High diversity and abundance of putative polyphosphate‐accumulating Tetrasphaera‐related bacteria in activated sludge systems

R E S E A R C H A R T I C L E

High diversityandabundanceof putativepolyphosphate-accumulatingTetrasphaera-related bacteria inactivatedsludge systemsHien Thi Thu Nguyen, Vang Quy Le, Aviaja Anna Hansen, Jeppe Lund Nielsen & Per Halkjær Nielsen

Department of Biotechnology, Chemistry, and Environmental Engineering, Aalborg University, Aalborg, Denmark

Correspondence: Per Halkjær Nielsen,

Department of Biotechnology, Chemistry, and

Environmental Engineering, Aalborg

University, Sohngaardsholmsvej 49, DK-9000

Aalborg, Denmark. Tel.: 145 9940 8503; fax:

145 9814 1808; e-mail: [email protected]

Received 18 July 2010; revised 22 December

2010; accepted 29 December 2010.

DOI:10.1111/j.1574-6941.2011.01049.x

Editor: Alfons Stams

Keywords

EBPR; PAO; P-removal; microautoradiography;

wastewater.

Abstract

The diversity of the putative polyphosphate-accumulating genus Tetrasphaera in

wastewater treatment systems with enhanced biological phosphorus removal

(EBPR) was investigated using the full-cycle rRNA approach combined with

microautoradiography and histochemical staining. 16S rRNA actinobacterial gene

sequences were retrieved from different full-scale EBPR plants, and the sequences

belonging to the genus Tetrasphaera (family Intrasporangiaceae) were found to

form three clades. Quantitative FISH analyses of the communities in five full-scale

EBPR plants using 10 new oligonucleotide probes were carried out. The results

showed that the probe-defined Tetrasphaera displayed different morphologies and

constituted up to 30% of the total biomass. It was shown that active uptake of

orthophosphate and formation of polyphosphate took place in most of the probe-

defined Tetrasphaera populations. However, aerobic uptake of orthophosphate

only took place after uptake of certain carbon sources under anaerobic conditions

and these were more diverse than hitherto assumed: amino acids, glucose, and for

some also acetate. Tetrasphaera seemed to occupy a slightly different ecological

niche compared with ‘Candidatus Accumulibacter’ contributing to a functional

redundancy and stability of the EBPR process.

Introduction

The enhanced biological phosphorus removal (EBPR) pro-

cess has been implemented in many wastewater treatment

plants (WWTPs) worldwide. When operated successfully,

the EBPR process is a relatively inexpensive and environ-

mentally sustainable option for phosphorus (P) removal;

however, the stability and reliability of the EBPR process can

be a problem (Seviour et al., 2003). EBPR is based on the

ability of polyphosphate-accumulating organisms (PAOs) to

take up P and accumulate it intracellularly as polyphosphate

when exposed to alternating anaerobic [O2 and nitrite/

nitrate absent] and aerobic conditions (Comeau et al.,

1986; Wentzel et al., 1986).

Many studies in both lab-scale and full-scale EBPR plants

have shown that ‘Candidatus Accumulibacter phosphatis’

(referred to as Accumulibacter hereafter) are important

PAOs and their enriched cultures generally behave as the

biochemical models predict (Hesselmann et al., 1999; Cro-

cetti et al., 2000; Liu et al., 2001; Zilles et al., 2002; Beer et al.,

2006; Garcia Martin et al., 2006). Under anaerobic condi-

tions, they take up low-molecular-weight organic acids (e.g.

acetate, propionate, and pyruvate) using polyphosphate as

an energy source. These substrates are stored as intracellular

polyhydroxyalkanoates via energy from hydrolysis of intra-

cellular polyphosphate and reducing power from glycolysis

of intracellular glycogen (Mino et al., 1998), the tricar-

boxylic acid cycle (Pereira et al., 1996; Louie et al., 2000;

Lemos et al., 2003), or both (Wexler et al., 2009; Zhou et al.,

2009). In the subsequent aerobic or denitrifying phase, the

PAO can use polyhydroxyalkanoates for growth and for

replenishing their polyphosphate and glycogen pools (van

Loosdrecht et al., 1997; Garcia Martin et al., 2006; Zhou

et al., 2009).

Besides Accumulibacter, Gram-positive Tetrasphaera-re-

lated organisms (Actinobacteria) are also putative PAOs that

are abundant in many full-scale EBPR plants (Eschenhagen

et al., 2003; Kong et al., 2005; Nielsen et al., 2010), often in

higher numbers than Accumulibacter. Studies of their eco-

physiology have, however, shown that they do not share all

typical PAO characteristics known from Accumulibacter.

They are able to take up phosphate and form polyphosphate

FEMS Microbiol Ecol ]] (2011) 1–12 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

MIC

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under aerobic conditions, but cannot take up short-chain

fatty acids under anaerobic conditions, and do not store

polyhydroxyalkanoates (Kong et al., 2005). They can take up

some amino acids, and some Tetrasphaera seem capable of

fermenting glucose (Kong et al., 2008). Furthermore, they

express extracellular surface-associated amylases for degrada-

tion of starch (Xia et al., 2008). These results suggest that the

ecophysiology of PAOs – and particularly Tetrasphaera-

related PAOs – is much more diverse than hitherto anticipated.

A few isolates of the genus Tetrasphaera are available.

