218
Bacterial community composition and function in sewage
treatment systems
Michael Wagner* and Alexander Loy
The application of modern molecular techniques has led to the
identification, in situ quantification, and partial ecophysiological
characterisation of bacteria responsible for bulking and
foaming or for nutrient removal in sewage treatment systems.
Unexpectedly, previously unrecognised, yet uncultured bacteria
were demonstrated to catalyse nitrogen and phosphorous
removal in activated-sludge and biofilm reactors. These
findings provide the basis for the development of novel
concepts for improving the efficiency and functional stability of
waste water treatment systems.
Addresses
Microbial Ecology Group, Lehrstuhl fr Mikrobiologie, Technische
Universitt Mnchen, Am Hochanger 4, D-85350 Freising, Germany
*e-mail: wagner@microbial-ecology.de
Current Opinion in Biotechnology 2002, 13:218227
0958-1669/02/$ see front matter
2002 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S0958-1669(02)00315-4
Abbreviations
EBPR enhanced biological phosphorous removal
FISH fluorescence in situ hybridisation
GAO glycogen-accumulating organism
MAR microautoradiography
PAO polyphosphate-accumulating organism
rRNA ribosomal ribonucleic acid
WWTP waste water treatment plant
Introduction
Communities of prokaryotic microorganisms present in
activated-sludge or biofilm reactors are responsible for
most of the carbon and nutrient removal from sewage and
thus represent the core component of every biological
waste water treatment plant (WWTP). By contrast, mass
occurrence of certain bacterial species can also be
detrimental for sewage treatment by negatively influencing
the settling properties of activated-sludge in the secondary
clarifiers, by contributing to the formation of foam or by
outcompeting microorganisms required for nutrient
removal. Consequently, a thorough knowledge of the
ecology of the microbial communities is required to reveal
factors influencing the efficiency and stability of biological
WWTPs and to develop promising strategies for improved
process performance.
Traditionally, microbial communities in WWTPs were
analysed either by light microscopic observation [1,2] or by
cultivation-dependent techniques [3,4]. These approaches
led to the postulation of model organisms for the most
important microbiologically driven processes in WWTPs [5],
but turned out to be inadequate for a proper description of
the composition and dynamics of the microbial communities
actually present [68]. During the past decade, a variety of
molecular approaches were developed and used to study
bacterial diversity in WWTPs in a cultivation-independent
manner (for a review see [9]). These studies demonstrated
that most of the suggested model organisms are of minor
relevance in situ and that other microorganisms, often not
yet culturable, are responsible for most key processes in
WWTPs (see below). Recently, first insights into the
ecophysiology of these microorganisms were obtained by
combining molecular methods for community composition
analysis with techniques to infer the function of the detected
microorganisms in situ. In this article, we review recent
advances in the field of microbial ecology of WWTPs. We
focus on aerobic or alternating aerobicanaerobic activated-
sludge and biofilm systems, but owing to space constraints
we do not cover the microbiology of anaerobic digesters.
Microbial diversity in waste water treatment
plants
Based on 16S ribosomal ribonucleic acid (rRNA) gene
library analyses, several general microbial diversity surveys
of activated-sludge and biofilm systems have been
performed since 1995 (Table 1). In total, more than 750 16S
rRNA gene sequences were analysed and sequences
affiliated with the Beta-, Alpha- and Gammaproteobacteria as
well as the Bacteroidetes and the Actinobacteria were most
frequently retrieved. These findings are consistent with
previous fluorescence in situ hybridisation (FISH) studies
of activated-sludge systems using group-specific rRNA-
targeted oligonucleotide probes (for a review see [9]).
Furthermore, the 16S rRNA data allow us to predict the
minimum number of bacterial species present in the
analysed systems to range from 17 to 268 (Table 1). Despite
the overall impressive number of 16S rRNA gene clones
that have been obtained from waste water treatment
reactors, however, there are insufficient data available for
the analysis of full-scale WWTPs. Furthermore, the actual
community composition of complex systems cannot be
inferred from 16S rRNA gene libraries alone, but must
be deduced by the use of quantitative FISH with
probes targeting the environmentally retrieved sequences.
Unfortunately, this so-called full-cycle rRNA approach has
rarely been used to at least partially characterise microbial
community structures in WWTPs [1012,13,14]. This is
probably because of the tediousness and technical difficulties
of microscopic counting of the different probe-labelled
microbial populations. The development of semi-automatic
digital image analysis routines [15,16] has significantly
accelerated quantitative FISH experiments and enabled
microbiologists for the first time to obtain encompassing,
high-resolution insights into the microbial community
composition of activated-sludge [13].
 Bacterial community composition and function in sewage treatment systems Wagner and Loy 219

Table 1

Summary of 16S rRNA-based diversity surveys of waste water treatment plants and reactors.
WWTPs and laboratory reactors

SBR1 + sodium acetate

High-load aeration basin

SBR2 unsupplemented

continuous flow reactor#

of a full-scale municipal

EBPR laboratory-scale

EBPR laboratory-scale

EBPR laboratory-scale

EBPR laboratory-scale

EBPR laboratory-scale
denitrifying industrial

Pilot-scale nitrifying
Full-scale nitrifying/

SBBR1

Average
reactor
plant*

plant

SBR
No. clones 62 94 96 97 92 51 92 150 92
No. OTUs 25 53 33 69 75 30 50 16 44
No. eOTUs** 32 83 42 144 268 59 109 17 94
Coverage (%) 77 64 78 48 28 51 46 93 61
Proteobacteria
Relative frequency of bacterial divisions in % (No.

