Une réévaluation de la methode d'incorporation de H14C03- pour mesurer la nitrification autotrophe et son application pour estimer des biomasses de bactéries nitrifiantes

Le processus de nitrification joue un rôle essentiel dans le cycle de l'azote dans les milieux aquatiques naturels. La mesure de l'activité nitrifiante est une étape obligée pour bien comprendre et quantifier les flux d'azote dans ces milieux. Ce travail présente une réévaluation de la méthode de mesure de l'activité nitrifiante autotrophe par la méthode d'incorporation de bicarbonate marqué au 14C et son application pour estimer des biomasses de bactéries nitrifiantes. La validité générale de la méthode a été démontrée par des tests menés sur des inhibiteurs de nitrification qui ont montré que l'utilisation combinée de N-serve (5 ppm) et de chlorate (10 mM) inhibait de manière complète et spécifique l'oxydation d'azote et l'incorporation de carbone des deux groupes de bactéries nitrifiantes. Un facteur de rendement (carbone incorporé par azote oxydé) de 0,1 mole C/mole N a également été déterminé sur des cultures pures de bactéries nitrosantes et nitratantes. Pour l'activité potentielle, en particulier, les conditions optimales pour la mesure d'activité nitrifiante ont également été établis: un pH entre 7 et 8, une température entre 20 et 30°C, une concentration en ammonium d'au moins 1 mmol/l et en oxygène d'au moins 6 mg/l. Une relation entre les mesures d'activité nitrifiante potentielle et la biomasse des bactéries nitrifiantes a été établie sur culture pure. Elle montre que dans les conditions de mesures de l'activité potentielle, 1 µg C de bactéries nitrifiantes oxyde 0,04 µmol N/hBy regenerating oxidised forms of nitrogen (nitrate), the nitrification process plays an important role in the nitrogen cycle of aquatic environments. The measurement of the activity and biomass of nitrifying bacteria is thus essential to understand and quantify the general nitrogen fluxes in those environments. Different methods of measuring the nitrifying activity exist. The first methods developed were based on the use of specific nitrification inhibitors: N-serve, allyl thio-urea, acetylene, methylfluoride and dimethyl ether, as most used. They consist in measuring differences of ammonium, nitrite and nitrate dynamics in an inhibited and control sample during time. These methods can be applied as long as the inhibitors are specific for nitrifying bacteria, and activities are high enough to allow the measurement of concentration variations during incubation times which are not too long. At the present time, the most used methods are dealing with isotopic tracers: 14C or 15N. 15N methods allow the direct measurement of the nitrifying activity, while 14C methods represent the measurement of a biomass production which can be converted into a substrate oxidation rate by the use of a yield factor. This factor is considered to be constant in the standard incubation conditions. The most frequently used enumeration methods of nitrifying bacteria are not very satisfactory. Classical culture techniques (most probable number) and immunofluorescence techniques are known to greatly underestimate the numbers of active organisms. Recently developed gene-probes techniques work well for the identification of particular strains, but are not yet useful for the numeration. A good alternative to these methods consists in the measurement of potential nitrifying activity which is correlated to the nitrifying biomass. This work presents a reassessment of the autotrophic nitrifying activity measurement by the 14C-bicarbonate incorporation method and its use to estimate the biomass of nitrifying bacteria. Several methods were used for our study: Continuous enrichment cultures of nitrifying bacteria were obtained from an inoculum coming from the Seine estuary (freshwater section). Pure cultures of Nitrosomonas europaea and Nitrobacter winogradskyi were obtained from the National Collection of Industrial and Marine Bacteria (Aberdeen, Scotland) and a continuous enrichment culture of mixed heterotrophic bacteria, without nitrifying organisms, was obtained with a freshwater inoculum by imposing a residence time of 2 hours (less than the generation time of nitrifying bacteria). Nitrifying cell numbers and size in the pure cultures were determined by epifluorescence with a microscope, after DAPI staining. Biovolumes