These include Tetrasphaera australiensis, Tetrasphaera japo-

nica (Maszenan et al., 2000), Tetrasphaera elongata (strain

LP2) (Hanada et al., 2002), T. elongata (strain ASP12) (Onda

& Takii, 2002), and the filamentous Tetrasphaera jenkinsii,

Tetrasphaera vanveenii, and Tetrasphaera veronensis (McKen-

zie et al., 2006). Storage of intracellular polyphosphate has

been found in all isolates, but only the filamentous species

are able to synthesize polyhydroxyalkanoates (McKenzie

et al., 2006). Interestingly, some of these cultured Tetra-

sphaera have some ecophysiological traits rather similar to

those of the uncultured Tetrasphaera-related PAO from full-

scale EBPR plants. For T. elongata (strain LP2), their aerobic

P uptake depends on an anaerobic uptake of glucose, and for

T. elongata strain ASP12, T. australiensis, and T. japonica, an

amino acid mixture (casamino acids) is required. However,

the strains are difficult to grow in pure culture, and more

detailed studies of their physiology are lacking.

The diversity of the genus Tetrasphaera is not well

described in full-scale EBPR plants. Surveys using FISH

applying probes targeting species from the Tetrasphaera

genus, Actino-221 and Actino-658 (Kong et al., 2005), have

shown that they usually grow as cocci in clusters of tetrads

and short rods in clumps. We have, however, occasionally

observed other morphologies of probe-defined Tetra-

sphaera, and so the entire diversity seems to be not yet well

described among these putative PAOs. Furthermore, it is

uncertain whether they all are PAOs capable of accumulating

intracellular polyphosphate in full-scale plants.

The aim of this study was to investigate the diversity, the

function, and the abundance of the genus Tetrasphaera in

full-scale EBPR WWTPs. A better understanding of their

ecophysiology, ecology, and abundances is needed to under-

stand the role of the Tetrasphaera-related PAOs in full-scale

EBPR plants and be able to manipulate the communities for

optimal P removal and troubleshooting.

Materials and methods

Activated sludge sampling

Activated sludge samples were collected from six Danish

full-scale WWTPs (Ejby Mølle, Hjørring, Abenra, Skive,

Aalborg East, and Odense), all of which were well-working

EBPR plants with nitrogen removal (nitrification and deni-

trification) and stable over several years. Effluent concentra-

tions are always below 1.0 mg total P L�1 and 8 mg total

N L�1. The C/P ratio in incoming wastewater was between

60 and 90 g total chemical oxygen demand g�1 total P for all

plants and 4 90% of the P was removed during treatment.

More information about the plants can be found elsewhere

(Nielsen et al., 2010). Samples from three plants (Ejby

Mølle, Hjørring, and Aalborg East) were used for studies of

the ecophysiology of Tetrasphaera. Two plants (Ejby Mølle

and Aalborg East) have a BioDeniphos configuration (Se-

viour & Blackall, 1999), and Hjørring WWTP has recirculat-

ing operation. Fresh samples from aeration tanks were stored

at 4 1C for o 24 h to carry out all the experiments. The

samples were diluted with filtered nitrate and nitrite-free

effluent to a final concentration of 1 g suspended solids L�1

before the experiments on ecophysiology were performed.

Clone library construction, clone screening,and sequencing

Community DNA was extracted from five activated sludge

samples using the PowerSoilTM DNA Isolation Kit (Mo Bio).

All extracted DNA were pooled and used for construction of

four clone libraries based on PCR amplification of 16S

rRNA genes with four different primer sets to get as many

different sequences as possible. Set A consisted of primers

27F and 1492R targeting Universal bacteria (Lane, 1991); set

B consisted of 27F and actino-1011R targeting T. japonica

(Liu et al., 2001); set C consisted of Actino-221F targeting

many Tetrasphaera (Kong et al., 2005) and 1492R, and set D

consisting of HGC236F targeting most Actinobacteria (Er-

hart et al., 1997) and 1492R. A touchdown PCR program

was applied: 5 min denaturing at 94 1C, followed by 10 cycles

of: (1) 94 1C for 30 s, (2) 1 min annealing at 60, 62, and 68 1C

(decreasing 1 1C per cycle) for primer sets A, B, and C,

respectively, and (3) 72 1C for 45 s, followed by 20 cycles

with the same denaturing and extension conditions, but

with 1 min annealing at 10 1C below the initial annealing

temperature, finalized with a 7-min extension at 72 1C. The

optimized template amount was 20–200 ng DNA per PCR

reaction. The PCR products were ligated into the pCRII-

TOPO vector (Invitrogen, Groningen, the Netherlands), and

clones with the correct 16S rRNA gene inserts (confirmed by

fragment size after PCR amplification) were sequenced with

the PCR primers by Macrogen (Seoul, Korea).

Phylogenetic analysis

The 16S rRNA gene sequences obtained were checked for

their chimeric properties using the MALLARD software (Ashel-

ford et al., 2006). Nearly full-length 16S rRNA gene

sequences were imported into the ARB software package

(Ludwig et al., 2004), aligned, and a phylogenetic tree was

FEMS Microbiol Ecol ]] (2011) 1–12c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

2 H.T.T. Nguyen et al.

constructed based on comparative analysis of aligned 16S

rRNA gene nucleotide sequences. Phylogenetic trees were

calculated using the neighbor-joining method with a Pois-

son correction model and a 100-replicate bootstrap analysis.

The branching pattern was compared with trees generated

using the neighbor-joining algorithm.

FISH, polyhydroxyalkanoate and 4,6-diamino-2-phenylindole (DAPI) staining

FISH was performed as detailed in Nielsen (2009). Lysozyme

[0.5 g L�1 in 100 mM Tris (pH 7.5) and 5 mM EDTA] was

used to increase the permeability of the cells. The oligonu-

cleotide probes EUBmix (equimolar concentration of

EUB338, EUB338II, and EUB338III) targeting most bacteria

(Amann et al., 1990; Daims et al., 1999) were labeled by

FLUOS [5(6)-carboxyfluorescein-N-hydroxy-succinimide

ester], whereas PAOmix (equimolar concentration of

PAO462, PAO651, and PAO846) targeting most Accumuli-

bacter (Crocetti et al., 2000), HGC69a targeting actinobacter-

ial high-G1C Gram-positive bacteria (Roller et al., 1994),

HGC236 targeting Actinobacteria (Erhart et al., 1997), acti-

no-1011 targeting T. japonica (Liu et al., 2001), Actino-221

and Actino-658 targeting potential Tetrasphaera-PAOs (Kong

et al., 2005) were labeled with either FLUOS or Cy3 (sulfoin-

docyanine dyes). Their specificities, hybridization require-

ments, and reference information are described in probeBase

(Loy et al., 2003). Twenty separate microscopy images were

taken for quantification FISH analysis, with the final results

reflecting the mean percentages of Tetrasphaera of the entire

bacterial community detected by EUBmix (Nielsen, 2009).