 3 (1) 26 (15) 5 (4) 13 (8) 17 (15) 16 (8) 4 (3) 5 (1) 11

 52 (9) 31 (9) 51 (11) 33 (20) 25 (13) 8 (2) 17 (8) 14 (1) 29


 18 (7) 22 (6) 8 (4) 10 (9) 8 (3) 5 (2) 7 (2) 10


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


 15 (1) 1 (1) 2 (2) 7 (5) 4 (2) 4
OTUs within the respective division)

Bacteroidetes 2 (2)
39 (17) 50 (7) 5 (5) 13 (9) 6 (1) 14
Acidobacteria 5 (3) 9 (6)5 (2) 3 (2) 7 (5) 2 (1) 4
Firmicutes 10 (5) 1 (1) 5 (1) 1 (1) 2
Actinobacteria 1 (1) 1 (1) 2 (2) 37 (3) 4 (2) 9 (2) 4 (3) 7
Nitrospira 2 (1) 8 (2) 2 (2) 3 (1) 3 (3) 2
Verrucomicrobia 2 (1) 3 (2) 1 (1) 4 (3) 1 (1) 2
Planctomycetes 12 (10) 9 (8) 8 (4) 3 (2) 13 (9) 6
Chorobi 1 (1) 2 (1) 3 (2) 1
Choroflexi 2 (1) 16 (8) 4 (3) 3 (3) 6 (3) 4
Fibrobacteres 9 (1) 1
Fusobacteria 3 (2) 2 (2) 1
OP11 1 (1) 0
Unaffiliated 3 (3) 0
*[11]; [13]; partially published in [48]; [88]; #[89]; [90]; [67]. **The number of total expected OTUs (eOTUs) was calculated according
to the formula: eOTUs = (number of OTUs 100%) x coverage1. Calculated according to the formula: coverage = [1 (n1 x N1)] 100%
with n1 = number of OTUs consisting of only one sequence and N = number of all clones in the library; SBR, sequencing batch reactor.
SBBR, sequencing biofilm batch reactor. Clones were assigned to different operational taxonomic units (OTUs) if they shared less than 97%
16S rRNA sequence similarity with each other (Table modified from [9]).

Filamentous bacteria distinguished morphologically) possess a Gram-positive cell

Although the presence of a certain number of filaments is envelope and are affiliated with the candidate division TM7
important for proper floc formation, the occurrence of large for which no cultured representatives are available.
filamentous bacterial populations is detrimental for sewage Furthermore, several research groups used micromanipulation
treatment as it causes foam formation or settling problems of to enrich or isolate filamentous microorganisms before the
the activated-sludge in the secondary clarifiers (bulking). determination of their 16S rRNA gene sequences [2023].
Many filamentous bacteria are recalcitrant to cultivation These studies clearly showed that distantly related bacteria
attempts and thus have only been provisionally described frequently possess a morphology not distinguishable by
on the basis of a few microscopically observable characteristics. light microscopy and, thus, strongly question the morpho-
More than 30 different filamentous morphotypes have been type concept for filament classification [24,25,26]. For
observed in domestic WWTPs and approximately 40 example, Nostocoida limicola-like filaments are members of
additional morphotypes were recently described in industrial at least five different bacterial divisions [20,22,2729]. The
activated-sludge plants [1,17,18]. This review period has importance of morphology-independent identification tools
seen significant progress in the phylogenetic assignment of is also illustrated by the accumulating evidence for the
filamentous morphotypes from activated-sludge (Figure 1). occurrence of non-filamentous growth forms of several
Hugenholtz and co-workers [19] demonstrated using filamentous bacteria within activated-sludge [30,31]. On the
transmission electron microscopy and the full-cycle rRNA basis of the 16S rRNA gene sequences of filamentous
approach that filaments matching the description of the bacteria, the set of rRNA-targeted oligonucleotide probes
Eikelboom types 0041 and 0675 (which cannot reliably be for direct, morphology-independent filament identification
220 Environmental biotechnology

Figure 1

Bacteroidetes
Chryseobacterium balustinum
Bergeyella zoohelcum
Eikelboom Type 1863
Betaproteobacteria
Eikelboom Type 1701
Leptothrix discophora
Leptothrix mobilis
Variovorax paradoxus
Eikelboom Type 0803
Eikelboom Type 1863
Moraxella osloensis
Moraxella catarrhalis
Acinetobacter baumannii
Eikelboom Type 1863
Acinetobacter calcoaceticus
Leucothrix mucor
Eikelboom Type 021N group I
Eikelboom Type 021N group II
Eikelboom Type 021N group III
Thiothrix defluvii
Thiothrix fructosivorans
Thiothrix ramosa
Thiothrix nivea
Thiothrix unzii
Gammaproteobacteria
Paracoccus denitrificans
Eikelboom Type 0411
Cytophaga lytica
Cytophaga columnaris Runellas lithyformis Planctomycetes
Eikelboom Type 0092
Gemmata obscuriglobus
Haliscomenobacter
hydrossis
Nostocoida limicola III
Chloroflexi and Thermomicrobia
Isosphaera pallida
Sphaerotilus natans Thermomicrobium roseum
Nostocoida limicola-like bacterium AHW4
Herpetosiphon aurantiacus
Chloroflexus aurantiacus
Eikelboom Type 1851
Janibacter thuringensis
Terrabacter tumescens
Nostocoida limicola II
Rhodococcus rhodochrous
Rhodococcus coprophilus
Skermania pinensis
Actinobacteria
Gordona amarae
Nocardia asteroides
Nocardia nova
"Microthrix parvicella"
Rhodospirillum MC2 Lactosphaera pasteurii
sodomense Nostocoida limicola I
Nostocoida limicola I
PPx3
EU34 Nostocoida limicola- Streptococcus suis
Firmicutes
like bacteria
10%
Alphaproteobacteria

Current Opinion in Biotechnology

16S rRNA-based phylogenetic tree showing the affiliation of filamentous bacteria from activated-sludge (white boxes). Multifurcation indicates that no
unambiguous branching order can be determined if different treeing methods are applied. The bar represents 10% estimated sequence divergence.

