Identity, abundanceand ecophysiologyof¢lamentousChloro£exispecies present inactivated sludge treatment plantsCaroline Kragelund1, Caterina Levantesi2, Arjan Borger3, Karin Thelen4, Dick Eikelboom5,Valter Tandoi2, Yunhong Kong1, Jaap van der Waarde6, Janneke Krooneman7, Simona Rossetti2,Trine Rolighed Thomsen1 & Per Halkjær Nielsen1
1Section of Environmental Engineering, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Aalborg,
Denmark; 2CNR, Water Res Institute, Rome, Italy; 3TNO MEP; Department of Environmental Engineering; Laan van Apeldoorn, The Netherlands;4VERMICON AG, Munich, Germany; 5ASIS vof, Zutphen, The Netherlands; 6SNV Kameroen, Koeriersdienst Ministerie van Buitenlandse Zaken,
Den Haag, The Netherlands; and 7BIOCLEAR Environmental Biotechnology, Groningen, The Netherlands
Correspondence: Per Halkjær Nielsen,
Section of Environmental Engineering,
Department of Biotechnology, Chemistry and
Environmental Engineering, Aalborg
University, Sohngaardsholmsvej 57, DK-9000
Aalborg, Denmark. Tel.:145 96358503;
fax145 96350558; e-mail: [email protected]
Received 6 March 2006; revised 6 October
2006; accepted 15 October 2006.
First published online 22 December 2006.
DOI:10.1111/j.1574-6941.2006.00251.x
Editor: Michael Wagner
Keywords
Chloroflexi ; filamentous bacteria; activated
sludge; ecophysiology.
Abstract
Filamentous Chloroflexi species are often present in activated sludge wastewater
treatment plants in relatively low numbers, although bulking incidences caused by
Chloroflexi filaments have been observed. A new species-specific gene probe for
FISH was designed and using phylum-, subdivision-, morphotype 1851- and
species-specific gene probes, the abundance of Chloroflexi filaments were mon-
itored in samples from 126 industrial wastewater treatment plants from five
European countries. Chloroflexi filaments were present in 50% of the samples,
although in low quantities. In most treatment plants the filaments could only be
identified with phylum or subdivision probes, indicating the presence of great
undescribed biodiversity. The ecophysiology of various Chloroflexi filaments was
investigated by a suite of in situ methods. The experiments revealed that Chloroflexi
constituted a specialized group of filamentous bacteria only active under aerobic
conditions consuming primarily carbohydrates. Many exo-enzymes were excreted,
e.g. chitinase, glucuronidase and galactosidase, suggesting growth on complex
polysaccharides. The surface of Chloroflexi filaments appeared to be hydrophilic
compared to other filaments present. These results are generally supported by
physiological studies of two new isolates. Based on the results obtained in this
study, the potential role of filamentous Chloroflexi species in activated sludge is
discussed.
Introduction
Members of the phylum Chloroflexi, formerly known as the
green nonsulphur bacteria, have primarily been associated
with extreme habitats, e.g. microbial mats in hot springs
(Boomer et al., 2002; Nubel et al., 2002) and hypersaline
environments (Nubel et al., 2001), where they are known as
(filamentous) anoxygenic photothophs (Hanada et al., 2002;
Hanada & Pierson, 2002; Nubel et al., 2002). Filamentous
members of the phylum Chloroflexi have also been found in
activated sludge wastewater treatment plants (WTP), and they
have occasionally been associated with bulking incidences
(Beer et al., 2002; Bjornsson et al., 2002; Schade et al., 2002).
A common filamentous microorganism, Eikelboom’s
morphotype 1851, has been micromanipulated, cultured,
and sequenced, which has revealed that this morphotype
belongs to the phylum Chloroflexi (Beer et al., 2002). A
species-specific gene probe for FISH demonstrated its pre-
sence in activated sludge systems. Bjornsson et al. (2002)
developed a phylum-specific as well as two subdivision-
specific gene probes for Chloroflexi based on five clones
originating from a sequencing batch reactor as well as
publicly available Chloroflexi sequences. Phylogenetic analy-
sis of the sequences revealed four subdivisions in total, but
target sites suitable for probes were only found for two
(div. 1 and 3). In subdivision 1, mainly clones from environ-
mental sources were located, e.g. from polluted habitats;
isolates obtained from activated sludge were also found here
(Juretschko et al., 2002; Yamada et al., 2005). In subdivision
3, most characterized isolates clustered, e.g. morphotype
1851 (Beer et al., 2002), Chloroflexi ssp., Oscillochloris ssp.,
Roseiflexus carstenholzii and Herpetosiphon ssp. The closest
FEMS Microbiol Ecol 59 (2007) 671–682 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
relative to morphotype 1851 was Roseiflexus carstenholzii
[however, it only shares 84% 16S rRNA gene sequence
similarity; Beer et al. (2002)]. Many Chloroflexi members,
at least those found in activated sludge, resemble morpho-
type 1851 as defined by Eikelboom & van Buijsen (1983) and
Jenkins et al. (1993). This morphotype is characterized as
having a cell diameter of 0.5–0.8 mm, length of filaments
4 200 mm, with rectangular cell shape and weak Gram-
positive and Neisser-negative stain. They possess an atypical
Gram-negative cell wall (Beer et al., 2002), and staining
results seem to some extent to depend on the dye used in
Gram solution A (carbol gentian violet vs. crystal violet)
(D.H. Eikelboom, personal communication). They are fre-
quently observed with epiphytic bacteria, especially in
municipal WTP, and are often found in bundles.
