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Antonie van Leeuwenhoek 81: 665680, 2002. 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Microbial community composition and function in wastewater treatmentplants

Michael Wagner

, Alexander Loy, Regina Nogueira, Ulrike Purkhold, Natuschka Lee & HolgerDaims Microbial Ecology Group, Lehrstuhl fr Mikrobiologie, Technische Universitt Mnchen, Am Hochanger 4, 85350Freising, Germany ( Author for correspondence; Tel.: +49-8161-71-5444; Fax: +49-8161-71-5475; E-mail:wagner@microbial-ecology.de)

Key words: FISH, microautoradiography, microbial ecology, nitrogen removal, phosphorus removal, wastewatertreatment

Abstract

Biological wastewater treatment has been applied for more than a century to ameliorate anthropogenic damage tothe environment. But only during the last decade the use of molecular tools allowed to accurately determine thecomposition, and dynamics of activated sludge and biolm microbial communities. Novel, in many cases yet notcultured bacteria were identied to be responsible for lamentous bulking and foaming as well as phosphorus andnitrogen removal in these systems. Now, methods are developed to infer the in situ physiology of these bacteria.Here we provide an overview of what is currently known about the identity and physiology of some of the microbialkey players in activated sludge and biolm systems.

Abbreviations: EBPR enhanced biological phosphorusremoval; FISH uorescence in situ hybridization; GAO glycogen-accumulating organism; PAO polyphosphate-accumulating organism; PHA polyhydroxyalkanoates;wwtp wastewater treatment plant

Introduction

Wastewater treatment is one of the most importantbiotechnological processes which is used worldwideto treat municipal and industrial sewage. This reviewfocuses on the microbiology of the activated sludgeprocess. In addition, we also cover activated sludgeand biolm nutrient removal plants in which anaer-obic and aerobic treatments are combined to allowfor complete nitrogen and/or biologically enhancedphosphorus removal. In all plant types, prokaryoticmicroorganisms dominate and are responsible for the

observed conversions. On the other hand, certain mi-croorganisms cause the most frequently encounteredproblems in wastewater treatment like activated sludgebulking and foaming. Consequently, the efciency androbustness of a wwtp mainly depend on the composi-tion and activity of its microbial community. Althoughbiological wastewater treatment has been used formore than a century, research on the microbiology

of this process suffered from severe methodologicallimitations (Wagner 1993) until a decade ago. Onlyafter the introduction of a set of different moleculartechniques in wastewater microbiology (for exampleWagner 1993, 1994; Bond 1995; Schramm 1996;Snaidr 1997; Juretschko 1998; Lee 1999; Purkhold2000), it has become possible to determine the com-position and dynamics of microbial communities inthese systems and to identify the microbial key play-ers for the different process types. It is the aim of this review to summarize these new insights and toprovide some outlook on how this knowledge could be

used to improve the performance of wwtps. It shouldbe noted that the bacterial nomenclature proposed inthe taxonomic outline (released 1, April 2001) of the second edition of Bergeys manual of systematicbacteriology (http://www.cme.msu.edu/bergeys/) wasused throughout this review. Furthermore, anaerobicammonium oxidizing bacteria, which were recentlydetected in wwtps (Schmid 2000, 2001), are not in-

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cluded in this review because a separate manuscriptdealing with these bacteria is included in the ISME 9special volume of Antonie van Leeuwenhoek (Strous2002).

Microbial diversity of wastewater treatment plants

The species richness of the microbial communitiesof ve laboratory-scale reactors and three wwtps hasbeen analyzed by 16S rRNA gene phylogenetic in-ventories established by using bacterial or universalprimers (Bond 1995; Snaidr 1997; Christensson 1998;Dabert 2001; Daims 2001; Liu 2001; Juretschko2002). Results of these surveys are summarized inTable 1. It is obvious that the number of plants ana-lyzed by the 16S rRNA approach is too low to inferultimate conclusions. However, already in the avail-able studies members of 13 bacterial divisions, of the36 divisions currently identied for the bacterial do-main (Hugenholtz 1998), were detected, indicatingconsiderable microbial diversity in wwtps. Consist-ent with previous quantitative FISH experimentsusinggroup-specic rRNA-targeted oligonucleotide probes(Wagner 1993, 1994; Manz 1994; Kmpfer 1996;Manz 1996; Neef 1996; Bond 1999; Liu 2001), Pro-teobacteria are abundant in each library and representmore than 50% of the clones in ve of the eightsurveys. With one exception (Christensson 1998), Betaproteobacteria are the most frequently retrievedmembers of this division. Apart from the Proteobac-

teria , molecular isolates afliated to the Bacteroidetes ,the Chloroexi and the Planctomycetes were retrievedin signicant numbers in several of the libraries. Onelibrary of a reactor designed for enhanced biologicalphosphorus removal in a continuous ow system isdominated by high-G+C Gram positive bacteria ( Ac-tinobacteria ), consistent with the proposed import-ance of these bacteria for phosphorus removal (seebelow) (Wagner 1994).