in activated-sludge was extended [23,25,30,31]. The nutrient-removal plants with dynamic anaerobic-aerobic
suitability of the FISH method to gain knowledge of conditions against most other activated-sludge bacteria. By
significant practical relevance was elegantly demonstrated applying the recently developed combination of FISH and
by determining threshold values of nocardioform actino- MAR [35], it is now possible to investigate the in situ
mycetes for foam formation and foam stability, parameters physiology of filamentous microorganisms and their
that should allow an objective evaluation of the effects of unicellular growth forms even in samples with genetically
control methods [31,32,33]. different filaments of the same morphotype. Using this
approach, the in situ physiology of Thiothrix spp. was inves-
In addition to an improved in situ identification and tigated in activated-sludge from an industrial WWTP [36].
quantification of filaments, the development of efficient
control strategies will require knowledge on the ecophysi- Bacteria responsible for nitrogen removal
ology of these microorganisms. Along this alley, Nielsen Ammonium (and urea that is hydrolysed to ammonium) is
and co-workers [34] used microautoradiography (MAR) the major nitrogen compound of sewage and is removed in
to investigate the in situ physiology of Microthrix parvicella, WWTPs by conversion to gaseous nitrogen via nitrification
one of the few filaments that can unambiguously be and denitrification. Nitrification, the aerobic oxidation of
identified on the basis of its morphology. This Gram- ammonium to nitrate via nitrite, is catalysed by two different
positive organism, which is difficult to maintain in pure groups of slow-growing, autotrophic bacteria the ammonia
culture, possesses a hydrophobic cell surface and is the oxidisers and the nitrite oxidisers. There is considerable
causative agent for worldwide foaming and bulking interest in understanding the ecology of nitrifying bacteria
problems in WWTPs with nutrient removal. MAR analyses because nitrification is the Achilles heel of many WWTPs
showed that M. parvicella, in contrast to most other activated- and the causes for nitrification breakdown events are not
sludge bacteria, is able to take up and store long-chain always obvious. Once nitrifiers have been washed out of a
fatty acids under anaerobic conditions and subsequently WWTP, recovery of the nitrification process can take very
metabolise them under aerobic conditions. This physiological long time periods owing to the slow growth rates of the
potential offers M. parvicella a competitive advantage in nitrifiers. Although standard textbooks [5] still report that
 Bacterial community composition and function in sewage treatment systems Wagner and Loy 221

Figure 2

No. of isolates Number of amoA clones No. of WWTPs in

/described which the
species MAS IAS FBR LBR respective clones
were detected
65

N. europaea/ N. eutropha lineage 4 4 11 37 9 61 10

36 Clones 25 Clones

36
Nc. mobilis lineage 4 0 9 23 0 32 5

20 Environmental lineage 1
0 1 0 18 1 20 6
(related to Nc. mobilis)
2 N. halophila lineage 2 0 0 0 0 0 0
6 N. communis lineage 1 1 0 3 1 5 3
2 Nitrosomonas sp. Nm 33 lineage 1 1 0 0 0 1 1
9 N. nitrosa lineage 1 0 8 0 0 8 1
7 Environmental lineage 2 0 5 0 0 2 7 4
3 Environmental lineage 3 0 0 0 3 0 3 1
7 Environmental lineage 4 0 3 0 0 4 7 4
1 Nitrosomonas sp. Nm 41 1 0 0 0 0 0 0
42
N. marina cluster 3 30 0 9 0 39 6

10 N. oligotropha lineage 1 4 0 1 4 9 4
2 N. ureae lineage 1 1 0 0 0 1 1
1 Nitrosomonas cryotolerans 1 0 0 0 0 0 0

14 Nitrosospira cluster 8 6 0 0 0 6 2

28 56 28 94 21 199

Current Opinion in Biotechnology

Schematic overview of the affiliation of amoA clones retrieved from WWTPs. MAS, municipal activated-sludge; IAS, industrial activated-sludge;
FBR, full-scale biofilm reactor; LBR, laboratory-scale biofilm reactor; , sum.

Nitrosomonas europaea and Nitrobacter spp. are the ammonium
and nitrite oxidisers in WWTPs, the actual picture is far
more complex.
Molecular tools for diversity analyses of ammonia oxidisers
are based on the amoA gene (which encodes for the active-
site subunit of the ammonia monooxygenase present in all
ammonia oxidisers) and/or 16S rRNA gene analysis.
Comparative sequence analyses of almost 200 amoA gene
fragments showed that a wide variety of different beta-
proteobacterial ammonia oxidisers occurs in nitrifying
WWTPs (Figure 2; for a review see [37]). In addition
to N. europaea, Nitrosomonas eutropha, Nitrosococcus mobilis,
members of the Nitrosomonas marina cluster, and four
phylogenetic lineages for which no cultured representative
exists are present. Furthermore, the amoA approach
revealed that ammonia oxidisers of the genus Nitrosospira,
in contrast to other ecosystems [38,39], are not important
in those full-scale nitrifying WWTPs analysed so far
(Figure 2). These findings are consistent with quantitative
FISH analysis of the ammonia oxidiser community
composition in WWTPs [6,14,4042]. It is important to
note, however, that physiologically inactive ammonia
oxidisers will also be detected by FISH as these bacteria
maintain high cellular ribosome contents under unfavourable
conditions [43,44]. The number of physiologically active
ammonia oxidisers can accurately be determined using
MAR-FISH with 14C-labelled bicarbonate as substrate
[35]. Recently, the molecular toolbox for ammonia oxidisers
was complemented by quantitative polymerase chain
222 Environmental biotechnology

Figure 3

Phylogeny and ecophysiology of Nitrospira-

(a) Leptospirillum spp. like bacteria. (a) 16S rRNA-based
Nitrospira sublineage IV
including N. marina phylogenetic tree showing the Nitrospira
Thermodesulfovibrio spp.
phylum. The bar represents 10% estimated
Nitrospira sublineage III sequence divergence. (b) Ecophysiological
analysis of Nitrospira-like bacteria in activated-
'Magnetobacterium bavaricum' Nitrospira sublineage II sludge by MAR-FISH. The large confocal
including N. moscoviensis laser scanning microscopic picture shows
and WWTP clones Nitrospira-microcolonies stained red with a
Nitrospira sublineage I specific probe and black silver granules
exclusively WWTP clones indicating the uptake of radioactive
10% bicarbonate. In the activated sludge,
bicarbonate was not only taken up by
Nitrospira-related cells but also by adjacent
(b) autotrophic ammonia oxidisers causing silver
granule formation beside the Nitrospira
microcolonies. Inset shows probe-specific
Nitrospira staining (left) and silver granule
detection (right) in separate frames.