Little is known about the physiology of filamentous
Chloroflexi in activated sludge. Only a few pure culture
studies have been conducted; one on the morphotype 1851
identified as Chloroflexi (Kohno et al., 2002) and another on
filamentous Chloroflexi isolated from mesophilic and ther-
mophilic methanogenic sludge granules (Yamada et al.,
2005). In the first study, five filamentous orange-pigmented
strains were isolated, characterized as morphotype 1851,
and named Kouleothix aurantiaca. Minor discrepancies were
seen between pure culture observations and the character-
ization manuals based on in situ observations, relating to
Gram staining, gliding motility on solid media and filament
length. The isolates grew mainly on sugars (e.g. glucose,
mannose, trehalose, and xylose) and pyruvate. Two of the
five strains were able to reduce nitrate to nitrite, and all
strains were able to ferment glucose (Kohno et al., 2002).
Three strains were also isolated from thermophilic and
mesophilic methanogenic sludge granules, and pure culture
investigations showed that they were strict anaerobes and
were specialized on carbohydrates (glucose, fructose and
sucrose). However, no growth was observed if yeast extract
was not added (Yamada et al., 2005). Recently, filamentous
Chloroflexi were identified in a nitrifying biofilm by applying
the phylum-specific gene probes (CFX1223 and GNSB941)
(Kindaichi et al., 2004). Studies of the ecophysiology using
FISH-micro-autoradiography (MAR) showed that they took
up N-acetylglucosamine and a mixture of amino acids, but
they were never observed to take up acetate.
Recent studies on the ecophysiology of filamentous
microorganisms in activated sludge wastewater treatment
plants (WWTP) suggest that it is impossible to make general
statements regarding their physiology (Wagner et al., 2002).
Properties such as substrate uptake capability and rates,
substrate affinity, storage abilities, surface properties, and
exo-enzyme activity depend on the species examined. Stu-
dies on Microthrix (Nielsen et al., 2002), Thiothrix (Nielsen
et al., 2000), filamentous Alphaproteobacteria (Kragelund
et al., 2006), Aquaspirillum-related filaments (Thomsen
et al., 2006), Skermania (Eales et al., 2005) and TM7-related
filaments (Thomsen et al., 2002) revealed that, generally,
two types of physiological strategies are exhibited by these
filamentous bacteria. Some filamentous bacteria are versatile
in substrate utilization, appearing as general consumers of
organic matter and exemplified by the filamentous Alpha-
proteobacteria (Kragelund et al., 2006). Others are very
restricted and thus consumers of only few specific organic
compounds such as lipids by Microthix (Andreasen &
Nielsen, 2000; Nielsen et al., 2002). Some filamentous
species are also able to take up substrates with electron
acceptors other than oxygen and have a large storage
capacity. Based on this, it appears that many filamentous
bacteria possess an unusual physiology and ecology. If
detailed knowledge about the ecophysiology of specific
filamentous bacteria were combined with the characteriza-
tion of WWTP process conditions, better and more efficient
WWTP control strategies could be developed to prevent
sludge bulking.
In this study, the identity, abundance, and ecophysiology
of Chloroflexi species in industrial and municipal WWTP
were investigated. For this purpose, a new species-specific
gene probe was designed and the ecophysiology of filamen-
tous Chloroflexi in industrial and municipal WWTPs was
investigated by applying several in situ methods in combina-
tion with FISH. Also included are physiological character-
istics of two new Chloroflexi isolates, which appear to be
closely related to K. aurantiaca.
Materials and methods
Activated sludge
Activated sludge samples used for the survey were fixed
directly at the WWTP in 50% ethanol or 3.6% formaldehyde
to preserve both Gram-positive and Gram-negative bacteria.
In total, 126 samples from different industrial WWTPs and
five samples from municipal WWTPs were used to monitor
the presence and abundance of filamentous Chloroflexi as
well as other filamentous species. Samples from many
different industrial plants were included [agro industry (6),
brewery (2), chemical (27), dairy (10), fish (3), food (11),
potato (12), pulp and paper (22), textile (5), tannery (4),
other (13), domestic (4), and unknown (6)]. Samples were
collected in Denmark, Italy, Poland, Germany and the
Netherlands. Of the 126 different industrial plants, 68 had
nitrification and 58 also had denitrification.
Ecophysiology experiments were carried out with acti-
vated sludge from industrial and municipal plants from the
Netherlands (TNO17 and TNO25), Italy (CNR1) and Den-
mark (Ega and Skagen). Plant descriptions are presented in
Table 1 for all plants except TNO25, for which data were not
available. All five WWTPs had nitrification, three had
FEMS Microbiol Ecol 59 (2007) 671–682c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
672 C. Kragelund et al.
denitrification and two had enhanced biological phosphorus
removal. The temperature of the process tank varied, and
industrial WTP were operated at a higher temperature than
most municipal WTP. A selector [a small compartmenta-
lized tank where raw influent is mixed with return sludge to
control filaments, see e.g. Martins et al. (2004)] was present
in TNO17.
