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Applied and Environmental Microbiology, March 2000, p. 1158-1166, Vol. 66, No. 3
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Quantitative Use of Fluorescent In Situ Hybridization To Examine Relationships between Mycolic Acid-Containing Actinomycetes and Foaming in Activated Sludge Plants

Russell J. Davenport,1,2 Thomas P. Curtis,1,2,* Michael Goodfellow,2,3 Fiona M. Stainsby,2,3 and Marc Bingley4

Departments of Civil Engineering1 and Agricultural and Environmental Science3 and Centre for Molecular Ecology,2 University of Newcastle, Newcastle upon Tyne, NE1 7RU, and Severn-Trent Water Ltd., Process Development, Finham Sewage Treatment Works, Coventry, CV3 6PR,4 United Kingdom

Received 7 September 1999/Accepted 30 November 1999

The formation of viscous foams on aeration basins and secondary clarifiers of activated sludge plants is a common and widespread problem. Foam formation is often attributed to the presence of mycolic acid-containing actinomycetes (mycolata). In order to examine the relationship between the number of mycolata and foam, we developed a group-specific probe targeting the 16S rRNA of the mycolata, a protocol to permeabilize mycolata, and a statistically robust quantification method. Statistical analyses showed that a lipase-based permeabilization method was quantitatively superior to previously described methods (P << 0.05). When mixed liquor and foam samples were examined, most of the mycolata present were rods or cocci, although filamentous mycolata were also observed. A nested analysis of variance showed that virtually all of the measured variance occurred between fields of view and not between samples. On this basis we determined that as few as five fields of view could be used to give a statistically meaningful sample. Quantitative fluorescent in situ hybridization (FISH) was used to examine the relationship between foaming and the concentration of mycolata in a 20-m3 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 × 106 cells ml-1 or 4 × 1012 cells m-2. We concluded that quantitative use of FISH is feasible and that quantification is a prerequisite for rational investigation of foaming in activated sludge.

* Corresponding author. Mailing address: Department of Civil Engineering, University of Newcastle, Newcastle upon Tyne, NE1 7RU, United Kingdom. Phone: 44-191-222-6690. Fax: 44-191-222-6669. E-mail: tom.curtis{at}ncl.ac.uk.

Applied and Environmental Microbiology, March 2000, p. 1158-1166, Vol. 66, No. 3
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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