Water Research 36 (2002) 469–481 Nitrifying and heterotrophic population dynamics in biofilm reactors:effectsofhydraulicretentiontimeandthepresenceof organic carbon Regina Nogueira a,b ,Lu ! ısF.Melo a , Ulrike Purkhold b , Stefan Wuertz c , Michael Wagner b, * a Department of Chemical Engineering, Faculty of Engineering of Porto, LEPAE, Porto, Portugal b Lehrstuhl fur Mikrobiologie, Technische Universit . at M . unchen, Am Hochanger 4, D-85350 Freising, Germany c Technische Universit . at M . unchen, Am Coulombwall, D-85748 Garching, Germany Received 29 September 2000; received in revised form 09 January 2001; accepted 1 February 2001 Abstract Two biofilm reactors operated with hydraulic retention times of 0.8 and 5.0h were used to study the links between population dynamics and reactor operation performance during a shift in process operation from pure nitrification to combined nitrification and organic carbon removal. The ammonium and the organic carbon loads were identical for both reactors. The composition and dynamics of the microbial consortia were quantified by fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes combined with confocal laser scanning microscopy, and digital image analysis. In contrast to past research, after addition of acetate as organic carbon nitrification performancedecreasedmoredrasticallyinthereactorwithlongerhydraulicretentiontime.FISHanalysisshowedthat this effect was caused by the unexpected formation of a heterotrophic microorganism layer on top of the nitrifying biofilm that limited nitrifiers oxygen supply. Our results demonstrate that extension of the hydraulic retention time mightbeinsufficienttoimprovecombinednitrificationandorganiccarbonremovalinbiofilmreactors. r 2002Elsevier Science Ltd. All rights reserved. Keywords: Nitrification; Biofilm; Organic carbon; Hydraulic retention time; Fluorescence in situ hybridization 1. Introduction Thecompetitionbetweenheterotrophicandnitrifying bacteriaforsubstrates(oxygenandammonia)andspace inbiofilmsisofmajorpracticalimportanceandthushas beenthesubjectofseveralpreviousstudies(forexample [1–3]). According to these investigations, competition in biofilms results in a stratified biofilm structure, the fast growingheterotrophicbacteriabeingplacedintheouter layers, where both substrate concentration and detach- ment rate are high, while the slow growing nitrifying bacteria stay deeper inside the biofilm. Thus a hetero- trophiclayercanformabovethenitrifiersinthebiofilm, whichconstitutesadisadvantagetothemwhenthebulk liquid oxygen concentration is low. In this case oxygen limitation resulting from consumption and resistance to mass transfer within the heterotrophic layer affects the nitrificationperformancenegatively.Aslongasthebulk oxygen concentration is high enough to preclude its depletion in the biofilm, however, the heterotrophic layer can also have a positive effect on the nitrifiers by protecting them from detachment [4]. Onepossibleapproachtominimizethecompetitionof heterotrophs and nitrifiers for oxygen is their spatial separation into a nitrifying biofilm population and a *Corresponding author. Tel.: +49-8161-71-5444; fax: +49- 8161-71-5475. E-mail address: wagner@mikro.biologie.tu-muenchen.de (M. Wagner). 0043-1354/02/$-see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII:S0043-1354(01)00229-9