The activated sludge was collected and sent to Aalborg
(Denmark) by express mail overnight. In situ experiments
were conducted immediately after arrival of the sample.
Undiluted sludge was used for exo-enzyme and surface
property experiments. In all other experiments the sludge
was diluted to 1 gSS L�1 with nitrate or nitrite-free super-
natant from the activated sludge.
Isolation, phylogenetic analysis and probedesign
Thee isolates of filamentous bacteria morphologically iden-
tified as morphotype 1851 (Strain EU25, Ver9Iso1 and
Ver9Iso2) were obtained by micromanipulation from acti-
vated sludge samples originating from industrial WWTP
treating pulp and paper waste and brewery waste. EU 25 was
isolated on a simple medium (MSV1acetate, MSV1A)
composed of MSV mineral base (Williams & Unz, 1989)
acetate (0.5 g CH3COO�L�1) as sole carbon source and
Eikelboom vitamin solution (1% v/v Eikelboom, 1975).
The rich R2A medium (Reasoner & Geldreich, 1985) was
used to isolate Ver9Iso1 and Ver9Iso2.
PCR, purification of products and sequencing were
performed as described in detail elsewhere (Levantesi et al.,
2004). The sequences were edited using Sequencer DNA
sequencing software (Gene Codes Inc., Ann Arbor, MI).
Checks for chimeric sequences were conducted using the
CHECK_CHIMERA program from Ribosomal Database Project
(http://rdp.cme.msu.edu) and the program BELLEROPHON
(Hugenholtz & Huber, 2003). 16S rRNA gene sequences
were compiled and aligned using the automatic nucleic acid
aligner in the ARB software package (Ludwig et al., 2004), and
alignments were refined manually. Aligned sequences were
used for calculation of trees by neighbour-joining, distance
matrix, parsimony, and maximum likelihood approaches
using default settings in the ARB software. Oligonucleotide
probes were designed using the probe design/match tools in
the ARB software package. To evaluate the formamide con-
centration for optimum stringency, the designed probe was
analyzed on ethanol-fixed EU25 culture applying hybridiza-
tion buffer containing 0–60% formamide (5% increments).
Identification and abundance
The filamentous bacteria present in industrial WWTPs were
morphologically identified using the Eikelboom classifica-
tion system and the filament index (FI) (Eikelboom, 2002).
FI determines the population size of filamentous bacteria
and ranges from 0 (no filaments) to 5 (very many).
Furthermore, FISH was applied with 16S rRNA gene-
targeted oligonucleotide probes targeting all Bacteria, the
Chloroflexi phylum, subdivisions and specific species within
Chloroflexi (Table 2). Further details on oligonucleotide
probes are available at probeBase (Loy et al., 2003). Oligo-
nucleotides were labelled with 5(6)-carboxyfluorescein-N-
hydroxysuccinimide ester (FLUOS) or with the sulphoindo-
cyanine dyes (Cy3 and Cy5) (Thermohybaid Interactive,
Ulm, Germany). A confocal laser scanning microscope,
CLSM (LSM 510, Carl Zeiss, Oberkochen, Germany)
equipped with a UV laser (351 and 364 nm), an Ar ion laser
(458 and 488 nm), and two HeNe lasers (543 and 633 nm)
were used to record fluorescent signals from the gene
probes.
The abundance of Chloroflexi species was determined in
126 industrial and five municipal WTP with different
process designs. The above-mentioned gene probes were
applied to estimate the filament abundance of the Chloro-
flexi phylum (probe CFX1223), subdivision 1 (probe
CFX784) and 3 (probe CFX109) of Chloroflexi, morphotype
1851 (probe Chl1851) as well as the K. aurantiaca-related
bacteria targeted by probe EU25-1238. Hybridizations
with any of the above-mentioned Chloroflexi probes for
the screening were done in combination with CFXMIX
(CFX12231GNSB941).
Table 1. Overview of wastewater treatment plants used for ecophysiological studies
Type of industry
AAE CNR1 Skagen Ega TNO17
Municipal Tannery Fish industry Municipal Paper
Nitrification 1 1 1 1 1
Denitrification 1 1 1 1 �Phosphorus removal (Biological/Chemical) B1Ch Ch B1Ch B1Ch Ch
Temperature of process tank ( 1C) 8–18 18–25 8–18 8–18 20
Sludge age (days) 25 15 25 25 30
Selector present� � � � � 1
�A small compartmentalized tank with a short residence time where raw influent is mixed with return sludge.
FEMS Microbiol Ecol 59 (2007) 671–682 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
673Ecophysiology of filamentous Chloroflexi species
In most cases, combinations of MAR-FISH, ELF-FISH,
and MAC-FISH (see below) were used for the studies of
ecophysiology, and hybridization was always done with
species-specific gene probes combined with the EUBMIX
(EUB3381EUB338-II1EUB338-III) targeting all Bacteria.