10 m
10 m 10 m

Current Opinion in Biotechnology

reaction (PCR) [45,46] and FISH [16] methods for inference
of absolute cell concentrations of these bacteria in complex
samples including activated-sludge. These methods, if
used in combination with primers or probes of appropriate
specificity (for a critical evaluation see [37]), could
become valuable tools to determine parameters important
for cost-effective design and operation of WWTPs.
The application of molecular methods revealed that yet
uncultured Nitrospira-like microorganisms and not Nitrobacter
spp., are the dominating nitrite oxidisers in most WWTPs
[6,14,46,47,48,49]. Two phylogenetically different groups of
these novel nitrite oxidisers, which frequently form tight
microcolonies with water-permeable channels, occur in
WWTPs (Figure 3a) [48]. According to recent research
these Nitrospira-like nitrite oxidisers are also of major impor-
tance in other ecosystems like drinking water distribution
systems [50] or soil [51]. By using MAR-FISH, Daims and
coworkers [48] investigated the ecophysiology of the uncul-
tured Nitrospira-like nitrite oxidisers in activated-sludge and
found that these bacteria are able to fix bicarbonate (Figure 3b)
and to simultaneously take up pyruvate. Nitrospira-like nitrite
oxidisers are probably K-strategists (with high substrate
affinities and low maximum activity or growth rate) for
oxygen and nitrite [47,52] and thus outcompete Nitrobacter
under substrate-limiting conditions in WWTPs. This hypo-
thesis would also explain why Nitrobacter and Nitrospira co-exist
in reactors with temporarily higher nitrite concentrations [40].
 Bacterial community composition and function in sewage treatment systems Wagner and Loy 223

In nutrient removal WWTP, nitrate produced by the Figure 4

nitrification process is converted to atmospheric nitrogen
by the activity of denitrifying bacteria. In contrast to
(a)
ammonia and nitrite oxidation, the capability to anaerobically
respire with nitrate (or nitrite) is widespread in the bacterial
and archaeal domains. Therefore, it is impossible to predict
from an environmentally retrieved 16S rRNA sequence
whether a microorganism is actually performing denitrifi-
cation. This difficulty is reflected by the fact that we still
do not know which microorganisms are important in situ
denitrifiers in WWTPs. Although a considerable number
of bacteria that are capable of denitrification in pure
culture were isolated from WWTPs [53,54] and shown to
be present in significant numbers in these systems
[12,55,56], their mere detection does not unequivocally
demonstrate that they are actually denitrifying. Future
attempts to identify and measure the activity of denitrifiers
in activated-sludge might rely on the use of genes and
transcripts of the nitrite reductase (nirS and nirK) as a
functional marker [5760]. Alternatively, MAR can be
applied to enumerate bacteria that are able to take up
acetate under denitrifying conditions in activated-sludge
[61]. By combining FISH with MAR denitrifiers can also be
(b)
identified in activated-sludge. Preliminary results showed
that Betaproteobacteria related to the Azoarcus-Thauera
complex are probably abundant denitrifiers in an industrial
WWTP (M Wagner, unpublished data).

The anaerobic oxidation of ammonium by deep-branching

Planctomycetes, which can be used for cost-effective and
space-effective nitrogen removal from high-strength waste
water, is not covered in this review as this topic was recently
reviewed in this journal [62]. Since then, anaerobic
ammonium oxidisers were also discovered and enriched
in Australia [63] and 16S23S rRNA spacer probes were
developed for in situ monitoring of activity changes of
anaerobic ammonium oxidisers in response to altered
environmental conditions [64].