The sample was stained with DAPI (0.2 mM final con-

centration) for 5 min after FISH, rinsed thoroughly with

sterile distilled water, and air-dried. The DAPI signal of

stained cells was examined using a Zeiss Axiovision epi-

fluorescence microscope (Carl Zeiss) equipped with a CCD

camera (Quantex, Photometrics). With this DAPI concen-

tration, the polyphosphate granules appear bright yellow,

while the cells remain pale blue (Kawaharasaki et al., 1999).

For FISH analysis, the same field examined for DAPI was

examined under the corresponding filter sets for fluores-

cence labels Cy3 and FLUOS.

Presence of polyhydroxyalkanoates as intracellular sto-

rage polymers was tested by Nile Blue staining largely as

described elsewhere (Kragelund et al., 2005). FISH was,

however, performed first followed by chemical staining and

relocation of the same microscopic field.

Design and testing of oligonucleotide probes

Tetrasphaera-specific probes were designed using the probe

design tool in the ARB software package (Ludwig et al., 2004).

The specificities of these probes were confirmed using the

CHECK PROBE program in the Ribosomal Database Project

(Maidak et al., 2001). The formamide concentration for

optimum probe stringency was determined by performing a

series of FISH experiments at 5% formamide increments

from 0% to 60% formamide on isolates when possible or on

activated sludge. The fluorescence intensities of single cells

from at least 20 images for each probe were evaluated by

image analysis using IMAGEJ software (Collins, 2007). The

specificities of new Tetrasphaera probes and the formamide

concentrations used are described in Table 1. Three reference

strains, T. elongata, T. jenkinsii, and T. australiensis (pro-

vided by R. Seviour, La Trobe University, Australia), were

used in this study. All strains were grown on GS or R2A

media (Maszenan et al., 2000).

Microautoradiography--FISH

A microautoradiography–FISH method was slightly mod-

ified from that described by Nielsen & Nielsen (2005) and

Kong et al. (2005). Briefly, 2 mL activated sludge samples

were incubated in 9 mL serum bottles with labeled and

unlabeled substrates under aerobic or anaerobic conditions.

All anaerobic preparations were carefully flushed with O2-

free N2. After incubation they were fixed by addition of 96%

ethanol and allowed to stand for 3 h at 4 1C, and they were

subsequently washed in a phosphate-buffered saline (PBS)

at 4 1C and finally resuspended in ice-cold PBS and ice-cold

96% ethanol (1 : 1). The samples were gently homogenized

between two gelatin-coated cover glasses (24� 60 mm).

FISH probing of microautoradiography-incubated bio-

mass, coating with emulsion, exposure, and development of

the hybridized FISH slides before being examined by micro-

scopy were carried out as described previously (Nielsen &

Nielsen, 2005). Each microautoradiography experiment was

repeated at least three times in different WWTPs. Controls

for nonradioactively induced silver grain formation (che-

mography) were always included, and no microautoradio-

graphy-positive cells were ever observed with any of the

nonradioactive chemicals used in this study.

The ability to take up labeled orthophosphate (33P) by

Tetrasphaera was investigated as follows. Before the incuba-

tions with 33P, sludge samples were incubated anaerobically

with different carbon sources (1 mM). Any unconsumed

carbon source was removed by washing the samples three

times with the filtered effluent water before labeled 33P and

nonlabeled P were added to a final concentration of 0.3 mM

at the very beginning of a 4-h incubation period with

oxygen. Samples for microautoradiography analyses were

taken at the end of the incubation.

In order to investigate the storage capacity of Tetra-

sphaera and Accumulibacter, it was investigated whether they

could continuously take up labeled acetate or glucose

(1 mM, 2 h incubation) after prolonged anaerobic conditions

(0, 3, 5, and 9 h) with unlabeled acetate present (2 mM).

FEMS Microbiol Ecol ]] (2011) 1–12 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

3Tetrasphaera-related bacteria in activated sludge systems

The sources and specifications of all the radioactive

chemicals in this study have been described previously

(Kong et al., 2005).

Nucleotide sequence accession numbers

The 16S rRNA gene sequences obtained in this study have

been deposited in the GenBank database under accession

numbers GU552242–GU552268.

Results

Clone library analysis

Four clone libraries were constructed based on the commu-

nity 16S rRNA genes amplified by four primer sets. A total of

189 16S rRNA gene sequences were retrieved, and 93

sequences in 10 different actinobacterial families were

identified. Nearly one-third of these sequences belonged to

the family Intrasporangiaceae in the Actinobacteria. Their

phylogenetic relationship with other closely related bacteria

is shown in the phylogenetic tree (Fig. 1). The clone

sequences grouped into three separate clades. Clade 1

includes clones related to sequences of T. elongata, which

was isolated from activated sludge (98.1–99.8% identity),

and Tetrasphaera duodecadis (97.6–98.9% identity). Clade 2

is related to T. jenkinsii (96.5–96.9% identity), T. australien-

sis (97.1–97.4% identity), ‘Candidatus Nostocoida limicola’

(96.3–96.7% identity), and T. veronensis (96.7–97% iden-

tity), all isolated from activated sludge. Clones in clade 3

shared 98.2–99.9% identity and were not related to any

cultured species. Besides these sequences, some clones were

related to T. japonica (97.1–97.5% identity) isolated from

activated sludge and some to Tetrasphaera sp. Ellin176

(96.1–97.0% identity) isolated from a soil sample. These

clones did not form other distinct clades or clusters.

