet d'eaux usées dans une rivière, la quantité de biomasse bactérienne hétérotrophe amenée par les effluents influence considérablement la cinétique de biodégradation de la matière organique dans la rivière et donc les caractéristiques du déficit d'oxygène généralement observé dans le milieu naturel en aval du rejet. La mesure de la biomasse bactérienne contenue dans un rejet domestique est donc nécessaire afin de bien comprendre la cinétique de biodégradation. Cette biomasse peut être estimée en microscopie à épifluorescence après coloration des cellules bactériennes par un fluorochrome. Cette technique appliquée aux eaux usées est néanmoins difficile et fastidieuse. Dans cette étude, une méthode alternative à l'estimation de la biomasse bactérienne dans les eaux usées a été testée ; elle consiste à mesurer l'activité exoprotéolytique potentielle (AEP) des bactéries. Nous avons montré qu'il existait, dans les eaux usées, une corrélation significative entre l'AEP et la biomasse bactérienne estimée en microscopie à épifluorescence ce qui permet d'utiliser l'AEP pour estimer facilement et rapidement la biomasse bactérienne dans ce type d'échantillon. Comme exemple d'application, des mesures d'AEP nous ont permis d'étudier l'impact de divers types de traitement dans plusieurs stations d'épuration sur la biomasse bactérienne hétérotrophe des effluents urbains. Sur base de ces mesures, les charges spécifiques en biomasse bactérienne (charge par habitant et par jour) des eaux brutes et traitées ont pu être calculées.Recent studies have shown that when wastewaters are discharged into a river, heterotrophic bacterial biomass in the effluent exerts considerable influence on the biodegradation kinetics of organic matter and provokes the oxygen deficits often observed downstream from outfalls. Quantification of bacterial biomass in wastewaters is required for a good understanding the biodegradation kinetics. Bacterial biomass can be estimated by epifluorescence microscopy after staining cells with fluorochrome. Applying this technique to wastewater samples, however, is painstaking and difficult. In this study, an alternative method for estimating bacterial biomass in wastewaters was tested; it consists of measuring the potential exoproteolytic activity (PEA) of bacteria. Previous studies on other types of aquatic systems have shown that bacterial biomass can be estimated from PEA. After optimising the PEA procedure for use with wastewater, we found a significant correlation between PEA and the bacterial biomass estimated by epifluorescence microscopy. This opens the way for PEA measurements for rapid and easy estimates of bacterial biomass in wastewaters. As an example of the application of this technique, the impact of wastewater treatment on bacterial biomass in effluent was investigated at various treatment plants. On the basis of our procedure, specific loads of bacterial biomass (load per inhabitant and per day) were calculated for raw and treated wastewaters

Biodegradable dissolved organic carbon removal during biological filtration on granular actived carbon