Bacteria catalysing phosphorous removal

WWTPs with enhanced biological phosphorous removal
(EBPR) are characterised by cycling the activated-sludge
through anaerobic and aerobic conditions. Through this
process regime, the growth of polyphosphate-accumulating
organisms (PAOs) is promoted. PAOs accumulate poly-
phosphate intracellularly in the aerobic stage and thus
allow efficient removal of the phosphorous with the excess
biomass. Although EBPR is widely applied, this process
still frequently suffers from failures and start-up problems.
During the review period, a significant advancement in
knowledge on the microbiology of this process has been
achieved. Molecular studies demonstrated more than
seven years ago that Acinetobacter, the traditional model
organism for EBPR, does not catalyse this process in
EBPR plants [8]; however, it was only recently that PAOs
were identified in laboratory-scale bioreactors as novel, yet
uncultured, Betaproteobacteria related to Rhodocyclus [65,66,67].
In contrast to Acinetobacter, the Rhodocyclus-related
20 m
Current Opinion in Biotechnology
In situ identification of PAOs by a combination of FISH and MAR after
incubation of activated-sludge with radioactive orthophosphate.
(a) The FISH picture and (b) the MAR picture of the same
microscopic field. Bacteria hybridising with the bacterial probe mix [87]
are labelled green. Rhodocyclus-like PAOs are stained yellow by
simultaneous hybridisation with a specific probe. White circles indicate
selected microcolonies of Rhodocyclus-like bacteria that took up
orthophosphate, microcolonies of these organisms that did not take up
orthophosphate are encircled in red. Black spots not matching with
Rhodocyclus-like bacteria indicate orthophosphate uptake by other
bacteria. Picture by Natuschka Lee.
bacteria, for which the provisional name Candidatus
Accumulibacter phosphatis has been proposed [65],
possess physiological characteristics expected for PAOs.
224 Environmental biotechnology
These Rhodocyclus-like bacteria also occur in significant
numbers in pilot and full-scale EBPR plants and contribute
to phosphorous removal [68,69]. Interestingly, however, in
these studies only a subpopulation of the Rhodocyclus-like
bacteria detectable with a specific probe accumulated
polyphosphates during aerobic growth (Figure 4). Further-
more, there is growing evidence that members of other
bacterial divisions than the Betaproteobacteria also contribute
to EBPR (Figure 4) [6769].
In systems with deteriorated EBPR, coccoid, non-
phosphate-accumulating bacteria occurring in tetrades
frequently accumulate (e.g. [70]). Originally, these bacteria
were termed G-bacteria, reflecting their dominance in an
acetate-fed reactor supplemented with glucose [70].
Subsequently, the designation glycogen-accumulating
organisms (GAOs) was used for these bacteria to reflect
the current perception that they synthesise glycogen (from
polyhydroxyalkanoates) in the aerobic phase and use it as
reducing power and energy to synthesise polyhydroxyalka-
noates from glucose and acetate in the anaerobic phase.
Bacteria with this physiological capacity would thus be
strong competitors for anaerobic substrate uptake for the
phosphate-accumulating bacteria in EBPR systems and
could contribute to EBPR failure [71,72]. Recently, a novel
group of uncultured Gammaproteobacteria that are GAO
candidates were identified by the full-cycle rRNA
approach in deteriorated EBPR systems [73]. It is important
to keep in mind, however, that various phylogenetically
different bacteria occur as cocci in tetrades in activated-
sludge and these bacteria also differ considerably in their
physiological traits, thus not all can be considered to be
GAOs [74,75].
Conclusions
Our understanding of the microbial community structure
in WWTPs continues to advance rapidly owing to the
ongoing development and application of molecular methods.
Today, for most of the important processes in WWTPs yet
uncultured prokaryotic key players have been identified.
The development of novel molecular techniques for in situ
analyses of the function of uncultured microorganisms
[35,64,76,77] has generated enormous opportunity for
investigating the ecophysiology of these biotechnologically
important microorganisms. The molecular toolbox can now
be applied to produce data that can be directly used for
modelling of activated-sludge and biofilm processes and to
improve bioaugmentation strategies [7880]. Ultimately,
these techniques should help to identify the links between
microbial community composition, function and process
stability. Such studies, which will have to include the
analyses of a substantial number of samples, will benefit
enormously from the application of high-throughput DNA
microarray techniques that are currently implemented in
microbial ecology research [60,8184]. Fundamental studies
to understand how the diversity of functionally important
prokaryotic groups in WWTPs can be influenced by plant
design and how these changes affect process stability
should be performed. In parallel, environmental genomic
techniques [85,86] will be applied to gain access to the
genome and transcriptome of uncultured, but functionally
important, bacteria in WWTPs.
Acknowledgements
We apologise to those authors whose work could not be included here due
to space constraints. The enthusiasm and hard work of Justyna Adamczyk,
Holger Daims, Matthias Horn, Stefan Juretschko, Natuschka Lee,
Angelika Lehner, Ulrike Purkhold, Markus Schmid, Stefan-Schmitz
Esser, and Kilian Stcker (some current or former members of the Microbial
Ecology Group) is greatly acknowledged. We would like to thank Per H Nielsen
and Mike Jetten for long-term collaboration and Sibylle Schadhauser for
excellent technical assistance. This work was supported by a grant from the
Deutsche Forschungsgemeinschaft (WA 1558/1-1).
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industrial wastewater treatment system as determined by 16S
rRNA analysis. Appl Environ Microbiol 2000, 66:1167-1174.
 Bacterial community composition and function in sewage treatment systems Wagner and Loy
13. Juretschko S, Loy A, Lehner A, Wagner M: The microbial community
composition of a nitrifying-denitrifying activated sludge from an