However, the relatively low coverage values (Gio-vannoni 1995; Singleton 2001, Juretschko 2002) of some of these libraries demonstrate that the number

of clones analyzed were too low in these studies to ad-equately represent the diversity in the established lib-raries (Table 1). Furthermore, the 16S rRNA approachsuffers from numerous biases introduced in the DNAextraction, PCR amplication, and cloning proced-ures (Meyerhans 1990; Suzuki & Giovannoni 1996;Juretschko 1998; Suzuki 1998). Therefore quantitat-ive data on the microbial community composition can

only be obtained if the 16S rRNA approach is com-bined with quantitative dot blot or in situ hybridizationtechniques.

So far, the microbial community structures of ac-tivated sludges from only two wwtps have been in-vestigated using the full-cycle rRNA approach which

includes the establishment of a 16S rRNA gene clonelibrary, the design of a set of clone-specic oligo-nucleotide probes for FISH, and the determination of the abundance of the respective bacterial populationsby quantitative FISH. Snaidr et al. analyzed a high-load aeration basin of a large municipal wwtp (Amann1996; Snaidr 1997) while Juretschko et al. studiedan intermittently aerated industrial wwtp containinga nitriying and denitrifying microbial community(Juretschko 1998; Juretschko 2002). The results fromthe respective 16S rRNA gene clone libraries areshown in Table 1. Snaidr and colleagues designedprobes for a few selected clones and found a highmicrodiversity of bacteria of the beta1 group of Beta- proteobacteria in the municipal activated sludge. Fur-thermore, 3 and 4% of all microbial cells in this systemcould be assigned to Sphingomonas - and Arcobacter -related populations, respectively.

The composition of the microbial community inthe industrial plant was investigated in more detailusing semi-automatic quantitative FISH (Juretschko2002). Hybridization with group-specic probesdemonstrated that the Betaproteobacteria made up al-most half of the total biovolume of those bacteriadetectable with the bacterial probe set. Other in situ

important groups were the Alphaproteobacteria , the Nitrospira -phylum, the Planctomycetes , and the Chlo-roexi . The composition of the Betaproteobacteriawithin this system was further analyzed using clone-specic probes for quantitative FISH. Bacteria relatedto Zoogloea ramigera and Azoarcus sensu lato werethe most abundant members of this class in situ ,and accounted for 36 and 34% of the biovolume of the Betaproteobacteria . In addition, signicant num-bers of the ammonia-oxidizer Nitrosococcus mobilis(which was not present in the clone library), Alcali-genes latus -, and Brachymonas denitricans -relatedmicroorganisms were recorded. In total only 2% of the Betaproteobacteria detectable in situ could not beassigned to a specic genus.

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T a b l e 1

. S u m m a r y o f

1 6 S r R

N A g e n e - b a s e d

d i v e r s

i t y s u r v e y s o f w a s t e w a t e r t r e a t m e n t p l a n t s a n

d r e a c t o r s .

S B R = s e q u e n c i n g

b a t c h r e a c t o r .

S B B R = s e q u e n c i n g

b i o

l m b a t c h r e a c t o r .

C l o n e s w e r e a s s i g n e d t o d i f f e r e n t O T U s

( o p e r a t i o n a l t a x o n o m

i c u n

i t s )

i f t h e y s h a r e d

l e s s t h a n

9 7 % 1 6 S r R

N A g e n e s e q u e n c e s i m

i l a r

i t y w

i t h e a c h o t

h e r

( a d a p t e d

f r o m

A . L

o y , 2

0 0 2 )

w w t p s a n

d l a b - r e a c t o r s

N o . o f

C o v e r a g e

B a c t e r i a l p h y l a 9

( N o . o f

O T U s

i n r e s p e c t i v e p h y l u m

)

C l o n e s

O T U s

C 1 0 [ % ]

P r o t e o

b a c t e r

i a 8

s e q u e n c e

d

B a c t e r o i d e t e s

A c i d o b a c t e r i a

F i r m i c u t e s

A c t i n o b a c t e r i a

N i t r o s p i r a

V e r r u c o m i c r o b i a

P l a n c t o m y c e t e s

C h l o r o b i

C h l o r o e x i

F i b r o b a c t e r e s

F u s o b a c t e r i a

O P 1 1

U n a f l i a t e d

H i g h - l o a d a e r a t i o n

b a s i n

o f a

6 2

2 5

7 7

3 ( 1 ) 5 2 ( 9 )

1 8 ( 7 )

1 5 ( 1 )

1 0 ( 5 )

2 ( 1 )

2 ( 1 )

f u l l

- s c a

l e m u n

i c i p a l p

l a n t

1

N i t r i

f y i n g /

d e n i t r i f y i n g

9 4

5 3

6 4

2 6 ( 1 5 ) 3 1 ( 9 )

2 ( 2 )

5 ( 3 ) 1 ( 1 )

1 ( 1 ) 2 ( 1 ) 3 ( 2 ) 1 2 ( 1 0 ) 1 ( 1 ) 1 6 ( 8 )

i n d u s t r i a l p l a n t 2

N i t r i

f y i n g

S B B R 1 3

9 6

3 3

7 8

5 ( 4 ) 5 1 ( 1 1 ) 2 2 ( 6 ) 4 ( 4 )

1 ( 1 )

2 ( 2 )

5 ( 2 )

1 ( 1 ) 8 ( 2 )