Design, testing, and optimization ofoligonucleotide probes

A number of oligonucleotide probes with different specifi-

cities were designed to investigate morphologies and abun-

dances and for the determination of important

ecophysiological traits (Table 1). Ten probes were designed

to target most, but not all, of the clones belonging to the

family Intrasporangiaceae retrieved in this study and 24

other partial and full sequences (Crocetti et al., 2000; Kong

et al., 2005). Attempts to design probes covering entire

clades (Fig. 1) or all Tetrasphaera sequences were not

successful.

The 10 probes were tested and optimized on samples

from five different EBPR WWTPs (Ejby Mølle, Hjørring,

Abenra, Skive, and Odense) and on pure Tetrasphaera

cultures. The sequences related to T. elongata (clade 1) were

targeted by probes Elo1-1250, Tet1-823, and Tet1-266. The

morphology of these bacteria was very diverse (Table 2).

Bacteria related to T. jenkinsii, T. australiensis, and T.

veronensis (clade 2) were targeted by five probes, and they

also showed large differences in morphology (Table 2 and

Fig. 2). Two morphotypes have not been described before:

the thin filaments (approximately 0.45mm in width and

25–60 mm in length) and the small branched rods. Clade 3

without cultured representatives was targeted by probes

Tet3-654 and Tet3-19. Probe Tet3-19-targeted bacteria were

cocci in clusters of tetrad, whereas Tet3-654-targeted bacter-

ia had four different morphologies (cluster of tetrads, small

cocci, branched rods, and short rods in clumps) (Fig. 2).

Probes targeting bacteria related to T. japonica and Tetra-

sphaera sp. Ellin176 were also designed, but the targeted cells

were present in only one of the treatment plants investigated

and only in very low numbers (o 1% of the EUB-targeted

cells, data not shown). Bacteria targeted by these probes

were not further investigated.

Table 1. Oligonucleotide probes designed for detection of Tetrasphaera in EBPR plants

Name Abbreviation Clade Target group Sequence (50–30) % Formamide

S-S-Elo1-1250-a-A-18 Elo1-1250 1 T. elongata and related clones CGCGATTTCGCAGCCCTT 20

S-S-Tet1-823-a-A-18 Tet1-823 1 T. elongata-related clones TGAGACCCGCACCTAGTT 30

S-S-Tet1-266-a-A-18 Tet1-266 1 Clone ASM31 CCCGTCGTCGCCTGTAGC 25

S-S-Tet2-842-a-A-18 Tet2-842 2 Clones related to T. jenkinsii, T. australiensis,

and T. veronensis

GCGGCACAGAACTCGTGA 30

S-S-Tet2-831-a-A-18 Tet2-831 2 T. australiensis, T. veronensis,

and uncultured Tetrasphaera

TCGTGAAATGAGTCCCAC 10

S-S-Tet2-892-a-A-18 Tet2-892 2 Clone ASM47 TAGTTAGCCTTGCGGCCG 5

S-S-Tet2-87-a-A-18 Tet2-87 2 Uncultured Tetrasphaera TCGCCACTGATCAGGAGA 10

S-S-Tet2-174-a-A-18 Tet2-174 2 T. jenkinsii, T. australiensis, T. veronensis,

and Candidatus N. limicola

GCTCCGTCTCGTATCCGG 20

S-S-Tet3-654-a-A-18 Tet3-654 3 Uncultured Tetrasphaera GGTCTCCCCTACCATACT 35

S-S-Tet3-19-a-A-18 Tet3-19 3 Clone ASM57 CAGCGTTCGTCCTACACA 0

Coverage is also shown in Fig. 1.

FEMS Microbiol Ecol ]] (2011) 1–12c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

4 H.T.T. Nguyen et al.

Ecophysiology determined bymicroautoradiography-FISH andchemical staining

All probe-defined Tetrasphaera showed intracellular DAPI-

positive granules indicating the presence of polyphosphate.

In order to investigate whether they had an active P uptake

when changing from anaerobic to aerobic conditions, a

number of experiments using 33P-labeled phosphate were

conducted. The five most abundant probe-defined popula-

tions were tested, and four of them could take up 33P under

aerobic conditions after a 2-h anaerobic preincubation

period with organic substrates (Table 3): clade 1 (targeted

by probe Tet1-266), clade 2 (Tet2-892 and Tet2-174), clade 3

(Tet3-654), but not those targeted by probe Tet3-19. Two

important observations were made: (1) the uptake

Uncultured bacteria, AF387312Uncultured bacteria, AF387313Uncultured bacteria, AF387307Tetrasphaera elongata, AB030911

Uncultured bacteria, AF387308Uncultured bacteria, AF387311

Clone ASM28, GU552252Clone ASM29, GU552253

Clone ASM31, GU552254 Clone ASM26, GU552251

Tetrasphaera duodecadis, AB072496Clone ASM14, GU552247

Clone ASM3, GU552243Clone ASM47, GU552259

Clone ASM12, GU552246Clone ska14, AY710276Clone ska15, AY710277

Clone ska20, AY710275Clone ska2, AY710287Clone ska22, AY710283Candidatus Nostocoida limicola, Y14596Tetrasphaera jenkinsii, DQ007321Tetrasphaera australiensis, AF125091

Tetrasphaera veronensis, Y14595Uncultured bacteria, AF387314

Clone ska9, AY710273Clone ASM24, GU552249

Clone ASM57, GU552268Clone ASM62, GU552263Clone ska3, AY710288Clone ASM59, GU552260

