Wat. Res. Vol. 35, No. 14, pp. 3325–3336, 2001 # 2001 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/01/$-see front matter PII: S0043-1354(01)00057-4 QUANTIFYING FILAMENTOUS MICROORGANISMS IN ACTIVATED SLUDGE BEFORE, DURING, AND AFTER AN INCIDENT OF FOAMING BY OLIGONUCLEOTIDE PROBE HYBRIDIZATIONS AND ANTIBODY STAINING D.B.OERTHER,F.L.DELOSREYESIII,M.F.DELOSREYESandL.RASKIN* Department of Civil and Environmental Engineering, University of Illinois at Urbana Champaign, 3221 Newmark Civil Engineering Laboratory, 205 North Mathews Avenue, Urbana, IL 61801, USA (First received 1 April 2000; accepted in revised form 1 January 2001) Abstract}Quantitative oligonucleotide probe hybridizations, immunostaining, and a simple foaming potentialtestwereusedtofollowanincidentofseasonalfilamentousfoamingattheUrbana-Champaign SanitaryDistrict,NortheastWastewaterTreatmentPlant.Apositivecorrelationwasobservedbetweenan increaseinfoamingpotentialandtheappearanceoffoamonthesurfacesofaerationbasinsandsecondary clarifiers.Inaddition,duringtheoccurrenceoffoaming,themassandactivityof Gordonia spp.increased as measured by fluorescence in situ hybridization, antibody staining, and quantitative membrane hybridization of RNA extracts. An increase in Gordonia spp. rRNA levels from 0.25 to 1.4% of total rRNA was observed using quantitative membrane hybridizations, whereas during the same period, the fractionofmixedliquorvolatilesuspendedsolidsattributedto Gordonia spp.increasedfrom4%tomore than32%ofthetotalmixedliquorvolatilesuspendedsolids.Theseresultsindicatethatboththeactivity and biomass level of Gordonia spp. in activated sludge increased relative to the activity and the biomass level of the complete microbial community during a seasonal occurrence of filamentous foaming. Thus, Gordonia spp. may represent a numerically dominant but metabolically limited fraction of the total biomass,andtheroleof Gordonia spp.infilamentousfoamingmaybelinkedmoretightlytothephysical presence of filamentous microorganisms than to the metabolic activity of the cells. # 2001 Elsevier Science Ltd. All rights reserved Key words}contact stabilization activated sludge, filamentous foaming, wastewater treatment, oligonucleotide hybridization probes, ribosomal RNA, antibody stain NOMENCLATURE ANOVA analysis of variance BOD 5 five-day biochemical oxygen demand, mgl 1 DO dissolved oxygen, mgl 1 FISH fluorescence in situ hybridization FITC fluorescein isothiocyanate F/M food to microorganism ratio, g BOD 5 [g suspended solids day] 1 MLSS mixed liquor suspended solids, mgl 1 RAS return activated sludge RNA ribonucleic acid rRNA ribosomal ribonucleic acid SI scum index SRT solids retention time, day SVI 30 thirty-min sludge volume index, mlg 1 TRITC tetramethyl rhodamine isothiocyanate UCSD, Urbana-Champaign Sanitary District, North east Wastewater Treatment Plant NEWWTP WAS waste activated sludge INTRODUCTION Filamentous foaming, the formation of a ‘‘stable, viscous, chocolate-colored scum layer’’ (Soddell and Seviour, 1990) on the surfaces of activated sludge aerationbasinsandsecondaryclarifiers,isacommon problem in wastewater treatment plants. Research using classical microbiological techniques, such as microscopic observation and culture-based enumera- tion, has demonstrated a link between the predomi- nance of filamentous microorganisms and instances of foaming in laboratory-, pilot-, and full-scale wastewater treatment plants (Goddard and Forster, 1986; Pitt and Jenkins, 1990; Jenkins et al., 1993). The current understanding of filamentous foaming suggests that certain conditions experienced by the activated sludge (e.g., low dissolved oxygen concen- trations [DO], low food to microorganism ratios [F/M],orhighsolidsretentiontimes[SRTs])createa selective advantage for the growth of filamentous microorganisms, which are subsequently captured in the wastewater treatment plant through physical configurations (e.g., scum baffles in the secondary *Author to whom all correspondence should be addressed. Tel.: +1-217-333-6964; fax: +1-217-333-6968 or -9464; e-mail: lraskin@uiuc.edu 3325