Influence possible des protozoaires sur le taux de mortalité des bactéries autotrophes nitrifiantes Possible Effect of Protozoa on the Autotrophic Decay Rate Value

Résumé

Le modèle de l'IAWQ du processus de boues activées représente les mécanismes endogènes de la biomasse nitrifiante par le décès des micro-organismes (équation d'ordre 1 par rapport à la biomasse). La constante de décès, ou taux de mortalité b_A, est aujourd'hui encore mal connue, et en particulier les facteurs influants sur sa valeur. De récentes études ont montré que la prédation par la microfaune pourrait être un facteur déterminant sur la valeur de b_A. Cette étude se propose donc de quantifier l'effet de la prédation sur la valeur de b_A. Deux réacteurs maintenus sans alimentation en substrat ont été caractérisés en parallèle: l'un a reçu une dose d'antibiotique spécifique aux eucaryotes (cycloheximide) afin de diminuer la quantité d'organismes de la microfaune, alors que l'autre n'a reçu aucun antibiotique (témoin). Les résultats obtenus montrent que le cycloheximide inhibe la plupart des organismes de la microfaune sauf les amibes; celles-ci semblent plutôt stimulées par cet antibiotique. En ce qui concerne la nitrification, un ralentissement de la production de nitrate dans le réacteur traité à l'antibiotique est observé à partir du sixième jour. Cette diminution de production de nitrate est probablement causée par une réduction de l'azote nitrifiable (qui est mobilisé par les amibes) couplée à une prédation des organismes nitrifiants par les amibes. D'ailleurs, l'augmentation de la prédation par les amibes à partir du jour 6 a diminué l'activité nitrifiante également mesurée par respirométrie (rO₂ Nmax). Cette diminution du taux de respiration indique une augmentation du taux de mortalité (bA) des organismes nitrifiants. En effet, la valeur du taux de mortalité mesurée dans le réacteur témoin est de 0.08 d^-1 alors que selon la microfaune présente dans le réacteur inhibé au cycloheximide, la valeur de ce taux de mortalité a varié entre 0.05 d^-1 et 0.15 d^-1.

Mots-clés:

• Protozoaires,
• prédation,
• nitrification,
• taux de mortalité,
• eaux usées,
• respirométrie,
• boue activée

Abstract

Designing biological wastewater treatment plants with the aid of the model developed by the IAWQ requires the knowledge of biological kinetic parameters. For nitrifying activated sludge, these parameters are related to nitrifying bacteria: maximum autotrophic growth rate µ_Amax, yield coefficient YA and the autotrophic decay rate b_A. Although variables influencing µ_Amax and Y_A values are well known, this is not the case for bA. MARTINAGE and PAUL (2000) have recently shown that the bA value is strongly influenced by the influent quality, leading to the assumption that influent quality has a strong effect on microfauna composition, and consequently on the grazing rate of microfauna on nitrifying bacteria. In fixed-film processes, protozoan grazing reduces the bacterial population considerably (NATUSCKA and WELANDER, 1994). However, although many data are available concerning the grazing rates of different protozoa, the effect of microfaunal grazing on nitrification is still a matter of debate (RATSAK et al., 1994) and its effect on the b_A value is still unknown. These two topics are investigated here.

Nitrifying activated sludges were grown in two identical batch reactors, but in one, cycloheximide was added to inhibit eucaryotic growth (MAURINES CARBONEILL et al., 1998). Microfauna organism numbers were quantified in both reactors by microscopic observations of flagellated protozoa (>8 µm), amoebae, ciliates, rotifers and higher invertebrates (Fig. 3). Microbial counts were then correlated with the b_A value. The latter was determined using the procedure proposed by SALZER (1992) which consists of characterising the time behaviour of the maximum nitrification rate measured by respirometry of activated sludge under substrate starvation. Under these conditions bacteria die and organic nitrogen is released into the bulk phase. This nitrogen is ammonified, and nitrifying bacteria use this substrate to produce nitrate, and then autotrophic bacterial growth occurs. This method takes this growth into account by characterising nitrate production during the experiment (Fig. 2).

The effect of cycloheximide on nitrification was first determined to make sure that this compound is not inhibitory toward nitrifiers. Results obtained (Table 1) show that cycloheximide was not inhibitory toward nitrate production or the maximum nitrification oxygen uptake rate (rO₂ N) after 4 hours of contact with nitrifying biomass. Cycloheximide addition in the activated sludge had an important impact on rotifers and flagellates but no effect on ciliates; it also seemed to stimulate amoebae growth. In both reactors, flagellates were mainly Peranama, attached ciliates were mainly Opercularia and Epistylis and a few Vorticella. Free ciliates like Aspidisca and Euplotes were found in both reactors.

Variation with time of the abundance of microfauna organisms is shown in Figures 4 and 5 for both reactors. In the reference reactor the number of microfauna organisms decreased with time (Fig. 4) probably due to substrate starvation. Microfauna composition remained however diversified. For the inhibited reactor (Fig. 5), three periods were observed. During period I, the microfauna was mainly composed of ciliates and the number of microfaunal organisms decreased rapidly. During period II, an important growth of amoebae was observed. Cycloheximide was then added during this period to reduce their number. This growth of amoebae seems to be caused by the resistance of these micro-organisms toward inhibiting compounds (SRIKANTH et BERK, 1993). During period III, the number of microfaunal organisms was lower than during period II, and microfauna was mainly composed of ciliates.

Nitrate concentration behaviour, necessary for bA calculation, is shown on Figure 6. In the reference reactor, nitrate concentrations varied linearly. For the inhibited reactor, the linear pattern was not observed during period II. This result was probably caused by an important nitrogen assimilation need of amoebae (ELDRIGE and JACKSON, 1993). Because organic nitrogen released by bacterial decay is consumed by amoebae assimilation, less nitrogen is available for the ammonification process and therefore for nitrification. Ammonia concentrations remained below 0.2 mg N·l^-1 during all the experiment for both reactors. When amoebae disappeared from the inhibited reactor (period III) nitrate concentration varied linearly again.

Variations of the maximum nitrification oxygen uptake rate (rO₂ N_max) with time are presented in Figure 7 (A&B) for both reactors. Two curves are plotted on each figure. Empty squares represent the measured rO₂ N and black points represent the maximum nitrification rate that would have been measured if no growth on ammonification products had occurred. For the reference reactor (Fig. 7A), a value for b_A of 0.08 d^-1 can be calculated and can be considered constant for a constant microfauna composition.

Three b_A values can be estimated for the reactor inhibited with cycloheximide (Fig. 7B), corresponding to the three periods observed for microfauna composition. During period I, the bA value is 0.05 d^-1 : a decrease in the microfaunal organism numbers implies a decrease of the b_A value. During period II, when a development of amoebae is observed, the bA value increases and reaches 0.15 d^-1. During period III with reduced grazing, the b_A value is 0.13 d-1. Since during periods I and III the microfauna is mainly composed of ciliates, this difference between b_A values is likely due to the observed difference in floc size between periods I and III.

The results obtained during this study tend to prove (1) that the use of cycloheximide reduces microfaunal populations but can lead to a development of amoebae, and (2) that microfauna grazing seems to have an influence on the b_A value, which can vary from 0.05 to 0.15 d^-1 depending on microfaunal composition and abundance.

Keywords:

• Protozoa,
• grazing,
• nitrification,
• decay rate,
• wastewater,
• respirometry,
• activated sludge