Une réévaluation de la methode d'incorporation de H14C03- pour mesurer la nitrification autotrophe et son application pour estimer des biomasses de bactéries nitrifiantes A reassessment of the H14C03- incorporation method for measuring autotrophic nitrification and its use to estimate the biomass of nitrifying bacteria

Résumé

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/h

Mots-clés:

• Activité nitrifiante,
• biomasse nitrifiante,
• incorporation de ¹⁴C-bicarbonate,
• inhibiteurs de nitrification,
• N-serve,
• chlorate

Mots-clés:

• Nitrifying activity,
• nitrifying biomass,
• ¹⁴C-bicarbonate incorporation,
• nitrification inhibitors,
• N-serve,
• chlorate

Abstract

By 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.