E B P R l a b - s c a l e

S B R 1 +

9 7

6 9

4 8

1 3 ( 8 ) 3 3 ( 2 0 )

8 ( 4 ) 3 ( 3 )

2 ( 2 )

5 ( 5 )

3 ( 2 ) 1 ( 1 )

4 ( 3 ) 3 ( 3 ) 1 ( 1 ) 1 3 ( 9 )

4 ( 3 )

3 ( 2 )

3 ( 3 )

s o d i u m a c e t a t e 4

E B P R l a b - s c a l e

S B R 2

9 2

7 5

2 8

1 7 ( 1 5 ) 2 5 ( 1 3 ) 1 0 ( 9 ) 1 ( 1 )

7 ( 5 ) 1 3 ( 9 )

7 ( 5 )

2 ( 2 ) 2 ( 2 ) 1 ( 1 )

9 ( 8 )

3 ( 3 )

2 ( 2 )

u n s u p p

l e m e n t e d 4

E B P R l a b - s c a l e c o n t

i n u o u s

5 1

3 0

5 1

1 6 ( 8 )

8 ( 2 )

8 ( 3 ) 4 ( 2 )

4 ( 2 )

6 ( 1 )

2 ( 1 )

3 7 ( 3 )

8 ( 4 )

2 ( 1 )

6 ( 3 )

o w r e a c t o r 5

E B P R l a b - s c a l e

S B R

6

9 2

5 0

4 6

4 ( 3 ) 1 7 ( 8 )

5 ( 2 ) 3 ( 3 )

3 9 ( 1 7 ) 9 ( 6 )

4 ( 2 ) 3 ( 1 ) 4 ( 3 )

3 ( 2 )

3 ( 2 )

1 ( 1 )

E B P R l a b - s c a l e r e a c t o r 7

1 5 0

1 6

9 3

5 ( 1 ) 1 4 ( 1 )

7 ( 2 ) 1 ( 1 )

5 0 ( 7 )

5 ( 1 )

9 ( 2 )

9 ( 1 )

1 ( S n a

i d r

1 9 9 7 ) ;

2 ( J u r e t s c h

k o 2 0 0 2 ) ;

3 ( D a i m s

2 0 0 1 a ) ;

4 ( B o n

d 1 9 9 5 ) ;

5 ( C h r i s t e n s s o n

1 9 9 8 ) ;

6 ( D a b e r t 2 0 0 1 ) ;

7 ( L i u 2 0 0 1 ) ;

8 P r o t e o

b a c t e r

i a a r e p r e s e n t a t t h e c l a s s

l e v e

l d u e t o t h e

e x t e n s

i v e r e p r e s e n t a t i o n o f t h i s p h y l u m ; 9

R e l a t

i v e

i n c i

d e n c e o f p h y l u m - l e v e l r e p r e s e n t a t i v e s

i n r e s p e c t i v e s t u d

i e s ,

1 0 c o v e r a g e w a s c a

l c u l a t e d a c c o r d

i n g t o t h e

f o r m u

l a C =

[ 1 -

( n 1

N

1 ) ]

1 0 0 %

, n 1 = n u m

b e r o

f O T U s c o n s

i s t i n g o f o n

l y o n e s e q u e n c e ,

N = n u m

b e r o f a l

l s e q u e n c e s

i n t h e

1 6 S r R

N A g e n e c l o n e

l i b r a r y .

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The lamentous bacteria

The efciency of the activated sludge process isstrongly inuenced by the settleability of the sludgeocs in the succeeding sedimentation tanks in whichthe biomass is separated from the treated sewage.

Filamentous bacteria can dramatically decrease thesettleability of activated sludge ocs (sludge bulking)or cause oating and foam formation of the biomass(sludge foaming). Despite the inclusion of selectors inthe plant design, which helps in some cases to controlthe overgrowth of lamentous bacteria, sludge bulkingand sludge foaming are still a major problem in manyprimarily industrial wwtps. Therefore, there is consid-erable interest to identify the lamentous bacteria inwwtps and to characterize their physiological proper-ties in order to develop specic control strategies tosuppress their growth.

Traditionally, lamentous bacteria were analyzedin activated sludge by using standard light microscopy.For provisional identication of these microorgan-isms, keys were developed using: (i) the reaction of thelaments to Gram- and Neisser-staining, and (ii) themorphological characteristics of the laments (Eikel-boom 1975; Jenkins 1993) and type numbers were as-signed to the different laments (e.g. Eikelboom Type021N). Since then a considerable number of lamenttypes were enriched (mainly by micromanipulation)and their 16S rRNA gene sequences were determined(for example Blackall 1996; Bradford 1996; Seviour1997; Howarth 1999; Kanagawa 2000; Snaidr 2001).

The phylogenetic tree shown in Figure 1 presents anoverview on the phylogenetic afliation of those la-ments. Several lament types, for example Eikelboomtype 1863 or Nostocoida limicola , harbor phylo-genetically unrelated species. Today, many of thelaments can specically and rapidly be detected inactivated sludge using rRNA-targeted oligonucleotideprobes (Wagner 1994; Erhart 1997; de los Reyes1998; Kanagawa 2000). In situ probing demonstratedpolymorphism of several laments within activatedsludge (Wagner 1994), a fact which further complic-ates morphology-based identication. Furthermore,

application of the full-cycle rRNA approach revealedthat especially industrial wwtps harbor a variety of lamentous bacteria which can not be found in thetraditional identication keys (e.g. Juretschko 2002).

