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

Abstract

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 de...

The response of river nitrification to changes in wastewater treatment (The case of the lower Seine River downstream from Paris)

“…This water mass is subject to the inputs of organic matter, nitrogen nutrients and microorganisms from the SAV plant as estimated above, and the dynamics of nitrogen species and nitrifying microorganisms is represented according to the formalism developed for the RIVE model based on studies of cultivated bacterial populations and experiments with natural communities (Billen, 1991;Billen et al, 1994;Brion and Billen, 1998;Garnier et al, 2002Garnier et al, , 2005Garnier et al, , 2007Ce´bron et al, 2004). Table 3 (400 and 600 m 2 , respectively, for upstream and downstream of the confluence with the Oise River) and therefore only determined by the Seine flow at the Paris intra muros station (150 m 3 .s x1 ) and the Oise flow at the Conflans station (100 m 3 .s x1 ).…”

“…x .L x1 of KNO 2 (Brion and Billen, 1998), i.e., at least ten times the half-saturation constant values of ammonium-and nitrite-oxidizing microorganisms. To determine the ammonium and nitrite oxidation rates, triplicate tests (with substrates, no inhibitor) were prepared and two other sets of triplicate separate samples were inhibited for all stations.…”

“…To express these potential activity measurements in terms of biomass of the corresponding microorganism, we used the values proposed by Brion and Billen (1998) for the maximum specific activity of ammonium-and nitrite-oxidizing microorganisms, respectively 0.95 and 3 mgN.h .mgC x1 at 20 xC:…”

The influence of macrofaunal burrow spacing and diffusive scaling on sedimentary nitrification and denitrification: An experimental simulation and model approach

“…The constants v NO3 , K O2,NO3 , and K NH4,NO3 correspond to maximum reaction rate and half saturation constants for O 2 and NH 4 ϩ respectively. Maximum reaction, v NO3 , depends directly on local biomass (Brion and Billen, 1998). The second order kinetic form with fixed rate constant k NO3 , Eq.…”

Lagoon pollution and nutritive compounds change in urban tropical area

“…The dissimilatory reactions from NO 3 -leading to NO 2 -coupled by reduction to nitrate ammonification triggering the NH 4 + / NH 3 production (Castel et al, 1996;Souchu et al, 1998). We could not confirm the extent of nitrification activity in 14 C-tracing method since this test has been achieved (Brion and Billen, 1998). In Figure 4, Nitrating bacterial populations study is very diversified in phylogenetic terms particularly those of Nitrobacter genus, known to be widely distributed in aquatic environments may provide specific and confirm the presence of groups of bacteria responsible for these phenomena (nitrification and ammonification) (Cébron et al, 2003).…”