industrial sewage treatment plant analyzed by the full-cycle rRNA
approach. Syst Appl Microbiol 2002, 25:84-99.
This manuscript provides for the first time an almost complete overview on
the microbial composition of activated-sludge from a full-scale WWTP.
Almost 100 16S rRNA gene sequences retrieved from activated-sludge
were sequenced and many new specific probes for FISH analyses were
designed and applied.
14. Gieseke A, Purkhold U, Wagner M, Amann R, Schramm A:
Community structure and activity dynamics of nitrifying bacteria in
a phosphate-removing biofilm. Appl Environ Microbiol 2001,
67:1351-1362.
15. Schmid M, Twachtmann U, Klein M, Strous M, Juretschko S, Jetten M,
Metzger JW, Schleifer KH, Wagner M: Molecular evidence for genus
level diversity of bacteria capable of catalyzing anaerobic
ammonium oxidation. Syst Appl Microbiol 2000, 23:93-106.
16. Daims H, Ramsing NB, Schleifer KH, Wagner M: Cultivation
independent, semiautomatic determination of absolute bacterial
cell numbers in environmental samples by fluorescence in situ
hybridization. Appl Environ Microbiol 2001, 67:5810-5818.
Advanced image analysis techniques are presented that allow the determi-
nation of absolute cell concentrations of uncultured bacteria in complex
environments. Using this technique the in situ number of ammonia oxidisers
in a full-scale WWTP was determined.
17. Kmpfer P, Wagner M: Filamentous bacteria in activated sludge
current taxonomic status and ecology. In The Encyclopedia of
Environmental Microbiology. Edited by Bitton G. New York: John
Wiley & Sons, Inc.; in press.
18. Eikelboom DH, Geurkink B: Filamentous microorganisms observed
in industrial activated sludge plants. Water Sci Technol 2002,
in press.
19. Hugenholtz P, Tyson GW, Webb RI, Wagner AM, Blackall LL:
Investigation of candidate division TM7, a recently recognized
major lineage of the domain Bacteria with no known pure-culture
representatives. Appl Environ Microbiol 2001, 67:411-419.
Transmission electron microscopy and the full-cycle rRNA approach were
used in this very nice paper to investigate uncultured, but numerically
important, filaments in activated-sludge.
20. Liu JR, Burrell P, Seviour EM, Soddell JA, Blackall LL, Seviour RJ: The
filamentous bacterial morphotype Nostocoida limicola I contains
at least two previously described genera in the low G+C Gram
positive bacteria. Syst Appl Microbiol 2000, 23:528-534.
21. Bradford D, Hugenholtz P, Seviour EM, Cunningham MA, Stratton H,
Seviour RJ, Blackall LL: 16S rRNA analysis of isolates obtained
from Gram-negative, filamentous bacteria micromanipulated from
activated sludge. Syst Appl Microbiol 1996, 19:334-343.
22. Blackall LL, Seviour EM, Bradford D, Rossetti S, Tandoi V, Seviour RJ:
Candidatus Nostocoida limicola, a filamentous bacterium from
activated sludge. Int J Syst Evol Microbiol 2000, 50:703-709.
23. Beer M, Seviour EM, Kong Y, Cunningham M, Blackall LL, Seviour RJ:
Phylogeny of the filamentous bacterium Eikelboom Type 1851,
and design and application of a 16S rRNA targeted
oligonucleotide probe for its fluorescence in situ identification in
activated sludge. FEMS Microbiol Lett 2002, 207:179-183.
24. Howarth R, Unz RF, Seviour EM, Seviour RJ, Blackall LL, Pickup RW,
Jones JG, Yaguchi J, Head IM: Phylogenetic relationships of
filamentous sulfur bacteria (Thiothrix spp. and Eikelboom type
021N bacteria) isolated from wastewater-treatment plants and
description of Thiothrix eikelboomii sp. nov., Thiothrix unzii sp.
nov., Thiothrix fructosivorans sp. nov. and Thiothrix defluvii sp. nov.
Int J Syst Bacteriol 1999, 49:1817-1827.
25. Kanagawa T, Kamagata Y, Aruga S, Kohno T, Horn M, Wagner M:
Phylogenetic analysis of and oligonucleotide probe development
for Eikelboom type 021N filamentous bacteria isolated from
bulking activated sludge. Appl Environ Microbiol 2000,
66:5043-5052.
This paper shows that filaments of the Eikelboom type 021N form at least
three different phylogenetic lineages and presents probes for specific in situ
detection of these groups in activated-sludge.
26. Seviour EM, Blackall LL, Christensson C, Hugenholtz P,
Cunningham MA, Bradford D, Stratton HM, Seviour RJ: The
filamentous morphotype Eikelboom Type 1863 is not a single
genetic entity. J Appl Microbiol 1997, 82:411-421.
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27. Schade M, Beimfohr C, Lemmer H: Phylogenetic and physiological
characterization of a Nostocoida limicola-like organism isolated
from activated sludge. Third IWA International Specialised
Conference on Microorganisms in Activated Sludge and Biofilm
Processes. Edited by Tandoi V, Passino R, Blundo CM. Rome, Italy
2001: 54-60.
28. Snaidr J, Beimfohr C, Levantesi C, Rossetti S, van der Waarde J,
Geurkink B, Eikelboom D, Lemaitre M, Tandoi V: Phylogenetic
analysis and in situ identification of Nostocoida limicola-like
filamentous bacteria in activated sludge from industrial
wastewater treatment plants. Third IWA International Specialised
Conference on Microorganisms in Activated Sludge and Biofilm
Processes. Edited by Tandoi V, Passino R, Blundo CM. Rome, Italy
2001: 61-67.
29. Liu JR, McKenzie CA, Seviour EM, Webb RI, Blackall LL, Saint CP,
Seviour RJ: Phylogeny of the filamentous bacterium Nostocoida
limicola III from activated sludge. Int J Syst Evol Microbiol 2001,
51:195-202.
30. Liu JR, Seviour RJ: Design and application of oligonucleotide
probes for fluorescent in situ identification of the filamentous
bacterial morphotype Nostocoida limicola in activated sludge.
Environ Microbiol 2001, 3:551-560.
31. Davenport RJ, Curtis TP, Goodfellow M, Stainsby FM, Bingley M:
Quantitative use of fluorescent in situ hybridization to examine
relationships between mycolic acid-containing actinomycetes and
foaming in activated sludge plants. Appl Environ Microbiol 2000,
66:1158-1166.
32. Oerther DB, de los Reyes FL III, de los Reyes MF, Raskin L:
Quantifying filamentous microorganisms in activated sludge
before, during, and after an incident of foaming by oligonucleotide
probe hybridizations and antibody staining. Water Res 2001,
35:3325-3336.
33. de los Reyes FL III, Raskin L: Role of filamentous microorganisms
in activated sludge foaming: relationship of mycolata levels to