All the above-mentioned methods were examined by CLSM
before hybridization with gene probes, except MAR-FISH.
For MAR-FISH, hybridization with gene probes was per-
formed before applying the photographic emulsion. Posi-
tions of interest were recorded by an automatic stage
controller, and digital images of filaments were recorded.
Fresh samples used for ELF and MAC were fixed in 4%
paraformaldehyde for 1 h before FISH was conducted as
described by Amann (1995) and the stage control enabled
relocation of the microscopic field. Slight modification of
MAC-FISH and ELF-FISH were necessary, for details see
Kragelund et al. (2005).
Physiological characterization of isolates EU25and Ver9Iso2
The aerobic growth of EU25 and Ver9Iso2 was analyzed on a
range of carbon and nitrogen sources at 20 1C. Ver9Iso1 was
not further characterized. Tests were performed in triplicate,
and positive growth was determined against a negative
control without any carbon or nitrogen source. The inocu-
lum for these experiments was grown in liquid MSV1A
medium, and cultures were inoculated at 1% (v/v). MSV
mineral base with Eikelboom vitamin solution 0.1% (v/v)
was used for substrate utilization tests; carbon source
concentration was 0.5 g L�1 except alcohols and Tween 80
added at 1% (v/v). Acetate consumption during EU25
growth was ascertained by gas chromatography (Perkin
Elmer 8500, stationary phase Carbopack B-DA 80-120 4%
CW 20 M) measuring substrate concentration before inocu-
lum and after 10 days when growth was clearly visible.
Growth of isolates on different nitrogen sources (ammo-
nium, nitrate, nitrite, urea) was assessed by adding these
compounds (at 0.011% w/v of nitrogen) to basal medium
devoid of other nitrogen-containing compounds. For these
tests, acetate was used as substrate (0.5 g L�1), and Eikel-
boom vitamin solution 0.1% (v/v) was added to the media.
Autotrophic growth was tested in liquid MSV mineral
base with Eikelboom vitamin solution 0.1% (v/v) without
any added organic carbon source and with the addition
of NaHCO3 solution (0.42 g L�1) and NaS2O3 � 5H2O
(0.4 g L�1). Denitrification tests were carried out in tubes
containing liquid medium. R2A liquid medium was used for
Ver9Iso2 and MSV1A medium for EU25 with the addition
of 0.1% KNO3. The vials contained a Durham tube that
allowed visualization of any gas produced during incuba-
tion. Nitrite production was determined colorimetrically.
The ability to grow under anaerobic conditions was analyzed
using several different experimental strategies to obtain
anaerobiosis; anaerobiosis in a serum bottle with N2,
anaerobiosis in oxoid anaerobic bags (with oxygen indica-
tor), and anaerobiosis in an anaerobic chamber. The tem-
perature growth range of strains EU25 and Ver9Iso2 was
determined in liquid R2A medium incubated at 10, 15, 20,
25, 35 and 40 1C.
Nucleotide accession numbers
Partial length 16S rRNA gene sequences (1127 bp) of EU25,
Ver9Iso1 and Ver9Iso2 (1330 bp and 1252 bp, respectively)
were deposited in Genbank under the accession numbers
DQ232757, DQ812549 and DQ812550, respectively.
MAR and MAR-FISH
The micro-autoradiography experiments were performed
using 3H-labelled and 14C-labelled organic compounds and14C-labelled bicarbonate. Details of the procedure, which
includes incubation, fixation, and hybridization with gene
probes, addition of a radiosensitive emulsion, exposure,
processing, and microscopical investigations, are given else-
where (Andreasen & Nielsen, 1997; Lee et al., 1999; Nielsen
Table 2. Overview of specificity, sequences and hybridization conditions of oligonucleotide probes used
Probe name Specificity Probe sequence (50–30) % FA� Reference
EUB338 Most Bacteria GCTGCCTCCCGTAGGAGT 0–60 Amann et al. (1990)
EUB338-II Planctomycetales GCAGCCACCCGTAGGTGT 0–35 Daims et al. (1999)
EUB338-III Verrucomicrobiales GCTGCCACC CGTAGGTGT 0–60 Daims et al. (1999)
CFXMIX(CFX12231GNSB941) Phylum Chloroflexi CCATTGTAGCGTGTGTGTMG1
AAACCACACGCTCCGCT
35 Bjornsson et al. (2002),
Gich et al. (2001)
CFX109 Chloroflexi subdivision 3 CACGTGTTCCTCAGCCGT 30 Bjornsson et al. (2002)
CFX784 Chloroflexi subdivision 1a1b ACCGGGGTCTCTAATCCC 35 Bjornsson et al. (2002)
Chl1851 Filamentous bacterium
Eikelboom morphotype 1851
AATTCCACGAACCTCTGCCA 35 Beer et al. (2002)
EU25-1238 Kouleothix aurantiaca Isolate EU-25 CTGCGCATTGCCACCGACAT 35 This study
�Formamide concentration in hybridization buffer (v/v).
FEMS Microbiol Ecol 59 (2007) 671–682c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
674 C. Kragelund et al.
et al., 2000). In brief, two series of studies were conducted.