En production d'eau potable, la nature bactérienne de l'abattement du carbone organique dissous biodégradable (CODB) observé dans les filtres à charbon actif en grains (CAG) a été démontrée. Les performances de fonctionnement de ce type de contacteur biologique ont été principalement étudiées sur pilotes. Dans la présente étude, elles sont vérifiées et transposées en condition d'exploitation sur une usine de production d'eau potable de la. banlieue parisienne. La colonisation bactérienne du CAG a été suivie et montre que l'équilibre biologique est atteint après filtration d'environ 12500m3 d'eau/m3 de CAG. Durant cette phase de colonisation, la biodégradation se substitue progressivement à l'adsorption pour abattre le COD. Après colonisation, l'efficacité des filtres biologiques, exprimée en terme d'abattement de CODB, est fonction du temps de contact quelle que soit la vitesse de filtration (dans la gamme de 2 à 18 m/h). Les résultats de suivis de deux filtres sur deux ans montrent que l'efficacité a été globalement meilleure en 1989 qu'en 1990, cette différence s'explique par les fluctuations plus importantes de CODB dans l'influent en 1989. Un modèle mathématique, établi à partir des équations cinétiques des processus bactériens dans les filtres à CAG (modèle CHABROL), développé sur base d'observations antérieures, permet de simuler correctement les observations faites au cours de la présente étude. Avec la mesure du CODB, le modèle CHABROL constitue un outil très bien adapté pour contrôler les performances des contacteurs biologiques. Ils permettent, entre autre, de définir le temps de contact optimal de l'eau dans le filtre en fonction d'une température et d'une qualité d'eau donnée dans l'influent et d'une qualité d'eau souhaitée dans l'effluent.In drinking water production, filtration on granular activated carton (GAC) is generally used in order to remove by adsorption the dissolved organic matter. Nevertheless, the adsorption capacity of GAC is rapidly saturated and it is so necessary to regenerate the GAC. An interesting alternate has been applied in some treatment plants. It consists to use GAC filtration without regeneration taking benefit of the activity of the microbial community which colonize the GAC particles (RITTMAN and HUCK, 1989). In fact, this biological filtration offers the advantage to specially remove the biodegradable fraction of the dissolved organic carbon (BDOC), which is responsible for the problem of bacterial growth into the distribution networks.The bacterial nature of the BDOC removal achieved by the biological filtration on GAC has been now clearly demonstrated (SERVAIS et al., 1991) and some important results of the functioning of these filters has been obtained in studies conducted on pilots filters (BOUILLOT et al., 1990; SERVAIS et al., 1992). These studies have for example shown that only a very small part of the bacterial biomass produced in the filter is exported with the outflow.In the present study, biological filtration has been investigated in a full scale treatment line at Choisy-le-Roi in the Parisian suburbs and the results compared with those gained on pilot filters.The working conditions of the three GAC filter studied are presented in table 1 and compared with those of pilot filters used in a previous study conducted al Neuilly-sur-Marne (table 2). The microbial colonization has been followed in two of the liners. If lasted roughly 3 months to reach biological equilibration, it corresponds to a water volume filtrated of 12 500 m3 per m3 of GAC. Efficiency of the removal during this period is presented in figure 2. Progressively, biological processes take turn with adsorption (fig. 1).As already demonstrated by SERVAIS et al. (1992), the efficiency of biological filtration, calculated in percentage of BDOC removal, increases with increasing contact time whatever the filtration velocity could be in the range 2 m/h to 18 m/h (fig. 3). However, the percentage of BDOC, at similar temperature, is higher in the GAC filters at Choisy-le-Roi than at Neuilly-sur-Marne. The fixed bacterial biomass is also higher at Choisy-le-Roi (average 7.5 µgC/cm3) than at Neuilly-sur-Marne (average 2 µC/cm3).Following during two years the functioning of the n° 56 and 38 filters (tables 3, 4 and fig. 5, 7), it seems that the global efficiency of filtration is better in 1990 than in 1989. This can be linked to the greater fluctuations in BDOC in the influent water in 1989 than in 1990, as shown on figure 8. Fluctuations in the quality of the influent water requires a period to reach the equilibrium during which the effluent is charchacterized by a lower quality (fig. 8). This period is longer at low temperature. The mathematical modal based on the kinetics of the basic microbiological processes involved in biological filtration (the CHABROL model) has been previously developed (BILLEN et al., 1992) in order la simulate the performances of the filtration. It can be used to simulate the vertical profiles of BDOC and bacterial biomass in the filters of the Choisy-le-Roi treatment plant, with modifying only one parameter in the model, the average bacterial mortality “kd” (fig. 4). BDOC decreases versus empty bed contact time (EBCT) calculated by the modal are presented on figure 6 for the Choisy-le-Roi and Neuilly-sur-Marne treatment plants and for two temperatures.From a management point of view, the minimum BDOC is reached for contact time between 15 and 20 minutes at Neuilly-sur-Marne, while at Choisy-le-Roi it is rather between 10 and 15 minutes.In conclusion, BDOC measurements and CHABROL modal constitute powerful tools for management and design of biological GAC filters

Carbon Cycling of Lake Kivu (East Africa): Net Autotrophy in the Epilimnion and Emission of CO2 to the Atmosphere Sustained by Geogenic Inputs

We report organic and inorganic carbon distributions and fluxes in a large (>2000 km2) oligotrophic, tropical lake (Lake Kivu, East Africa), acquired during four field surveys, that captured the seasonal variations (March 2007–mid rainy season, September 2007–late dry season, June 2008–early dry season, and April 2009–late rainy season). The partial pressure of CO2 (pCO2) in surface waters of the main basin of Lake Kivu showed modest spatial (coefficient of variation between 3% and 6%), and seasonal variations with an amplitude of 163 ppm (between 579±23 ppm on average in March 2007 and 742±28 ppm on average in September 2007). The most prominent spatial feature of the pCO2 distribution was the very high pCO2 values in Kabuno Bay (a small sub-basin with little connection to the main lake) ranging between 11213 ppm and 14213 ppm (between 18 and 26 times higher than in the main basin). Surface waters of the main basin of Lake Kivu were a net source of CO2 to the atmosphere at an average rate of 10.8 mmol m−2 d−1, which is lower than the global average reported for freshwater, saline, and volcanic lakes. In Kabuno Bay, the CO2 emission to the atmosphere was on average 500.7 mmol m−2 d−1 (~46 times higher than in the main basin). Based on whole-lake mass balance of dissolved inorganic carbon (DIC) bulk concentrations and of its stable carbon isotope composition, we show that the epilimnion of Lake Kivu was net autotrophic. This is due to the modest river inputs of organic carbon owing to the small ratio of catchment area to lake surface area (2.15). The carbon budget implies that the CO2 emission to the atmosphere must be sustained by DIC inputs of geogenic origin from deep geothermal springs.AFRIVA