were estimated according to cell size and converted in biomasses according to a conversion factor determined experimentally with a carbon analyser. Ammonium was measured with the indophenol blue method, nitrate was reduced in nitrite on a cadmium bed and nitrite was measured with the sulfanilamide method. Bicarbonate was measured by acid titration in natural water samples, and with the evolution method for culture samples. C incorporation rates are measured by the incubation of samples with 14C-bicarbonate, the samples being filtered on 0.2 µm membranes, acidified and counted for radioactivity by liquid scintillation. The general validity of the method was demonstrated by experiments on nitrification inhibitors in enrichment cultures. These experiments consisted in measuring the effect of different combinations of N-serve, ethanol (the organic solvent of N-serve) and chlorate, on N-oxidation rates and C incorporation rates on samples of the two nitrifying enrichment cultures (ammonium- and nitrite-oxidising bacteria). The inhibitors effects were also determined on the C incorporation rates of heterotrophic bacteria. The results showed that the use of a combination of N-serve (5 mg/l, final concentration) and chlorate (10 mmol/l, final concentration) gave the best inhibition of ammonium- and nitrite-oxidation. However, the ethanolic solution of N-serve had an unwanted result on C incorporation. The organic solvent enhanced heterotrophic incorporation of C which totally masked out the autotrophic contribution of nitrifying bacteria. For this reason N-serve was added in the empty flask before the sample to allow the evaporation of the solvent. By acting this way, inhibition of autotrophic C incorporation by nitrifying bacteria was also complete, while heterotrophic incorporation was unaffected.To measure potential nitrifying activities, the optimal growth conditions of nitrifying bacteria were determined on enrichment cultures: a pH between 7 and 8, a temperature between 20 and 30 °C, an ammonium concentration over 1 mmol/l, and an oxygen concentration over 6 mg/l. An experience consisting in following N oxidation, C incorporation and cell growth in a pure culture of Nitrosomonas europaea and Nitrobacter winogradskyi in optimal conditions allowed us to determine a yield factor (incorporated C/oxidised N) of 0.09 and 0.02 molC/molN for the ammonia- and nitrite-oxidising bacteria respectively. The determined optimal growth rate was 0.05 h-1 for the two nitrifying species. The specific activity of nitrifying bacteria, which correspond to the maximum N-oxidation rate of 1 µg C of nitrifying bacteria, is given by the ratio between the growth yield and the growth rate of those organisms. This factors allowed us to establish a relationship between potential nitrifying activity measurements and nitrifying biomass: in optimal growth conditions, 1 µgC of ammonium-oxidising bacteria oxidised 0.05 µmolN/h and 1 µgC of nitrite-oxidising bacteria oxidised 0.21 µmolN/h.Our conclusion is that the results presented in this paper allow the validation of the 14C-bicarbonate incorporation method with and without inhibitors to measure the nitrifying activity. The main differences of our protocol to the original ones is that we propose the use of a combination of 2 inhibitors, N-serve and chlorate, and the elimination by evaporation of the organic solvent of N-serve (ethanol) to avoid any interference with the heterotrophic populations. The method can be used in in situ conditions, to allow real nitrifying activities measurements in samples. In this case, carbon incorporation rates can be converted in ammonium oxidation rates with the use of the conversion factor 0.11 µmoles incorporated C by µmoles oxidised N (0.09 molC/molN for ammonium oxidation and 0.02 for nitrite-oxidation). The method can also be used by placing the sample in optimal temperature, pH, oxygen and ammonium conditions for nitrifying bacteria, to allow potential nitrifying activity measurements. This potential activity can be used to estimate the nitrifying biomass by considering a conversion factor of 0.04 µmolN/h/µgC (0.05 µmolN/h/µgC for ammonium-oxidation and 0.21 µmolN/h/µgC for nitrite-oxidation). The rapidity of the method, itís sensitivity and the fact that no special equipment is needed, except the one for 14C detection, makes it a very useful method in aquatic ecology