Clone ska6, AY710272Clone ska16, AY710278

Clone ska5, AY710289Clone ska21, AY710284

Clone ska25, AY710284Clone ASM64, GU552265Clone Ska12, AY710274Clone ASM63, GU552264Clone ASM66, GU552267Clone ASM65, GU552266

Clone ska26, AY710285Clone ASM25, GU552250Clone ASM33, GU552255

Clone ASM8, GU552245 Clone ASM44, GU552257Clone ska7, AY710271Clone ASM61, GU552262

Clone ska28, AY710286Clone ASM6, GU552244

Clone ASM60, GU552261Tetrasphaera japonica, AF125092

0.01

Tet

1-82

3T

et1-

266

Tet

2-84

2

Elo

1-12

50T

et3-

654

Tet

3-19

Tet

2-83

1

Tet

2-87

Tet

2-17

4T

et2-

892

Cla

de 1

Cla

de 2

Cla

de 3

Clone ASM1, GU552242Clone ASM39, GU552256

Clone ASM45, GU552251Clone ASM16, GU552248

Clone ska19, AF710281Tetrasphaera sp. Ellin176, AF409018

85

60

87

95

54

57

8758

6856

88

7651

55

66

97

97

9867

8096

9669

82

82

4866

60

86

72

81

Out

grou

p

Ornithinicoccus hortensis, Y17869

Fig. 1. Distance matrix tree of the genus Tetrasphaera in the Actinobacteria showing high diversity of Tetrasphaera-related bacteria in an EBPR-

activated sludge system. Ten betaproteobacterial sequences were used as the outgroup. Boldface names indicate the sequences obtained in this study.

Each sequence represents one clone. The dotted parts of the bracket lines indicate that the sequences are not covered by probes. The bar indicates 1%

sequence divergence.

FEMS Microbiol Ecol ]] (2011) 1–12 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

5Tetrasphaera-related bacteria in activated sludge systems

depended on the type of organic substrate present during

the 2-h anaerobic preincubation and (2) only certain

morphotypes from each probe-defined population could

take up P.

When raw wastewater was added as substrate under

anaerobic conditions, all probe-defined morphotypes took

up 33P in the subsequent aerobic stage except some bacteria

from clade 3 targeted by probe Tet3-19, and the filamentous

morphotype of clade 2 (hybridizing with probe Tet2-174).

In control experiments without any added external organic

substrate in the anaerobic preincubation, none of the probe-

defined populations were able to take up 33P in the aerobic

period. This indicated that they were all able to accumulate

energy reserves during the anaerobic period to take up 33P

under aerobic conditions, presumably by taking up organic

substrates. The effect of specific organic substrates during

anaerobic preincubation on the subsequent 33P uptake by

the Tetrasphaera-PAO is shown in Table 4. The four sub-

strates tested (casamino acids, glutamic acid, glucose, and

acetate) were consumed by most Tetrasphaera. Thus, the

physiological traits shown here meet the criteria for PAOs,

and so the four probe-defined populations are called Tet1-

266-PAO, Tet2-892-PAO, Tet2-174-PAO, and Tet3-654-PAO

here.

The ability of Tetrasphaera-PAOs to take up acetate,

glucose, casamino acid, and glutamic acid was investigated

under both aerobic and anaerobic conditions (Table 4). The

uptake pattern was very similar for three of the probe-

defined populations (Tet2-892-PAO, Tet2-174-PAO, and

Tet3-654-PAO) as they could take up all four substrates

under both conditions. Only Tet1-266-PAOs were unable to

take up acetate and glutamic acid. Interestingly, Tet3-19-

defined populations could take up glucose and glutamic

acid under both anaerobic and aerobic conditions (Table 4),

but were not able to take up 33P (Table 3) despite the

presence of intracellular DAPI-positive granules.

None of the probe-defined nonfilamentous Tetrasphaera

bacteria had any intracellular polyhydroxyalkanoates as

tested by Nile Blue staining. Because most Tetrasphaera were

able to take up acetate and other organic compounds under

anaerobic conditions, it was investigated whether they could

continuously take up acetate under prolonged anaerobic

conditions, or whether they reached a saturation level

indicative of storage capacity. No Tetrasphaera-PAOs could

assimilate acetate after 3, 5, and 9 h of preincubation with

unlabeled acetate (Table 5). Therefore, under anaerobic

conditions, the storage capacity of Tetrasphaera-PAO was

saturated after 3 h. This indicated that no growth took place

Table 2. Abundance and morphology of Tetrasphaera and Accumulibacter in five full-scale EBPR plants as determined by quantitative FISH

Treatment Plant (% EUBmix� SD)

Probe Clade Morphology Relative abundance % Ejby Mølle Hjørring Abenra Skive Odense

Elo1-1250 1 Short rods in clumps 50 0 0 o 1 3.2� 1.3 1.2�1.0

Branched rods 50

Tet1-823 1 Short rods in clumps 50 o 1 3.4�1.6 0 0 0

Branched rods 50

Tet1-266 1 Clusters of tetrads 50 5.5� 2.0 4.2�1.2 4.1� 1.4 o 1 4.3�1.0

Branched rods 45

Other 5

Tet2-842 2 Cluster of tetrads 100 o 1 1.1�1.0 3� 1.3 o 1 o 1

Tet2-831 2 Cluster of tetrads 100 0 0 3.1� 1.5 0 0

Tet2-892 2 Cluster of tetrads 60 5.4� 1.3 4.4�1.0 4.5� 1.5 4.3� 1.0 4.4�1.0

Thin filaments 35

Other 5

Tet2-87 2 Cluster of tetrads 100 2.3� 1.7 1.2�1 o 1 o 1 o 1

Tet2-174 2 Cluster of tetrads 60 5.2� 1.3 4�1.0 2.9� 1.0 1.7� 1.0 4.5�1.2

Branched rods 25

Filaments 10

Other 5

Tet3-654 3 Cluster of tetrads 30 6.8� 2.0 5.7�1.1 5.5� 1.8 5.4� 2.0 5.2�1.4

Branched rods 35

Short rods in clumps 30

Other 5

Tet3-19 3 Cluster of tetrads 100 4.3� 1.5 4.1�1.0 3.2� 1.0 3.5� 1.0 4.3�1.0

PAO mix

(Accumulibacter)