The availability of FISH probes for many of the laments now allows to investigate their in situphysiology within activated sludge systems (Nielsen1998). Mircothrix parvicella was shown to be a spe-

cialized lipid consumer being able to take up longchain fatty acids (but no short chain fatty acids or gluc-ose) under anaerobic conditions and subsequently usethe storage material for growth when nitrate or oxygenis available as electron acceptor (Andreasen & Nielsen1997; Nielsen 2001). However, M. parvicella could

not take up phosphorus under aerobic conditions ex-cluding its importance for enhanced biological phos-phorus removal (see below). In addition, Thiothrix sp. laments in industrial wwtps were shown to bephysiologically very versatile since they incorporatedradioactively labeled acetate and/or bicarbonate underheterotrophic, mixotrophic and chemolithoautotrophicconditions. The Thiothrix laments were active un-der anaerobic conditions (with or without nitrate) inwhich intracellular sulphur globules were formed fromthiosulphate and acetate was taken up (Nielsen 2000).

Microorganisms responsible for enhancedbiological phosphorus removal and nitrogenremoval

The identication and characterization of those bac-teria responsible for phosphorus and nitrogen re-moval in wwtps is complicated by the fact that 16SrRNA sequence-based identication of a microorgan-ism does generally not allow to infer its functionalproperties. Phylogenetically closely related microor-ganisms may possess different metabolic potentialswhile on the other hand several physiological traits

like the ability to denitrify are found dispersed in manydifferent phylogenetic lineages. Therefore, the full-cycle rRNA-approach has to be supplemented withother techniques which allow a functional assignmentof the detected bacteria to identify the members of the most important physiological groups in wwtps. Indetail, the use of: (i) lab-scale reactors, inoculatedwith activated sludge, which select for the respectivefunctional microbial group (Burrell 1998; Hesselmann1999; Strous 1999; Crocetti 2000), (ii) functionalgenes coding for key enzymes of certain metabolicpathways as phylogenetic and physiological markers

for the respective guild (Juretschko 1998; Purkhold2000), (iii) the combination of FISH and micro-electrodes (Schramm 1996; Okabe 1999; Schramm1999, 2000), (iv) the recently developed combina-tion of FISH and microautoradiography (Lee 1999;Daims 2001b) allowed to identify the bacteria cata-lyzing important transformations of biological nutrientremoval.

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Figure 1. 16S rRNA-based tree showing the phylogenetic afliation of lamentous bacteria occurring in activated sludge (labeled in bold).It should be noted that the Thiothrix et al. group contains also at least three phylogenetically distinct lineages including laments of theEikelboom Type 021N (Kanagawa 2000). The tree was calculated using the neighbor-joining method with a 50% bacterial conservation lter.Multifurcations connect branches for which a relative order could not be unambiguously determined by applying different treeing methods. Thebar corresponds to 10% estimated sequence divergence.

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Figure 2. 16S rRNA-based tree showing the phylogenetic afliation of putative polyphosphate accumulating organisms (PAOs), glycogenaccumulating organisms (GAOs) and G-bacteria. The tree was calculated using the maximum-likelihood method with a 50% bacterial con-servation lter. The bar corresponds to 10% estimated sequence divergence. a 16S rRNA gene clones: AF109792, AF109793, AF124650,AF124655 (Liu 2000). b 16S rRNA gene clones: AJ225335, AJ225341, AJ225342, AJ225348, AJ225351, AJ225353, AJ225355, AJ225356,AJ225360, AJ225364, AJ225370, AJ225376-AJ225379, Y15796, AF255628, AF255629 (Christensson 1998; Liu 2001). c 16S rRNA geneclones: AF093777, AF093778, AF093780, AF093781, AF314424, AF124652, AF124656 (Nielsen 1999; Liu 2000; Dabert 2001). d 16S rRNAgene clones: AJ224947, AF255631, AF255641, AF204244, AF204245, AF204247, AF204248, AF314417, AJ225350, AJ225358, X84620(Bond 1995; Christensson 1998; Hesselmann 1999; Crocetti 2000; Dabert 2001; Liu 2001).

Microorganisms of importance for enhanced biological phosphorus removal

Phosphorus removal from wastewater is important toprevent eutrophication and is therefore an integral partof modern municipal and industrial nutrient removalwwtps. Phosphorus can be precipitated by the ad-dition of iron or aluminum salts and subsequentlybe removed with the excess sludge. Chemical pre-cipitation is a very reliable method for phosphorusremoval but increases signicantly the sludge produc-tion and thus creates additional costs. Furthermore,the use of chemical precipitants may introduce heavymetal contamination into the sewage and increases thesalt concentration of the efuent. Alternatively, phos-

phorus removal can be achieved by microbiologicalmechanisms in a process called EBPR, for reviews see(Jenkins & Tandoi 1991; van Loosdrecht 1997; Mino1998, 2000). This process is characterized by cyclingthe activated sludge through alternating anaerobic andaerobic conditions. In the anaerobic stage the bacteriaresponsible for EBPR are supposed to gain energyfrom polyphosphate hydrolysis accompanied by sub-