Distribution of nitrifying activity in the Seine River (France) from Paris to the estuary

The distribution of nitrification has been measured with the H14CO3- incorporation method in the Seine River and its estuary during summer conditions. The Seine River below Paris receives large amounts of ammonium through wastewater discharge. In the river itself, this ammonium is only slowly nitrified, while in the estuary nitrification is rapid and complete. We show that this contrasting behavior is related to the different hydrosedimentary conditions of the two systems, as nitrifying bacteria are associated with suspended particles. In the river, particles and their attached bacteria either rapidly settle or have a sestonic behavior. Because of the short residence times of the water masses, the dow growing nitrifying population has no time to develop sufficiently to nitrify the available ammonium. The estuary is characterized by strong tidal dynamics. Particles settle and are resuspended continuously with the strong current inversions of ebb and hood. As a result of these dynamics, particles and their attached nitrifying bacteria experience longer residence times in a temporary suspended state than the water masses themselves, providing to slow growing nitrifying bacteria the opportunity to develop a large population capable of nitrifying all the available ammonium

Mission critical database for SPS accelerator measurements

In order to maintain efficient control over the hadron and lepton beams in CERN¹s SPS accelerator, measurements are of vital importance. Beam parameters such as intensities, positions and losses need to be rapidly available in the SPS control room to allow the operators to monitor, judge and act on beam physics conditions. For the 1994 SPS startup, a completely new and redesigned measurement system based on client and server C-programs running on UNIX-workstations was introduced. The kernel of this new measurement system is an on-line ORACLE database.The NIAM method was used for the database design as well as a technique to tag synchronized data with timeslots instead of timestamps. A great attention was paid to proper storage allocation for tables and indices since this has a major impact on the efficiency of the database, due to its time-critical nature. Many new features of Oracle7 were exploited to reduce the surrounding software.During the 1994 SPS physics run, this new measurement system was commissioned successfully and the infrastructure proved to be acceptably reliable. Hence, for the 1995 startup, the size of the measurement system was increased drastically to fulfill a variety of measurement needs. This proliferation of measurements beyond the initial scope showed the correct design of the system, as well as the performance limitations within the actual hardware configuration. This paper describes the overall design and discusses performance issues of this critical system

The nitrogen cycle in the Seine and Scheldt estuaries

The Seine and Scheldt estuaries are both located in the same geographical area and they represent important tributaries of the North Sea. Due to their high population density and large agricultural areas, the Seine and Scheldt estuaries receive extremely high nitrogen loads, mainly originating from wastewater and land fertilisers. The way this nitrogen is transformed in the system and exported to the North Sea depends on the characteristics of each system. The Seine and Scheldt estuaries are the outlet of river systems with respectively 78600 km² and 21500 km² watershed area, 420 and 108 m³/s average discharge, 46 and 40 % of agriculture area, and 195 and 425 inh./km² population density. Major difference between the estuaries lays in their hydrological characteristics. While the Seine estuary is typically channelled over most of its length with very reduced intertidal areas and short residence times (in the order of 1 week on the average), the Scheldt estuary has a typical funnel shape with large intertidal areas and long residence times (in the order of 1 month). The influence of these characteristics on major N-transformation processes (inorganic N assimilation by plankton, organic N mineralisation, nitrification, and denitrification) is presented and discussed, and the importance of both estuaries as sources of N for the North Sea is be evaluated

The LHC Logging Service : Handling terabytes of on-line data

Based on previous experience with LEP, a long-term data logging service for the LHC was developed and put in place in 2003, several years before beam operation. The scope of the logging service covers the evolution over time of data acquisitions on accelerator equipment and beam related parameters. The intention is to keep all this time-series data on-line for the lifetime of LHC, allowing easy data comparisons with previous years. The LHC hardware commissioning has used this service extensively prior to the first beams in 2008 and even more so in 2009. Current data writing rates exceed 15TB/year and continue to increase. The high data volumes and throughput rates, in writing as well as in reading, require special arrangements on data organization and data access methods

Eignung der Gammaspektrometrie zum Kartieren der Mächtigkeit und der C-Vorräte von Moorböden

Moore stellen einen der großen variablen Bodenspeicher für organisches C dar. Es wird geprüft werden, ob die Gammaspektrometrie zum Kartieren von Moorböden in Südwestdeutschland anwendbar ist. Im Ergebnis lassen sich grundsätzlich mit der Gammaspektrometrie Torfmächtigkeiten bis zu einem Meter in Abhängigkeit der Strahlungseigenschaften des unterliegenden Substrates differenzieren. Allerdings ist in Landschaftsräumen mit unterschiedlicher Geologie auch eine getrennte Kalibrierung dieses Verfahrens erforderlich sind

Generation of angular-momentum-dominated electron beams from a photoinjector

Various projects under study require an angular-momentum-dominated electron beam generated by a photoinjector. Some of the proposals directly use the angular-momentum-dominated beams (e.g. electron cooling of heavy ions), while others require the beam to be transformed into a flat beam (e.g. possible electron injectors for light sources and linear colliders). In this paper, we report our experimental study of an angular-momentum-dominated beam produced in a photoinjector, addressing the dependencies of angular momentum on initial conditions. We also briefly discuss the removal of angular momentum. The results of the experiment, carried out at the Fermilab/NICADD Photoinjector Laboratory, are found to be in good agreement with theoretical and numerical models.Comment: 8 pages, 7 figures, submitted to Phys. Rev. ST Accel. Beam