Coccobacillus in

microcolonies

5.4� 1.0 3.1�1.3 4.8� 1.5 4.5� 1.0 5.4�1.2

The relative abundance of each morphology within each probe-targeted group is estimated along with their contribution to the total bacteria, which

was estimated using EUBmix.

FEMS Microbiol Ecol ]] (2011) 1–12c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

6 H.T.T. Nguyen et al.

under anaerobic conditions and that they only had a limited

uptake/storage capacity. However, all Tetrasphaera-PAOs

could take up glucose after 9 h of incubation with unlabeled

acetate, indicating that fermentation took place.

Abundance of Tetrasphaera in full-scaleEBPR plants

The distribution of Tetrasphaera was investigated in five full-

scale, well-functioning EBPR plants with nitrogen removal

(nitrification and denitrification). According to the micro-

autoradiography–FISH results, most of the probe-defined

Tetrasphaera were also PAOs. Only species from clade 3

targeted by probe Tet3-19 were either not active or not

PAOs. The probe-defined Tetrasphaera-PAOs were present

in high abundance (18–30% of all Bacteria targeted by

EUBmix) in all treatment plants investigated (Table 2). This

high abundance was supported by Gram staining of the

samples, showing that 20–30% of the biomasses were indeed

Gram positive. This was much higher than the abundance of

Accumulibacter (3.1–5.4%; Table 2). The abundance of each

probe-defined population varied in the different treatment

plants. Approximately half of the probe-defined populations

[targeted by the specific probes Tet1-266 (clade 1), Tet2-892,

and Tet2-174 (both clade 2), Tet3-19 and the broader Tet3-

654 (clade 3)] were present in all WWTP investigated with

relatively high abundances (4 5%). The others were domi-

nant only in one or a few plants (e.g. Tet1-823-PAOs in

Hjørring WWTP). When probes Actino-221 and Actino-

658 were applied, only approximately 50% of the Tetra-

sphaera were detected because these probes do not target

clade 1 and a large part of clade 2. The new probes cover

most Tetrasphaera in the five plants investigated, and nearly

all cells positive with broad actinobacterial probes (HGC69a

and HGC236) were targeted by one of the new probes. On

the other hand, the broad actinobacterial probes did not

Fig. 2. FISH and microautoradiography images

of Tetrasphaera-PAO in activated sludge. FISH

images (a, b) show bacteria hybridizing with the

bacterial probes EUBmix (green) and probe Tet3-

654 (a, red) or probe Tet2-892 (b, red). Yellow

microcolonies and cells are overlay of red and

green. Arrow indicates new thin filaments tar-

geted by probe Tet2-892 (b). (c) FISH image

showing the branched rods (circles) and filament

(square) hybridizing with probe Tet2-174. (d)

Bright-field microautoradiography image show-

ing dark silver grains on top of the branched rods

indicating that they took up 33P aerobically after

an anaerobic preincubation with casamino acids,

but the filaments did not. FISH image (e) and

microautoradiography image (f) show the bac-

teria targeted by probe Tet3-654 (yellow) taking

up labeled glucose anaerobically. The bacteria

indicated with circles (e) have positive microau-

toradiography signals (f). Scale bar (10 mm) for all

images.

FEMS Microbiol Ecol ]] (2011) 1–12 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

7Tetrasphaera-related bacteria in activated sludge systems

target all Tetrasphaera (e.g. the branched rods and the thin

filaments) targeted by the new probes and thus under-

estimated the total abundance of Tetrasphaera (and Actino-

bacteria). Interestingly, the thin filaments and the branched

rods were also not targeted by the EUBmix probes.

Discussion

No further studies of Tetrasphaera-related bacteria have

been made since they were first shown to have PAO behavior

and found in high numbers in full-scale EBPR systems

(Kong et al., 2005, 2008). In this study, we have conducted

a more detailed investigation and demonstrated a high

phylogenetic and physiological diversity. Furthermore, their

high abundance indicates that they play an important role in

full-scale EBPR plants, are perhaps more important than

Accumulibacter, and have a different metabolism.

Phylogeny and identification of Tetrasphaera byoligonucleotide probes

The phylogenetic analysis of clone sequences obtained from

full-scale EBPR plants showed the presence of three phylo-

genetically distinct clades in the genus Tetrasphaera in the

family Intrasporangiaceae. Two of these clades are repre-

sented by described Tetrasphaera isolates, although only

relatively distantly for most (96–98% identity), and they

are all showing a significantly higher diversity of Tetra-

sphaera-related bacteria in full-scale EBPR plants than

described previously (Kong et al., 2005).

Clade 1 (T. elongata-related sequences) was covered by

probes Elo1-1250, Tet1-823, and Tet1-266. Bacteria targeted

by probe Tet1-266 were most abundant. Interestingly, the

probe was designed to target only clone ASM31 (with two

mismatches to any other sequence), but it still showed two

phenotypes (branched rods and cocci in tetrads). Both

phenotypes were PAOs based on uptake of 33P so the probe

may target ecotypes with different morphologies or there is

still an undescribed diversity. This sequence is not targeted

by probe Actino-221 or Actino-658 designed by Kong et al.