sequent P i release for uptake of short-chain fatty acidsand their storage in form of PHA. Two different mod-els were postulated for the production of the reducing

equivalents for this anaerobic metabolism (Comeau1986; Mino 1987) In the subsequent aerobic stage, thePAOs possess a selective advantage compared to othermicroorganisms which were not able to take up fattyacids under the preceding anaerobic conditions by util-izing the stored PHA in an otherwise carbon-poorenvironment. In parallel, PAOs restore their polyphos-phate pools by aerobic uptake of available phosphatefrom the wastewater. After sedimentation in the sec-ondary clarier, a part of the biomass is recycled tothe anerobic stage and mixed with new wastewaterwhile the excess sludge containing the intracellularpolyphosphates is removed from the system.

In contrast to chemical precipitation, EBPR plantshave been frequently reported to fail. This raised in-terest in the microbiology of the process. Traditionally,based on cultivation experiments Gammaproteobac-teria of the genus Acinetobacter were believed to bethe only PAOs (Fuhs & Chen 1975; Ltter & Murphy1985; Bayly 1989). However, today it has become

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apparent that Acinetobacter can accumulate polyphos-phate but does not possess the above described PAOmetabolism (e.g. van Loosdrecht 1997). Furthermore,cultivation-independent methods like uorescent an-tibody staining (Cloete & Steyn 1987), respiratoryquinone proles (Hiraishi 1989), and FISH with a

genus-specic probe (Wagner 1994; Kmpfer 1996)demonstrated that the relative abundance of Acineto-bacter in EBPR systems was dramatically overestim-ated due to cultivation biases further conrming that Acinetobacter is not of importance for EBPR.

Several other bacteria isolated from EBPR reactorshave been suggested as PAO candidates. Microlunatus phosphovorus , a high-G+C Gram-positive bacteriumaccumulates aerobically polyphosphate and consumesit under anaerobic conditions but fails to take up acet-ate or accumulate PHA under anaerobic conditions(Nakamura 1991, 1995). FISH with a probe spe-cic for Microlunatus phosphovorus demonstrated thepresence of this organism in an EBPR plant (2.7% of the total bacteria) (Kawaharasaki 1998) but no directindications for the importance of this bacterium forEBPR are available Furthermore, Lampropedia spp.were shown to possess the basic metabolic features of a PAO but their acetate-uptake-phosphate-releaseratiowas much lower that EBPR models predict, and noadditional data regarding the abundance or activity of these morphologically conspicuous bacteria in EBPRsystems have been published.

Compared to these cultivation-based attempts, thehunt for PAOs was more successful using molecu-

lar tools for analyses of EBPR systems. Betaproteo-bacteria and high-G+C Gram-positive bacteria ( Ac-tinobacteria ) increased in number after addition of acetate to the raw sewage of a EBPR-full-scale wwtpsuggesting that these groupsbenet from the enhancedavailability of short chain fatty acids in the anaerobicbasin and thus represent candidates for PAO (Wagner1994). Additional support for the importance of bothgroups for EBPR stems from FISH experiments in anefcient EBPR laboratory-scale sequencing batch re-actor (Bond 1999) and respiratory quinone proling ina laboratory-scale EBPR system (Liu 2000). Recently, Actinobacteria related to the suborder Micrococcineaewere reported to be abundant in EBPR systems andthus might be important for EBPR (Christensson1998; Crocetti 2000; Liu 2001) (Figure 2). Whilethe function of Actinobacteria as PAOs still has tobe proven, evidence is available that Betaproteobac-teria of the family Rhodocyclaceae (Figure 2) areimportant PAOs in the so far investigated EBPR sys-

tems. These bacteria, for which the name CandidatusAccumulibacter phosphatis was suggested (Hessel-mann 1999) were present in signicant numbers inEBPR systems (Bond 1995; Hesselmann 1999; Cro-cetti 2000; Dabert 2001; Lee 2001). Furthermore,phosphorus accumulation by these bacteria in the aer-

obic phase was demonstrated by sequential FISH andpolyphosphate staining (Crocetti 2000; Liu 2001). Inaddition, acetate uptake in the anaerobic phase andphosphorus uptake under aerobic conditions could bedemonstrated for Candidatus Accumulibacter phos-phatis using FISH and microautoradiography (Lee2001).

A potential reason for the failure of EBBR plantsis the presence of bacteria which use previously storedcompounds such as glycogen (also referred to asGAOs) to compete with the PAOs for substrate up-take under anaerobic conditions (Satoh 1992; Cech &Hartman 1993; Liu 1997). Cech and Hartmann (Cech& Hartman 1993) described Gram-negative cocci inclumps and packages of tetrads in activated sludgeand called these morphotypes G-bacteria, since theirnumbers increased after glucose addition. Mino et al.(1998) suggested that bacteria with a similar mor-phology are GAOs. However, recent molecular andphysiological analyses of various G-bacteria isolatedfrom wwtps revealed a high phylogenetic diversity of this morphotypeand provided no support for their roleas GAOs (Seviour 2000) (Figure 2). These results sug-gest that GAOs should be dened by their phenotypeand not by their cell morphology. Recently, molecu-

lar community analyses of deteriorated EBPR reactorsrevealed the predominance of a novel bacterial groupwithin the Gammaproteobacteria (Figure 2; Nielsen1999), which are good GAO candidates since theyprobably accumulate PHA, and store little or no poly-phosphates (Nielsen 1999; Liu 2001; Crocetti 2001).

