Denitrification, activity of bacterial flocs, and growth of a filamentous bacterium in relation with the bulking of activated sludge

Activated sludge with poor settling characteristics is caught under the term "bulking" sludge. Various types of bulking activated sludge can be distinguished.1) Sludge containing an abundance of filamentous microorganisms.2) Sludge, characterized by excessive amount of bacterial slime.3) Flotating sludge caused by denitrification.

The excessive growth of filamentous bacteria, sometimes occurring in activated sludge plants, is difficult to understand. One of the hypotheses is that the filamentous bacteria protruding from floes have a better competitive ability as compared with unicellular microorganisms which occur mostly in floes, in which the growth conditions are assumed to affect adversely the development of these bacteria.

As activated sludge floes are difficultly to dispers without damaging the cells, it was decided to study the effect of the aggregative condition on the bacterial activity by using floc-forming strains of which the floes were easily dispersable by adding the enzyme cellulase. Most experiments were carried out with Zoogloea ramigera, strain I-16-M, and an Alcaligenes sp., strain 15.

As floc formation turned out to be a rather unreproducible and unpredictable phenomenon, it was often difficult to obtain sufficient amounts of floes. For this reason it was necessary to study floc formation by some pure cultures. In chapter I it was shown that floc formation of Zoogloea ramigera can be strongly stimulated by adding small amounts of NaCl or Na 2 HPO 4 /NaH 2 PO 4 to suspended cells. Unless the cells were very old, the growth phase of the culture was unimportant in respect to flee formation. The stimulatory effect of the salts could not satisfactorily be explained.

By studying the activities of cells in floes and in suspension, it was shown (chapter II) that the oxygen uptake rate of cells in floes and of cells in suspension, in the presence of a high level of substrate, represents a zero order reaction above oxygen concentrations in the medium of approx. 1.5 ppm. and approx. 0.1 ppm., respectively. In activated sludge, where only part of the floes consists of living bacteria, the rate-limiting oxygen concentration is approx. 0.5 ppm. Glucose respiration becomes rate-limiting when the glucose concentration is below approx. 20 ppm. with cells in floes and below approx. 8 ppm. with cells in suspension. In the same chapter it was shown that the protein and poly-beta-hydroxybutyrate syntheses of cells in floes of slowly shaken cultures were strongly retarded as compared with these activities in cells occurring in suspension.

The rate-limiting concentrations of oxygen and substrate (glucose) for the uptake of these compounds by cells in flocs are in the same range as the values found in many reactors of activated sludge plants.

To compare the competitive ability of Haliscomenobacter hydrossis, a filamentous bacterium, with that of the unicellular Zoogloea ramigera, continuous culture experiments were carried out. Zoogloea ramigera was the most abundant organism present in the mixed cultures at all tested dilution rates (using a complex medium). The filamentous microorganism was shown to have a low affinity towards the uptake of glucose. Both, amino acids and glucose were used as energy and C sources by this organism (chapter III).

Floc flotation, caused by N 2 gas bubbles resulting from the dissimilatory reduction of nitrate, is another aspect of the phenomenon of bulking sludge. Denitrification occurs only under anaerobic conditions. These conditions were shown to occur within flocs at relatively low oxygen concentrations of the surrounding medium. In chapter IV dissimilatory nitrate reduction in the flocs of the Alcaligenes sp. strain 15, was shown to take place as soon as the supply of oxygen within parts of the flocs was inadequate. By studying the denitrifying ability of this strain, the dissimilatory nitrate reduction turned out to be even independent of the presence of oxygen when the cells had been subjected to a special treatment. Upon aerobic precultivation of strain 15 with NH4+-N or NO3--N as nitrogen source, aerobic nitrate respiration of the washed cells was measured after nitrate addition as soon as the oxygen uptake was inhibited. This inhibition was caused by NO, the reductionproduct of nitrite (chapter V). The same aerobic dissimilatory nitrate reduction was observed with anaerobically precultivated cells which afterwards had been aerated for some hours in a nitrogen-free medium. It was shown that in these cells duringthe aeration period the NO reductase and N 2 O reductase were inactivated/broken down giving rise to the accumulation of NO and N 2 O during subsequent anaerobic nitrate respiration. When the cells were re-aerated, even very small amounts of the produced NO were sufficient to inhibit the oxygen uptake, resulting in a continued aerobic nitrate respiration.

This type of aerobic dissimilatory nitrate reduction as found with strain 15 was also found with a number of different denitrifying strains, isolated from activated sludge. The oxygen uptake of activated sludge itself could not be inhibited for a long time upon addition of NO as the presence of many NO-reducing bacteria (denitrifying bacteria) readily removed the inhibiting agent (chapter V).

In the aeration tank of an activated sludge plant, the oxygen concentration sometimes drops temporarily to such a level that the interior parts of the flocs become anaerobic. Denitrification in these anaerobic innerparts starts only when the denitrifying bacteria present possess the enzymes for the reduction reactions. In chapter VI it was proved that a number of denitrifying bacteria have the ability to synthesize the dissimilatory nitrate reductase in the presence of oxygen and ammonium ions so that these bacteria are able to start dissimilatory nitrate reduction as soon as all the oxygen has been consumed.

A more detailed study of the "constitutive" character of the synthesis of the dissimilatory nitrate reductase in strain 15 and another denitrifying strain, N4, showed that the synthesis of the enzyme by strain 15 was strongly repressed when the growth medium was over-satisfied with oxygen or upon the addition of chlorate. The synthesis of the dissimilatory nitrate reductase in strain N4 was hardly affected by the mentioned repressing agents. The repressing effect of chlorate on the synthesis of the dissimilatory nitrate reductase in cells of strain 15 was explained by the redox potential-increasing effect of this agent assuming that the redox potential is the regulating factor in the synthesis of this enzyme, rather than oxygen.