Quantitative use of fluorescent in situ hybridization to examine relationships between mycolic acid-containing actinomycetes and foaming in activatedsludge plants

The formation of viscous foams on aeration basins and secondary clarifiers of activated sludge plants is a common and widespread problem. Foam formati on is often attributed to the presence of mycolic acid-containing actinomyc etes (mycolata). In order to examine the relationship between the number of mycolata and foam, we developed a group-specific probe targeting the 16S r RNA of the mycolata, a protocol to permeabilize mycolata, and a statistical ly robust quantification method, Statistical analyses showed that a lipase- based permeabilization method was quantitatively superior to previously des cribed methods (P << 0.05), When mixed liquor and foam samples were examine d, most of the mycolata present were rods or cocci, although filamentous my colata were also observed. A nested analysis of variance showed that virtua lly all of the measured variance occurred between fields of view and not be tween samples. On this basis we determined that as few as five fields of vi ew could be used to give a statistically meaningful sample. Quantitative fl uorescent in situ hybridization (FISH) was used to examine the relationship between foaming and the concentration of mycolata in a 20-m(3) completely mixed activated sludge plant, Foaming occurred when the number of mycolata exceeded a certain threshold value. Baffling of the plant affected foaming without affecting the number of mycolata, We tentatively estimated that the threshold foaming concentration of mycolata was about 2 x 10(6) cells ml(- 1) or 4 x 10(12) cells m(-2). We concluded that quantitative use of FISH is feasible and that quantification is a prerequisite for rational investigat ion of foaming in activated sludge.