foaming initiation and stability. Water Res 2002, 36:445-459.
Very thorough study that elegantly demonstrates how molecular techniques
can be used to better understand foaming problems in WWTPs.
34. Nielsen PH, Roslev P, Dueholm TE, Lund Nielsen J: Microthrix
parvicella, a specialized lipid consumer in anaerobic-aerobic
activated sludge plants. Water Sci Technol 2002, in press.
An excellent paper that demonstrates the potential of the MAR method to
reveal the in situ physiology of microorganisms that are difficult to culture.
The authors provide an interesting hypothesis for the competitive advantage
of M. parvicella in nutrient removal plants.
35. Lee N, Nielsen PH, Andreasen KH, Juretschko S, Nielsen JL,
Schleifer K-H, Wagner M: Combination of fluorescent in situ
hybridization and microautoradiography a new tool for
structure-function analyses in microbial ecology. Appl Environ
Microbiol 1999, 65:1289-1297.
36. Nielsen PH, de Muro MA, Nielsen JL: Studies on the in situ
physiology of Thiothrix spp. present in activated sludge. Environ
Microbiol 2000, 2:389-398.
37. Purkhold U, Pommering-Rser A, Juretschko S, Schmid MC,
Koops H-P, Wagner M: Phylogeny of all recognized species of
ammonia oxidizers based on comparative 16S rRNA and amoA
sequence analysis: implications for molecular diversity surveys.
Appl Environ Microbiol 2000, 66:5368-5382.
This paper provides an encompassing framework for 16S rRNA gene and
amoA gene based diversity surveys of ammonia-oxidising bacteria and
discusses limitations of existing probes and primers for the detection of
these bacteria.
38. Kowalchuk GA, Stephen JR: Ammonia-oxidizing bacteria: a model
for molecular microbial ecology. Annu Rev Microbiol 2001,
55:485-529.
39. Webster G, Embley TM, Prosser JI: Grassland management
regimens reduce small-scale heterogeneity and species diversity
of beta-proteobacterial ammonia oxidizer populations. Appl
Environ Microbiol 2002, 68:20-30.
40. Daims H, Purkhold U, Bjerrum L, Arnold E, Wilderer PA, Wagner M:
Nitrification in sequencing biofilm batch reactors: lessons from
molecular approaches. Water Sci Technol 2001, 43:9-18.
41. Nogueira R, Melo LF, Purkhold U, Wuertz S, Wagner M: Nitrifying and
heterotrophic population dynamics in biofilm reactors: effects of
hydraulic retention time and the presence of organic carbon.
Water Res 2002, 36:469-481.
226 Environmental biotechnology
42. Liebig T, Wagner M, Bjerrum L, Denecke M: Nitrification
performance and nitrifier community composition of a chemostat
and a membrane-assisted bioreactor for the nitrification of sludge
reject waters. Bioprocess Biosyst Eng 2001, 24:203-210.
43. Wagner M, Rath G, Amann R, Koops H-P, Schleifer K-H: In situ
identification of ammonia-oxidizing bacteria. Syst Appl Microbiol
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44. Morgenroth E, Obermayer A, Arnold E, Brhl A, Wagner M,
Wilderer PA: Effect of long-term idle periods on the performance
of sequencing batch reactors. Water Sci Technol 2000,
41:105-113.
45. Hermansson A, Lindgren PE: Quantification of ammonia-oxidizing
bacteria in arable soil by real-time PCR. Appl Environ Microbiol
2001, 67:972-976.
46. Dionisi HM, Layton AC, Harms G, Gregory IR, Robinson KG,
Sayler GS: Quantification of Nitrosomonas oligotropha-like
ammonia-oxidizing bacteria and Nitrospira spp. from full-scale
wastewater treatment plants by competitive PCR. Appl Environ
Microbiol 2002, 68:245-253.
47. Schramm A, de Beer D, van den Heuvel JC, Ottengraf S, Amann R:
Microscale distribution of populations and activities of
Nitrosospira and Nitrospira spp. along a macroscale gradient in a
nitrifying bioreactor: quantification by in situ hybridization and the
use of microsensors. Appl Environ Microbiol 1999, 65:3690-3696.
48. Daims H, Nielsen JL, Nielsen PH, Schleifer KH, Wagner M: In situ
characterization of Nitrospira-like nitrite-oxidizing bacteria active
in wastewater treatment plants. Appl Environ Microbiol 2001,
67:5273-5284.
The authors developed oligonucleotide probes for the in situ detection of
the Nitrospira-related nitrite oxidisers and used them together with MAR
to investigate the ecophysiology of these uncultured nitrite oxidisers in
activated sludge.
49. Burrell PC, Keller J, Blackall LL: Microbiology of a nitrite-oxidizing
bioreactor. Appl Environ Microbiol 1998, 64:1878-1883.
50. Regan JM, Harrington GW, Noguera DR: Ammonia- and nitrite-
oxidizing bacterial communities in a pilot-scale chloraminated
drinking water distribution system. Appl Environ Microbiol 2002,
68:73-81.
51. Bartosch S, Hartwig C, Spieck E, Bock E: Immunological detection
of Nitrospira-like bacteria in various soils. Microb Ecol 2002,
in press.
52. Schramm A, De Beer D, Gieseke A, Amann R: Microenvironments
and distribution of nitrifying bacteria in a membrane-bound
biofilm. Environ Microbiol 2000, 2:680-686.
A nice paper that provides supporting data for the hypothesis that
Nitrobacter outcompetes Nitrospira in the presence of high oxygen and
nitrite concentrations.
53. Gumaelius L, Magnusson G, Pettersson B, Dalhammar G:
Comamonas denitrificans sp. nov., an efficient denitrifying
bacterium isolated from activated sludge. Int J Syst Evol Microbiol
2001, 51:999-1006.
54. Khan ST, Hiraishi A: Isolation and characterization of a new
poly(3-hydroxybutyrate)-degrading, denitrifying bacterium from
activated sludge. FEMS Microbiol Lett 2001, 205:253-257.
55. Neef A, Zaglauer A, Meier H, Amann R, Lemmer H, Schleifer K-H:
Population analysis in a denitrifying sand filter: conventional and
in situ identification of Paracoccus spp. in methanol-fed biofilms.
Appl Environ Microbiol 1996, 62:4329-4339.
56. Lemmer H, Zaglauer A, Neef A, Meier H, Amann R: Denitrification in
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the bacterial community in biofilms. Water Res 1997,
31:1903-1908.
57. Braker G, Zhou J, Wu L, Devol AH, Tiedje JM: Nitrite reductase
genes (nirK and nirS) as functional markers to investigate
diversity of denitrifying bacteria in pacific northwest marine
sediment communities. Appl Environ Microbiol 2000,
66:2096-2104.
58. Braker G, Ayala-del-Rio HL, Devol AH, Fesefeldt A, Tiedje JM:
Community structure of denitrifiers, bacteria, and archaea along