Nitrifying bacteria

The nitriers encompass two groups of microorgan-isms, the ammonia- and the nitrite-oxidizing bacteria,which catalyze the oxidation of ammonia to nitrite and

of nitrite to nitrate, respectively. Since most of thenitrogen in the inuent of a wwtp is present eitherin form of urea (which is hydrolyzed to ammonia)or ammonium/ammonia, the nitrifying bacteria play acentral role in nitrogen removal in wwtps. It is import-ant to lower the ammonia concentrations in the efuentof wwtps since this compound is toxic to aquaticlife and promotes eutrophication in the receiving wa-

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Figure 3. Diversity of amoA clones retrieved from different nitrifying wwtps. A schematic AmoAbased phylogenetic classication of the beta-proteobacterial ammonia oxidizing bacteria is shown. Multifurcations connect branches for which a relative order could not be unambiguously

determined by applying different treeing methods. The cluster designations were adopted from those of Purkhold et al. (2000). The height of each tetragon indicates the number of sequences in the cluster. Gammaproteobacterial ammonia-oxidizers are not included since they currentlyhave not been detected by FISH nor by amoA -analyses in nitrifying wwtps (adapted from A. Loy, 2002).

ters. Nitrifying bacteria are extremely slow-growingmicroorganisms and are recalcitrant to cultivation at-tempts. Due to the sensitivity of nitrifying bacteria todisturbances like pH- and temperature shifts, break-down of the nitrication process is frequently reportedfrom municipal and especially industrial wwtps.

According to textbooks the model ammonia-oxidizer is Nitrosomonas europaea . However, FISH

analyses in nitrifying activated sludge and biolmsshowed that other ammonia-oxidizers are more im-portant. In an industrial nitrifying/denitrifying plantthe dominant ammonia-oxidizer was Nitrosococcusmobilis , a bacterium which was previously consideredto occur in brackish water only (Juretschko 1998).Subsequently, N. mobilis was also detected in signic-ant numbers in a nitrifying sequencing batch biolm

reactor (Daims 2001a). In contrast, Nitrosospira -related ammonia-oxidizers were found to be dominantin situ in a laboratory scale uidized bed reactor(Schramm 1998). Although Nitrosospira was also re-ported in a PCR-based study as important ammonia-oxidizer genus in wwtps (Hiorns 1995), this ndingcould not be conrmed by FISH analyses of vari-ous wwtps and by a large amoA -based ammonia-

oxidizer diversity survey in wwtps (Purkhold 2000).Today it is generally accepted that nitrosomonads (in-cluding Nitrosococcus mobilis ) and not nitrosospiras(encompassing the genera Nitrosospira , Nitrosolobusand Nitrosovibrio ) are important for ammonia oxid-ation in wwtps. This perception is also reected inFigure 3 which shows the afliation of 199 amoAclones retrieved from various nitrifying wwtps. Only

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Figure 4. Phylogenetic tree of the genus Nitrospira based on comparative analysis of 16S rRNA sequences. The basic tree topology wasdetermined by maximum likelihood analysis of all sequences longer than 1300 nucleotides. Shorter sequences were successively added withoutchanging the overall tree topology. Branches leading to sequences shorter than 1000 nucleotides are dotted to point out that the exact afliationof these sequences cannot be determined. Black spots on tree nodes symbolize high parsimony bootstrap support above 90% based on 100iterations. The scale bar indicates 0.1 estimated changes per nucleotide. Clones from wwtps and reactors as well as sequences that belong toisolated strains are depicted in bold. The four sublineages of the genus Nitrospira are boxed and marked by the numbers I to IV.

6 amoA clones from these systems cluster with thenitrosospiras while the remaining 193 clones are af-liated with the nitrosomonads. Figure 3 also showsthat almost all recognized lineages of betaproteobac-terial ammonia-oxidizers can be found in wwtps. Nu-

merically, the Nitrosomonas europaea/Nitrosomonaseutropha -lineage, the Nitrosococcus mobilis -lineage,and the Nitrosomonas marina cluster are most fre-quently detected.

In conclusion, wwtps harbor a diversity of ammonia-oxidizers of the Betaproteobacteria , whichwas enormously underestimated previously. Most of

these ammonia-oxidizers are, based on comparativeamoA sequence analyses, relatively close relativesof described ammonia-oxidizer species. Interestingly,quantitative FISH results indicate that some nitrifyingwwtps are dominated by a single ammonia-oxidizer

species (Juretschko 1998) while other plants harborat least ve different co-existing ammonia-oxidizerpopulations which are present in signicant numbers(Daims 2001a).