In the first series, various potential substrates were tested for
uptake under aerobic conditions to see whether gene probe-
defined representatives of Chloroflexi (probe-positive phy-
lum filaments (CFX1223), probe-positive subdivision 3
filaments (CFX109), probe-positive Type 1851 filaments
(Chl1851) or probe-positive K. aurantiaca-like filaments
(EU25-1238) were specialized or general consumers of
organic substrates. For this, a selection of substrates was
chosen representing short and long chain fatty acids, sugars,
alcohols, and amino acids. In the second series, potential use
of electron acceptors other than O2 was tested by studying
uptake of the same positively tested organic substrates with
nitrate or nitrite present as electron acceptor or under
anaerobic conditions (no oxygen, nitrate, or nitrite present).
In all experiments, 2 mL diluted activated sludge (1 gSS L�1)
was transferred to glass serum vials with a final substrate
concentration of 2 mM, and the labelled fraction was 10 mCi
per vial. Incubation time was 3 h (5 h for bicarbonate). In all
anaerobic incubations with nitrate or nitrite as e-acceptor
and strict anaerobic experiments, a preincubation step of 2 h
was included with unlabelled organic substrate (2 mM, oleic
acid 0.5 mM). In this way, only bacteria able to take up large
amounts of the substrate under these conditions (for storage
or growth) would be MAR-positive (Andreasen & Nielsen,
2000). After the preincubation period, labelled (10mCi per
vial) and unlabelled substrate was added to a final substrate
concentration of c. 2 mM (oleic acid 0.5 mM). The samples
were incubated for 3 h. All vials for anaerobic incubation
with either nitrate or nitrite as e-acceptor and strict anaero-
bic incubations were closed with a gas-tight rubber stopper
and flushed with ultrapure nitrogen gas before incubation.
When thiosulphate or nitrate was added, a final concentra-
tion of 2 mM was used (nitrite only 0.5 mM). A minimum of
30 filaments of each gene probe-defined organism were
investigated in each incubation to determine potential
uptake. In most experiments, MAR-positive and MAR-
negative filaments were assessed by comparing silver grains
on top of filaments to the background level. As a control for
chemography, sludge was pasteurized at 70 1C for 10 min
just before incubation under defined conditions. Light
microscopy was used to detect silver grains from MAR.
[3H]N-acetylglucosamine was purchased from American
Radiolabelled Chemicals Inc. (Bio Nuclear AB, Sweden).
Details of other radiochemicals used in this study can be
found elsewhere (Kragelund et al., 2005).
The isolate EU25 was grown in R2A medium. It was
washed thee times in liquid MSV media before MAR
incubation to remove residual carbon substrate. MAR
experiments were performed as described above. Additional
experiments were designed to test aerobic the photoauto-
trophic or photoheterotrophic ability of EU25. For this
purpose, labelled bicarbonate was used in combination with
unlabelled substrates (acetate and thiosulphate), and incu-
bations were carried out in both light and darkness. An
anaerobic experiment was carried out to determine the
potential uptake of labelled bicarbonate together with
acetate and thiosulphate in the presence of light.
Enzyme-labelled fluorescence (ELF), ELF-FISH
The presence of exo-enzyme activity was determined using
enzyme-labelled fluorescence (ELFs-97, Molecular Probes,
Eugene, OR), where substrates upon enzymatic cleavage
form a fluorescent precipitate (excitation 345 nm/emission
530 nm) on the surfaces of bacteria or microcolonies within
flocs (Van Ommen & Geesey, 1999; Nielsen et al., 2002). The
enzymatic activities of chitinase, esterase, galactosidase,
glucuronidase, lipase, and phosphatase activities were in-
vestigated. An optimized protocol can be found elsewhere
(Kragelund et al., 2005, 2006).
MAC, MAC-FISH
Surface properties were investigated using microsphere
adhesion to cells (MAC) where sulphate-modified micro-
spheres with hydrophobic characteristics and a diameter of
0.02 mm were applied (Molecular Probes). Details of micro-
spheres and protocol have previously been described (Niel-
sen et al., 2001; Kragelund et al., 2005).
Results
Phylogenetic analysis and gene probe design
Isolate EU25 and Ver9Iso2 shared 98% sequence similarity.
EU25 clustered together with the Eikelboom morphotype
1851 sequence obtained by Beer et al. (2002) in subdivision
3, along with most of the other isolates of Chloroflexi sp. The
isolate EU25 was closely related (99.1–99.9% 16S rRNA gene
sequence similarity) to the published sequences for K.
aurantiaca (GenBank AB079637-41). The sequence similar-
ity of isolate EU 25 and Eikelboom Type 1851 (Beer et al.,
2002) was 94.7%. Ver9Iso 1 and Ver9Iso2 shared 99%
sequence similarity. The K. aurantiaca sequences and Ver9-
Iso2 shared 98%. Ver9Iso2 and Eikelboom Type 1851 (Beer
et al., 2002) shared c. 93% sequence similarity.