(2005), and so these bacteria have not been detected

previously.

Clade 2 contains four isolated species: T. jenkinsii, T.

australiensis, T. veronensis, and the filamentous ‘Candidatus

Nostocoida limicola’. Most abundant were bacteria targeted

Table 3. Uptake of P by different probe-defined Tetrasphaera under aerobic conditions as determined by microautoradiography

Uptake of Pi with following organic substrates used in anaerobic preincubation:

Probe defined organisms Morphology Wastewater Acetate Glucose Casamino acids Glutamic acid No addition

Tet1-266 Cluster of tetrads, small cocci,

branched rods, short rods in clumps

1 � 1 1 � �

Tet2-892 Cluster of tetrads, 1 1 1 1 1 �Short rods in clumps 1 1 1 1 1 �Branched rods 1 1 1 1 1 �Thin filaments 1 � � � � �

Tet2-174 Branched rods 1 1 1 1 1 �Small cocci 1 1 1 1 1 �Cluster of tetrads 1 1 � � 1 �Filaments � � � � � �

Tet3-654 Cluster of tetrads, branched rods,

small cocci, short rods in clumps

1 1� 1 1 1 �

Tet3-19 Cluster of tetrads � � � � � �

Effect of different organic substrates during anaerobic preincubation on uptake of 33P under subsequent aerobic conditions.

1, 4 90% of probe-defined cells were microautoradiography positive; 1�, 50–90% of probe-defined cells were microautoradiography positive;

� , o 1% of probe-defined cells were microautoradiography positive.

Table 4. Uptake of organic substrates by probe-defined Tetrasphaera

under anaerobic (Anae) and aerobic (Ae) conditions in Hjørring and Ejby

Mølle WWTPs as investigated by microautoradiography

Tet1-266 Tet2-892 Tet2-174 Tet3-654 Tet3-19

Anae Ae Anae Ae Anae Ae Anae Ae Anae Ae

Acetate

Hjørring � � 1 1 1 1 1� 1� � �Ejby Mølle � � 1 1 1 1 1� 1� � �

Glucose

Hjørring 1 1 1 1 1 1 1 1 1 1

Ejby Mølle 1 1 1 1 1 1 1 1 1 1

Casamino acids

Hjørring 1 1 1 1 1 1 1 1 � �Ejby Mølle 1 1 1 1 1 1 1 1 � �

Glutamic acid

Hjørring � � 1 1 1 1 1 1 1 1

Ejby Mølle � � 1 1 1 1 1 1 1 1

1, 4 90% of probe-defined cells were microautoradiography positive;

1�, 50–90% of probe-defined cells were microautoradiography posi-

tive; � , o 1% of probe-defined cells were microautoradiography

positive.

FEMS Microbiol Ecol ]] (2011) 1–12c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

8 H.T.T. Nguyen et al.

by Tet2-892 (targeting only one clone) and the probe

targeting the isolates (Tet2-174). Probe Actino-221 covers

largely the same as Tet2-87 and Tet2-892, but Tet2-174 does

not, and so these have been overlooked in previous studies.

Tet2-174 hybridized with several bacteria exhibiting mor-

photypes also described by the four isolates in clade 2.

Clade 3 contains only sequences from uncultured clones,

and probes Tet3-654 and Tet3-19 were designed to cover all

sequences as it was not possible to design a single probe

targeting all. Probe Tet3-19 (targeting clone ASM57) hybri-

dized only with cocci in tetrads and these are likely not PAOs

(Table 3). Probe Tet3-654 targets almost the same sequences

as Actino-658 but Tet3-654 covered more morphotypes than

Actino-658 and a higher number of Tetrasphaera. When the

two probes were used simultaneously with different fluor-

ochromes, only Tet3-654 targeted certain morphotypes,

such as the branched rods. These results show that Tet3-654

is more comprehensive and should be used to target Tetra-

sphaera-PAOs in clade 3.

At least six different morphotypes were found in the

Tetrasphaera clades 1–3 (short rods, branched rods, small

cocci, cocci in tetrads, filaments, and thin filaments). It

shows that the diversity is much greater than previously

known. The morphology of the rod-shaped cells seems very

similar to that of T. elongata, strains ASP12 (Onda & Takii,

2002) and LP12 (Hanada et al., 2002). The typical clusters of

tetrads are very similar to T. australiensis and T. japonica

(Maszenan et al., 2000). The filamentous morphology of

Tet2-174-targeted bacteria was similar in both size and

morphology to ‘Candidatus Nostocoida limicola’ targeted

by probe NLIMII175 (Liu & Seviour, 2001). However, the

thin filaments, branched rods, and small cocci have not been

described previously for Tetrasphaera. Interestingly, the thin

filaments observed in clade 2 (putative PAOs based on

carbon and 33P-uptake) did not hybridize with the EUBmix

or the broad actinobacterial probes (HGC69a and

HGC236), and so they have so far been overlooked. They

are usually hidden within the activated sludge floc, where

they can be quite abundant.

In order to detect the most important Tetrasphaera-PAOs

in full-scale EBPR plants, we propose to apply probe Tet1-

266 for clade 1, Tet2-892 and Tet2-174 for clade 2, and Tet3-

654 for clade 3.

Ecophysiology of Tetrasphaera

Most probe-defined Tetrasphaera were shown to be putative

PAOs as they contained polyphosphate and could actively

take up labeled P under aerobic conditions, provided they

had an organic substrate they could take up in the previous

anaerobic phase. Only bacteria targeted by probe Tet3-19 in

clade 3 and most filamentous morphotypes did not demon-

strate a PAO phenotype.

So far, most probe-defined Tetrasphaera in full-scale

EBPR plants can take up different amino acids and glucose

(Kong et al., 2005, 2008) under both anaerobic and aerobic

conditions, and this was confirmed in this study. This is very

similar to most isolated T. elongata strains in clade 1.