Traditionally, Nitrobacter was considered as themost important nitrite-oxidizer in wwtps (Henze1997). Therefore, the nding that Nitrobacter could

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Figure 5. Analyses of the in situ physiology of Nitrospira like bacteria. Uptake of radioactive bicarbonate by Nitrospira -like bacteria in abiolm from a sequencing batch reactor under aerobic incubation conditions. (a) shows Nitrospira cells stained by a specic probe (b) showsonly the radiographic lm to visualize the microautoradiographic signal at the position of the Nitrospira cells (indicated by white lines). Othermicroautoradiographic signals were caused by CO 2 -xing bacteria, which were not detected by the Nitrospira -specic probe. (c, d) No uptakeof acetate by Nitrospira -like bacteria in the same biolm under aerobic incubation conditions. (c) shows Nitrospira cells stained with the specicprobe. (d) shows the micrograph of the radiographic lm at the same position. The localization of the Nitrospira microcolonies is indicated bywhite borderlines.

not be detected by FISH with specic 16S rRNA-targeted probes in various nitrifying wwtps cameas a surprise (Wagner 1996). Using the full cyclerRNA approach the occurrence of novel, yet uncul-tured Nitrospira -like nitrite-oxidizing bacteria in nitri-fying wwtps could be demonstrated (Juretschko 1998;Okabe 1999; Daims 2000, 2001b; Gieseke 2001).The importance of these microorganisms for nitrite-oxidation in wwtps was also conrmed by reactorenrichment studies (Burrell 1998). Today, four dif-

ferent phylogenetic lineages, two of them containing16S rRNA gene clones of wwtps, within the genus Nitrospira have been recognized (Figure 4; Daims2001b) and phylum- as well as genus-specic probessuitable for FISH are available (Daims 2001b). Com-bination of FISH and microautoradiography showedthat the Nitrospira -like nitrite-oxidizers in activatedsludge x CO 2 and can also grow mixotrophically us-ing pyruvate but not acetate, butyrate, and propionate(Figure 5; Daims 2001b).

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It has been postulated that the predominanceof Nitrospira -like bacteria over Nitrobacter in mostwwtps is a reection of their different survivalstrategies. While Nitrospira -like nitrite-oxidizers are,according to data extracted from microelectrode-FISHanalyses, K -strategists and thus may possess a low

max but are well-adapted to low nitrite and oxy-gen concentrations, Nitrobacter is postulated to be arelatively fast-growing r -strategist with low afnitiesto nitrite and oxygen (Schramm 1999). Since nitrite-concentrations in most reactors from wwtps are low, Nitrospiras will outcompete Nitrobacter in these sys-tems. In plants with temporally or spatially elevatednitrite concentrations, for example in nitrifying se-quencing batch reactors, both nitrite-oxidizers shouldbe able to co-exist. Consistent with this hypothesis co-occurrence of Nitrobacter and Nitrospira -like bacteriahas been observed by FISH in a nitrifying sequencingbatch biolm reactor (Daims 2001a). We recently star-ted to investigate the competition between Nitrospiraand Nitrobacter in controlled chemostat experiments.Two chemostats were inoculated with the same nitrify-ing biolm containing Nitrospira -like nitrite-oxidizersand operated under identical conditions (oxygen, tem-perature, pH, and liquid retention time). After additionof Nitrobacter sp. to the chemostats, the nitrite con-centration in the inuent of one of the reactors wasincreased such that nitrite peaks (up to 80 mg l 1) inthe efuent of this reactor were detectable. Consistentwith the above mentioned K / r -hypothesis, this perturb-ation stimulated the growth of Nitrobacter while in

the undisturbed control reactor Nitrospira dominated(Figure 6; R. Nogueira, unpubl.). Interestingly, thedominance of Nitrobacter over Nitrospira caused bythe elevated nitrite concentrations could not be rever-ted by lowering the available nitrite concentration tothe original level. One possible explanation for thisresult is that Nitrobacter if present at a certain celldensity is able to inhibit the growth of Nitrospira -likenitrite oxidizers.

Denitrifying bacteria

Denitrication is used in wastewater treatment toconvert the product(s) of nitrication into gaseousnitrogen compounds (mainly dinitrogen) and thusto remove them from the sewage. Most attemptsto identify and enumerate denitriers in activatedsludge are based on cultivation-dependentapproaches.Members of the genera Alcaligenes , Pseudomonas , Methylobacterium , Bacillus , Paracoccus and Hypho-

microbium were isolated as part of the denitrifyingmicrobial ora from wwtps (Sperl & Hoare 1971; At-twood & Harder 1972; Knowles 1982; Schmider &Ottow 1986; Vedenina & Govorukhina 1988). Thelatter genus was also detected microscopically byits typical cell morphology in denitrifying activated

sludge (Timmermans & van Haute 1983; Nyberg1992). However, little is known about whether theabove listed bacterial genera are representative for thein situ active denitriers in wwtps. Neef et al. usingspecic FISH probes detected signicant numbers of Paracoccus spp. and Hyphomicrobium spp. in a de-nitrifying sand lter supplemented with methanol asreduced carbon compound for nitrate reduction. Butboth genera were present in numbers below 0.1% of the total cell counts in a non-denitrifying sand lterrun in parallel without addition of methanol, indir-ectly suggesting an active participation of both generain the denitrication process (Neef 1996). Molecularstudies of the community composition of denitrifyingbacteria are difcult to perform since the denitrify-ing phenotype can not be inferred from the phylogenyof a microorganism. However, the combination of FISH and microautoradiography (Lee 1999) allowsidentication of denitriers in situ by performing twotypes of experiments in parallel. In the rst experi-ment the wwtp sample is incubated under anaerobiccondition in absence of nitrate or nitrite with radio-actively labeled substrates which are typically usedas electron donors for denitrication. In the secondexperiment the sample is incubated with the same