The phylogenetic tree based on all publicly available 16S
rRNA gene sequences of primarily activated sludge clones
including isolate EU25 and Ver9Iso1 and 2 belonging to the
Chloroflexi phylum is shown in Fig. 1. Subdivisions 1–4
defined by Bjornsson et al. (2002) are marked with digits. All
sequences included are targeted by the phylum-specific
probes (CFXMIX), and the subdivision probes are also
denoted with a digit [subdivision 1 (CFX784) and subdivi-
sion 3 (CFX109), respectively]. All sequences within
FEMS Microbiol Ecol 59 (2007) 671–682 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
675Ecophysiology of filamentous Chloroflexi species
subdivision 3 were a perfect match to CFX109. Within this
subdivision, the probe Chl1851 designed for morphotype
1851 in 2002 by Beer et al. also targeted all K. aurantiaca
strains as well as isolates EU25, Ver9Iso1 and Ver9Iso2. One
16S rRNA gene-targeted oligonucleotide probe (EU25-
1238) in this project was designed in 2001 to target EU25
and was applied on all industrial samples collected in this
study. The probe sequence for EU25-1238 was 50-
CTGCGCATTGCCACCGACAT-30, and the optimal forma-
mide concentration was determined as 35%. Isolates EU25,
Ver9Iso1, Ver9iso2 and K. aurantiaca strains were a perfect
match to the probe EU25-1238.
Morphology of Chloroflexi filaments
Morphological characteristics such as filament length,
width, and cell shape were noted for filaments included in
the ecophysiological study, and measurements were carried
out on FISH-labelled filaments rather than on fresh samples.
This was done to locate the filaments otherwise hidden
inside floc material and, thus, cell diameter of the filaments
might be slightly biased due to the fixation procedure and
the FISH protocol. Only minor differences were observed
between the different gene probe-defined Chloroflexi sp.
Variable diameters were observed, ranging from 0.5 to
1.0 mm and variable Gram staining results. Moreover, epi-
phytic bacteria were absent from most of the Chloroflexi
filaments in industrial samples, whereas heavy growth of
epiphytic bacteria was noted in municipal WTP. All repre-
sentatives had a rectangular cell shape, were relatively short
and contained small PHA granules. Most Chloroflexi fila-
ments targeted by any of the above-mentioned probes in the
survey also hybridized with the probe for all Bacteria
(EUBMIX). However, only about half of the CFX109-positive
populations in the WWTP used for ecophysiological studies
showed a positive EUBMIX signal, indicating that the EU-
BMIX is unable to hybridize with all Chloroflexi species.
Abundance
The filament index of the 126 industrial samples screened
exceeded 1.5 for 104 samples and, of these, 92% had FI
greater than 2.5. Industrial WWTP often contained co-
occurring filamentous populations and this was found in
CFX784
0.10
EU25-1238
Chl1851
CFX109
CFX1223 +GNSB-941
33
2244
11
Fig. 1. 16S rRNA gene tree showing the phylogenetic affiliation of strain EU25, isolates Ver9Iso1 and Ver9Iso2 within the Chloroflexi phylum. The tree
was calculated by the neighbour-joining method using ARB default settings. The scale bar corresponds to 10% estimated sequence divergence. Perfect-
match organisms targeted by gene probes are illustrated with grey boxes. Acinetobacter was chosen as out-group (not shown). Numbers correspond to
subdivisions defined by Bjornsson et al. (2002).
FEMS Microbiol Ecol 59 (2007) 671–682c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
676 C. Kragelund et al.
c. 75 of the WWTPs, 35% of which contained Chloroflexi
with a filament index 4 1.5. Chloroflexi were identified in
63 of the WWTPs; of these, 52% contained Chloroflexi
populations larger than FI4 1.5. Only in 16 WWTPs were
Chloroflexi present as FI4 2.5. In general, no industrial
waste particularly favoured the presence of filamentous
Chloroflexi; they were detected in all types of industries as
well as in all the municipal plants. Filamentous Chloroflexi
were found in WWTP with carbon removal, nitrification
and denitrification. In 32 of the Chloroflexi- containing
WWTP samples it was not possible to identify the filament
beyond phylum level (CFX 1223), indicating the presence of
many yet unidentified Chloroflexi species. In seven samples
probe CFX1223 positive filaments were higher than
FI4 1.5. Subdivision 3 affiliated Chloroflexi positive by
probe CFX109 were responsible for the high filament index
(FI4 1.5) in four WWTPs, probe positive EU25-1238
targeted filaments in two WWTPs, probe-defined CFX784
filaments and probe Chl1851 positive filaments each in one
plant. Approximately 50% of the probe EU25-1238 defined
filaments did not hybridize with the probe Chl1851,
although the sequence of EU25 should have a perfect match
to probe Chl1851. This could indicate an undescribed
phylogenetic diversity. The gene probe-defined filaments
were often located in bundles within the floc material,
rendering a morphological identification difficult. They
were rarely found outside flocs, except if filament index
exceeded 2, as was the case in most samples used for
ecophysiological studies.