However, the ecophysiology of Tetrasphaera from clades 2

and 3 (probe Tet2-892, Tet2-174, and Tet3-654) seems more

diverse and more similar to that of T. australiensis and T.

japonica (Maszenan et al., 2000): all grow on complex media

and utilize all four substrates tested in this study (glucose,

acetate, casamino acids, and glutamic acid). The pattern of

acetate uptake of Tet3-654-defined PAOs was rather com-

plex. The microautoradiography-positive fraction varied

from plant to plant and even from year to year in the same

plant. Therefore, we have tested several old fixed microau-

toradiography samples from earlier studies with the same

varying results (data not shown). The varying uptake of

substrates may be explained by the broad coverage of probe

Tet3-654 allowing significant undetected temporal changes

of subpopulations within the targeted populations. This

complexity of Tet3-654-PAOs in acetate uptake can also

explain why bacteria targeted by probe Actino-658 (which

does not cover all Tet3-654-PAOs) were not observed to take

up short-chain fatty acids.

Table 5. Uptake of labeled acetate or labeled glucose by probe-defined Tetrasphaera and Accumulibacter under anaerobic conditions after prolonged

anaerobic pre-incubation (3, 5 and 9 h) with unlabeled acetate

Probe-defined

organisms

No preincubation

uptake of acetate

3, 5 or 9 h preincubation

uptake of acetate

9 h preincubation

uptake of glucose

Tetrasphaera

Tet1-266 � � 1

Tet2-892 1 � 1

Tet2-174 1 � 1

Tet3-654 1� � 1

Accumulibacter

PAOmix 1 � �

1, 4 90% of probe-defined cells were microautoradiography positive; 1�, 50–90% of probe-defined cells were microautoradiography positive;

� , o 1% of probe-defined cells were microautoradiography positive.

FEMS Microbiol Ecol ]] (2011) 1–12 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

9Tetrasphaera-related bacteria in activated sludge systems

Also, as observed in the present study, all Tetrasphaera

were able to consume glucose anaerobically, but they did not

all take up P in the following aerobic period. In clade 2, the

filamentous morphotype (very similar to ‘Candidatus Nos-

tocoida limicola’) hybridizing with probe Tet2-174 did not

actively take up P aerobically and may not be active PAOs

despite the presence of intracellular P.

Physiology of Tetrasphaera-PAOs compared withEBPR biochemical models

The physiology of Tetrasphaera-PAOs is more complex and

in several aspects very different from Accumulibacter and the

general biochemical models used to explain the behavior of

PAOs (Garcia Martin et al., 2006; Oehmen et al., 2007; Zhou

et al., 2009).

Most surprising is the ability of Tetrasphaera-PAOs to

take up acetate and other substrates in the anaerobic period,

but their lack of ability to form polyhydroxyalkanoates as a

storage compound. Staining with Nile Blue or Sudan Black

did not detect polyhydroxyalkanoates, and so the presence

and identity of any intracellular compound is still unknown.

Interestingly, the anaerobic uptake capacity of acetate was

exhausted after 3 h, very similar to that of Accumulibacter

(Kong et al., 2005), strongly indicating the presence of an

unknown storage compound. Glucose, on the other hand,

was taken up after 9 h preincubation with acetate and

presumably fermented. Tetrasphaera can take up glucose

even after several days under anaerobic conditions in the

presence of glucose (Kong et al., 2008). In any case, the

anaerobic uptake of substrate, either as acetate, amino acids,

or glucose, was decisive for the subsequent uptake of P in the

aerobic phase. However, for the thin filamentous bacteria

hybridizing with Tet2-892, only wastewater could provide

the right, so far unknown, organic substance during the

anaerobic period.

Role of Tetrasphaera-PAO in full-scaleEBPR systems

Four probe-defined groups in the three clades of Tetra-

sphaera-PAO constituted 18–30% of the total bacterial

biomass present in five well-working EBPR plants with

nitrogen removal. A high number of Tetrasphaera has also

been reported in German EBPR plants (Eschenhagen et al.,

2003), and many Gram-positive bacteria (up to 35% of the

biomass) have been reported in Australian EBPR plants

(Beer et al., 2006), although their identities have not been

investigated in detail. Their abundance is often substantially

greater than that of Accumulibacter, indicating that Tetra-

sphaera-PAO may play an important role in full-scale EBPR

plants.

Tetrasphaera-PAOs seem to occupy a slightly different

ecological niche than Accumulibacter. They are more diverse

in substrate uptake, can take up glucose (but not mannose

or galactose, Kong et al., 2008), and probably also ferment

under anaerobic conditions. Whether they can grow under

anaerobic conditions is, however, uncertain (Kong et al.,

2008). As they excrete extracellular amylases (Xia et al.,

2008), they appear to be specialized degraders and consu-

mers of starch and other polysaccharides in the wastewater.

A certain functional redundancy among the bacteria in

WWTPs is seen for most important processes, for example

among denitrifiers (Thomsen et al., 2007; Hesselsoe et al.,

2009), ensuring high resilience and a stable function. The

same seems to be the case for PAOs with several ecotypes of

Accumulibacter (He et al., 2007), and, as shown in this study,

considerable diversity of Tetrasphaera. The most important

factors determining the presence of either Tetrasphaera-

PAOs or Accumulibacter can be the availability of glucose

and amino acids as well as the anaerobic residence time in

the anaerobic reactor, where the ability to ferment will be

beneficial the longer the residence time.

Acknowledgements

This study was funded by Viborg Energy, Danish Council for

Independent Research, and Aalborg University. We thank

Prof. Robert Seviour for providing Tetrasphaera cultures

and Marianne Stevenson and Artur Tomasz Mielczarek for

technical help.

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