labeled substrates under anaerobic conditions but inthe presence of nitrate or nitrite. Bacterial species,identied by FISH, which take up substrate underanaerobic conditions exclusively in the presence of nitrate or nitrite are most likely denitriers. The useof this technique in combination with the full-cyclerRNA approach revealed that novel, still uncultured Azoarcus -related bacteria are important denitriers inan industrial nitrifying/denitrifying wwtp (Juretschko2000).

Conclusions and future perspectives

During the last decade, the application of moleculartools in wastewater microbiology has revolutionizedour view on the microbial ecology of these systems.Different groups of still not culturable bacteria areidentied and shown to be responsible for sludge bulk-ing, enhanced biological phosphorus removal, nitrite

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Figure 6. Competition between Nitrospira and Nitrobacter in chemostats. Two parallel chemostats were inoculated with nitrifying activatedsludge containing Nitrospira -like bacteria. Subsequently, Nitrobacter was added (arrows). In one chemostat nitrite peaks in the efuent (up to80 mg l 1 ) were induced by temporarily elevating the nitrite concentrations in the inuent (B; upper panel). The other chemostat served ascontrol reactor which always had nitrite efuent concentrations of below 5 mg l 1 (A, upper panel). The relative biovolume of Nitrospira and Nitrobacter (to the biovolume of all cells detectable by FISH) was determined by quantitative FISH in the chemostats for a period of 98 days(A, B; lower panels).

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oxidation, and denitrication. Surprisingly, the modelorganisms described in text books for these processesand for ammonia oxidation are shown to be gener-ally not of importance for wastewater treatment. Itis important to note that signicant diversity existsin each of these functional groups of bacteria. In

the next research phase in wastewater microbiology,more detailed knowledge on the biology of the abovementioned non-cultured bacteria needs to be gained.Therefore, an increased effort on the developmentof suitable cultivation strategies for these bacteria isneeded. In parallel, the use of techniques referred to asenvironmental genomics should allow to investigatethe genome composition of these bacteria without theneed of cultivation (Schleper 1998).

Regarding application, the most obvious benet of the progress described is that it provides a basis fora more knowledge-driven treatment of wwtp failures.One strategy to improve a particular aspect of processperformance in a wwtp, for example during start-upor after its breakdown, is the addition of special-ized microorganisms or activated sludge from anotherwwtp (Rittmann & Whiteman 1994). This operationaltool which is called bioaugmentation does howeverfrequently fail (e.g. Bouchez 2000 and referencestherein). Such failure is typically caused by addition of the wrong microorganisms, for example the modelorganisms for nitrication, which can not competesuccessfully with the autochthonous bacteria in theplant and are thus eliminated or washed-out. There-fore, it was (and still is) important to identify those mi-

croorganisms responsible for nitrogen or phosphorusremoval in a functioning wwtp. If problems arise theseresults can be used as guidance to select the appropri-ate bacterial additive (for culturable microorganisms)or a well-suited activated sludge from a neighboringwwtp containing a comparable microbial ora. Oncethe appropriate bacteria have been selected they needto be protected for example by polymer embeddingfrom grazing by protozoa (Bouchez 2000).

Compared to curing failure of a certain process ina wwtp by bioaugmentation, protecting the plant fromprocess deterioration is a more sustainable strategy.For this purpose we need to understand the linksbetween the diversity of a functionally important bac-terial group and the stability of the catalyzed process.Preliminary observations indicate that plants with lowfunctional redundancy due to the presence of a lowdiversity of bacteria of a certain functional group aremore sensitive to failure of the respective process thanplants harboring a high diversity of the same bacterial

group. If increase in diversity can indeed be provento cause process stabilization then it will be import-ant to learn how plant design and process parametercontrol can be optimized to increase the diversity of functionally important bacterial groups. To answerthese ecologically and economically important ques-

tions it will be necessary to determine the microbialcommunity composition of a large number of differentsamples obtained from tightly controlled reactor stud-ies as well as full scale wwtps. Due to the tediousnessof many of the established molecular methods thiskind of research will greatly benet from the imple-mentation of modern high throughput techniques likeDNA microarrays for measuring microbial communitycomposition in complex samples (Guschin 1997).

Acknowledgements

We acknowledge Justyna Adamczyk, Matthias Horn,Stefan Juretschko, Angelika Lehner, Markus Schmid,and Stefan Schmitz-Esser from the Microbial Eco-logy group (www.microbial-ecology.de) at the Tech-nische Universitt Mnchen for their contributionsand enthusiasm as well as Sibylle Schadhauser, andJutta Elgner for excellent technical assistance. Sup-port by the Deutsche Forschungsgemeinschaft (projectWA1558/1) is gratefully acknowledged.

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