Different morphologies were targeted by the Chloroflexi
probes in the FISH survey; CFX1223 targeted primarily
typical morphotype 1851 as well as some without epiphytic
bacteria. Also, some filaments were very thin (0.5 mm), and
others with a diameter of 2.0 mm were targeted. A few
showed similarities to a thick morphotype 0041, and others
looked like morphotype Type 021N. CFX 109 targeted
mainly thin filaments without attached growth. The probes
Chl1851 and EU25-1238 both primarily targeted morpho-
type Type 1851, although some differences were seen with
respect to epiphytic bacteria and Gram staining results.
Apart from filamentous bacteria, single cells were targeted
by the phylum-specific probes (CFX1223 and CFXMIX) as
well as the subdivision-specific probes (CFX784 and
CFX109).
Pure culture physiology
Isolates Ver9Iso2 and EU25 grew preferentially on carbohy-
drates (glucose, fructose), organic acid (acetate, pyruvate)
and yeast extracts; no alcohols were utilized (Table 3). Minor
differences were observed between the two isolates, in
particular in propionate usage. However, these differences
might account for the longer incubation time of strain
Ver9Iso2, where it was observed that a larger selection of
substrates could be utilized by increasing the incubation
time from 40 to 90 days. Uptake of acetate was validated for
isolate EU25 by gas chromatography. Here, acetate concen-
tration decreased to 24% of initial concentration after c. 10
days of incubation. None of the isolates was able to grow
chemo-autotrophically. Neither isolate EU25 nor Ver9Iso2
was able to denitrify. The capability to grow anaerobically
could not be verified due to replica failure. However, only
scarce growth was observed whenever positive anaerobic
growth replicas were obtained. The temperature growth
range was identical for both isolates, where 15 1C was the
minimum temperature sustaining growth, and 35 1C the
maximum temperature.
The pure culture EU25 was tested for uptake of several
substrates (Table 4) using MAR, and only glucose and
mannose were taken up; uptake of fatty acids, amino acids
or ethanol was not observed. Minute traces of glucose were
taken up under conditions where nitrate served as
e-acceptor. No aerobic photoautotrophic or photochemo-
trophic behaviour was observed for EU25. Anaerobic in-
cubation with labelled bicarbonate together with acetate and
thiosulphate did not result in a positive MAR signal (data
not shown).
Table 3. Physiology of isolates EU25 and Ver9Iso2 in pure culture and
Kouleothix aurantiaca data from Kohno et al. (2000)
Metabolism EU25 Ver9Iso2 Kouleothix aurantiaca
Chemoautotrophic � � ND
Denitrification � � �Reduction of NO3
�-NO2� � � 1 (two strains)
Anaerobic growth 1� � 1� � 1 (glucose and fructose)
Substrate utilized
for growth
1 1 1
Glucose 1 1 ND
Fructose 1 1 1
Lactose ND 1 ND
Galactose 1 1 ND
Acetate 1 1 1
Pyruvate � 1� ND
Propionate ND 1 ND
Lactate � ND ND
Oxalacetate � � ND
Citrate 1 1 ND
Yeast extract ND 1 ND
Casaminoacids � � ND
Ethanol � � ND
Propanol � ND ND
Butanol � ND ND
Tween 80
Temperature growth
range
15–35 1C 15–35 1C 25–30 1Cw
�Growth after 90 days of incubation.wOptimum growth temperature. ND, not determined; 1� , failure
between replica; 1, growth; � , no growth.
FEMS Microbiol Ecol 59 (2007) 671–682 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
677Ecophysiology of filamentous Chloroflexi species
Ecophysiology
Substrate assimilation profile
A number of WWTPs were selected for detailed studies of
the ecophysiology of various probe-defined Chloroflexi
species. Type 1851 (positive with Chl1851) was present in
one treatment plant. A closely related species positive with
the probe EU25-1238, but not probe Chl1851, was present
in another two plants. Some filaments, not positive with the
specific probes, but positive with the broader probe CFX109,
were also studied as well as some filaments only positive
with the phylum probe CFX1223. Uptake of various carbon
substrates by the probe-defined Chloroflexi under aerobic in
situ conditions is shown in Table 4. All substrates tested were
taken up by some floc-forming bacteria and single cells
during all incubations, serving as positive controls.
Type 1851 (positive with Chl1851 or EU25-1238) mainly
consumed glucose and N-acetylglucosamine among the
substrates tested. They did not consume a range of other
substrates such as acetate, ethanol and amino acids. How-
ever, some filaments also took up butyrate and pyruvate; this
was largely in agreement with the pure culture studies.
Other Chloroflexi species positive with the broader probes
(subdivision 3-targeted filaments and phylum-specific fila-
ments) also all consumed glucose and, to a varying degree,
the other substrates. Some filaments only positive with the
phylum probe (CFX1223) showed a slightly broader uptake
spectrum, which reflects the existence of one or more new
Chloroflexi species in the plant. Uptake of substrates under
denitrifying or anaerobic conditions was never observed for
any Chloroflexi (data not shown).
Surface properties and exo-enzymatic activity
The distribution of hydrophobic and hydrophilic surfaces
was investigated in the different sludges by MAC (Table 5).