redox gradients in Pacific Northwest marine sediments by
terminal restriction fragment length polymorphism analysis of
amplified nitrite reductase (nirS) and 16S rRNA genes. Appl
Environ Microbiol 2001, 67:1893-1901.
59. Michotey V, Mejean V, Bonin P: Comparison of methods for
quantification of cytochrome cd1-denitrifying bacteria in
environmental marine samples. Appl Environ Microbiol 2000,
66:1564-1571.
60. Wu L, Thompson DK, Li G, Hurt RA, Tiedje JM, Zhou J: Development
and evaluation of functional gene arrays for detection of selected
genes in the environment. Appl Environ Microbiol 2001,
67:5780-5790.
61. Nielsen JL, Nielsen PH: Enumeration of acetate-consuming
bacteria by microautoradiography under oxygen and nitrate
respiring conditions in activated sludge. Water Res 2002,
36:421-428.
62. Jetten MS, Wagner M, Fuerst J, van Loosdrecht M, Kuenen G,
Strous M: Microbiology and application of the anaerobic
ammonium oxidation (anammox) process. Curr Opin Biotechnol
2001, 12:283-288.
63. Toh SK, Webb RI, Ashbolt NJ: Enrichment of autotrophic anaerobic
ammonium-oxidizing consortia from various wastewaters. Microb
Ecol 2002, in press.
64. Schmid M, Schmitz-Esser S, Jetten M, Wagner M: 16S23S rDNA
intergenic spacer and 23S rDNA of anaerobic ammonium-
oxidizing bacteria: implications for phylogeny and in situ
detection. Environ Microbiol 2001, 3:450-459.
65. Hesselmann RPX, Werlen C, Hahn D, van der Meer JR, Zehnder AJB:
Enrichment, phylogenetic analysis and detection of a bacterium
that performs enhanced biological phosphate removal in
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66. Crocetti GR, Hugenholtz P, Bond PL, Schuler A, Keller J, Jenkins D,
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organisms and design of 16S rRNA-directed probes for their
detection and quantitation. Appl Environ Microbiol 2000,
66:1175-1182.
A nice paper which, together with the data presented by Hesselmann et al.
[65], showed that Rhodocyclus-related bacteria are PAOs.
67. Liu WT, Nielsen AT, Wu JH, Tsai CS, Matsuo Y, Molin S: In situ
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68. Lee N, Jansen J, Aspegren H, Dircks K, Henze M, Schleifer K-H,
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phosphorus-accumulating bacteria in wastewater treatment
plants with enhanced biological phoshorus removal operated
with and without nitrogen removal. Water Sci Technol 2002,
in press.
69. Zilles JL, Hung C-H, Noguera DR: Presence of Rhodocyclus in a
full-scale wastewater treatment plant and their participation in
enhanced biological phoshorus removal. Water Sci Technol 2002,
in press.
70. Cech JS, Hartman P: Glucose induced breakdown of enhanced
biological phosphate removal. Environ Technol 1990, 11:651-656.
71. Liu W-T, Mino T, Nakamura K, Matsuo T: Role of glycogen in acetate
uptake and polyhydroxyalkanoate synthesis in anaerobic-aerobic
activated sludge with a minimized polyphosphate content.
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72. Liu W-T, Mino T, Nakamura K, Matsuo T: Glycogen accumulating
population and its anaerobic substrate uptake in anaerobic-
aerobic activated sludge without biological phosphorus removal.
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73. Nielsen AT, Liu WT, Filipe C, Grady L, Molin S, Stahl DA:
Identification of a novel group of bacteria from a deteriorated
biological phosphorus removal reactor. Appl Environ Microbiol
1999, 65:1251-1258.
74. Tsai C-S, Liu W-T: Phylogenetic and physiological diversity of
tetrad-forming organisms in deteriorated biological phosphorus
removal systems. Water Sci Technol 2002, in press.
75. Seviour RJ, Maszenan AM, Soddell JA, Tandoi V, Patel BK, Kong Y,
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76. Oerther DB, Pernthaler J, Schramm A, Amann R, Raskin L: Monitoring
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 Bacterial community composition and function in sewage treatment systems Wagner and Loy
77. Radajewski S, Ineson P, Parekh NR, Murrell JC: Stable-isotope
probing as a tool in microbial ecology. Nature 2000, 403:646-649.
The authors introduce in this excellent paper a new and very elegant method
to investigate the function of microorganisms in environmental samples
without cultivation.
78. Bouchez T, Patureau D, Dabert P, Juretschko S, Dor J, Delgens P,
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79. Muttray AF, Yu ZT, Mohn WW: Population dynamics and metabolic
activity of Pseudomonas abietaniphila BKME-9 within pulp mill
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80. Watanabe K, Miyashita M, Harayama S: Starvation improves
survival of bacteria introduced into activated sludge. Appl Environ
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81. Urakawa H, Noble PA, El Fantroussi S, Kelly JJ, Stahl DA: Single-
base-pair discrimination of terminal mismatches by using
oligonucleotide microarrays and neural network analyses. Appl
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82. Guschin DY, Mobarry BK, Proudnikov D, Stahl DA, Rittmann BE,
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83. Liu WT, Mirzabekov AD, Stahl DA: Optimization of an oligonucleotide
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84. Small J, Call DR, Brockman FJ, Straub TM, Chandler DP: Direct
detection of 16S rRNA in soil extracts by using oligonucleotide
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227
85. Beja O, Suzuki MT, Koonin EV, Aravind L, Hadd A, Nguyen LP,
Villacorta R, Amjadi M, Garrigues C, Jovanovich SB et al.:
Construction and analysis of bacterial artificial chromosome
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86. Beja O, Aravind L, Koonin EV, Suzuki MT, Hadd A, Nguyen LP,
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An outstanding paper demonstrating how much can be learnt from
environmental genomics.
87. Daims H, Brhl A, Amann R, Schleifer K-H, Wagner M: The
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89. Christensson M, Blackall LL, Welander T: Metabolic transformations
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90. Dabert P, Sialve B, Delgenes JP, Moletta R, Godon JJ:
Characterisation of the microbial 16S rDNA diversity of an
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