Sludge flocs from all industrial WWTPs showed parts of
the flocs covered with microspheres and other parts with-
out microspheres, thus acting as control. All gene probe-
defined filaments tested hydrophilic, as no hydrophobic
microspheres attached to their surface. Although probe
EU25-1238-defined filaments from TNO25 appeared
slightly less hydrophilic with few microspheres attached,
other filamentous species present in the sludge were more
Table 4. Uptake of substrates by the different filamentous Chloroflexi species under aerobic conditions as investigated by MAR
Probe WTP
CFX1223 CFX109 Chl1851 EU25-1238
CNR1 AAE Ega Skagen TNO17 Skagen TNO25
Pure culture
EU25
Formate � � � � � � � �Acetate � 1 � � � � � ND
Propionate � ND � � ND � � �Butyrate � 11 � 11 ND � 1 �Pyruvate � (1) � � 11 � � �Oleic acid � � � � � � � �Glucose 11 (1) 1/11 1/11� 11 11 1 11
Mannose 11 ND � � 1 � � 1
Galactose 1 ND � � � � � �Leucine � ND � � � (1) � �Glycine � � � � ND � � �Thymidine ND 11 ND ND ND ND ND ND
Ethanol � ND � � ND � � �N-acetyl-glucosamine ND 11 � � ND 11 ND ND
Bicarbonate1thiosulphate � ND � � ND � � �
�Not all filaments are positive.
ND, not determined; � , No silver grains (no substrate uptake); (1), some filaments slightly positive, some silver grains; 1, few silver grains, but clearly
positive; 11, positive, many silver grains.
Table 5. Surface properties and exo-enzymatic activity of the Chloro-
flexi species determined by MAC and ELF, respectively
WTP
Gene probe
defined
filament
Surface
properties
(MAC)
Exo-enzyme
activity (ELF)
CNR1 CFX1223 � CHIT/GLU
AAE CFX1223 ND CHIT/GLU
TNO17 Chl1851 � EST/GLU
TNO25 EU25-1238 (� ) EST/GAL
Pure culture EU25 EU25-1238 � EST/GAL
ND, not determined; � ,no hydrophobic beads attached; (� ), few
hydrophobic beads attached; CHIT, chitinase; EST, esterase; GAL, galac-
tosidase; GLU, glucuronidase.
FEMS Microbiol Ecol 59 (2007) 671–682c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
678 C. Kragelund et al.
hydrophobic. The pure culture EU25 was also characterized
as hydrophilic.
The presence of exo-enzyme activity in the sludge flocs
and on the filament surfaces was determined by enzyme-
labelled fluorescence assays (Table 5). Sludge flocs from all
plants exhibited exo-enzyme activity for all enzymes tested,
although some enzyme activity was low and some very high,
e.g. lipase and esterase, respectively. Esterase activity was
observed for all Type 1851 (Chl1851 or EU25-1238-positive
filaments) and for the pure culture. Glucuronidase and
galactosidase activity was also found for some Type 1851.
The filaments that were positive only with the phylum probe
CFX1223 exhibited chitinase and glucuronidase activity.
Discussion
This study presents a comprehensive investigation of fila-
mentous Chloroflexi species present in both municipal and
industrial WWTP. At present, very little is known about the
physiology of phylum representatives of Chloroflexi present
in wastewater systems, where only in situ data from an
autotrophic nitrifying biofilm (Kindaichi et al., 2004) and
two pure culture studies exist (Kohno et al., 2002; Yamada
et al., 2005). This paper reports the first investigation from
WWTP where information on identity, abundance and
ecophysiology is combined.
Identity and abundance
Phylogenetic analysis of activated sludge clones belonging to
the Chloroflexi phylum showed that these are found in
subdivisions 1 and 3, as defined by Bjornsson et al. (2002).
The isolate EU 25 and different K. aurantiaca strains sharing
between 99.1 and 99.9% 16S rRNA gene similarity are
located in subdivision 3. The isolated morphotype 1851
identified by Beer et al. (2002) and isolates EU 25 and
Ver9Iso2 from this study most likely belong to two different
species with 94.7% and 93% 16S rRNA gene similarity, but
this should be confirmed by DNA : DNA hybridization.
Filamentous Chloroflexi were present in half of the 126
industrial WTP investigated in this comprehensive study
and thus verify the observations by Beer et al. (2002) and
Bjornsson et al. (2002) that Chloroflexi is a normal member
of the activated sludge microbial community. In 33 WWTP
samples, Chloroflexi filaments were present as FI4 1.5,
indicating potential bulking. In c. 12% of the WWTP
samples, high amounts of Chloroflexi filaments were found
(FI4 2.5), identifying them as important filamentous bac-
teria involved in bulking incidences. We found that a large
fraction of the samples had unidentified Chloroflexi species
that only hybridized with the phylum probe, not with
subdivision- or species-specific probes. The new probe
designed in this study hybridized with almost the same
sequences as probe Chl1851 (Beer et al., 2002). It was
designed before Beer’s probe was published and was there-
fore used in the survey. However, when Chl1851 subse-
quently was used on the same sludge samples, it appeared
that there was not a complete overlap of the two probes on
Chloroflexi filaments, indicating that the diversity is still
poorly described in activated sludge. Probe Chl1851-posi-
tive filaments were identified in 22% of the Chloroflexi
positive population. The probe EU25-1238 targeted an
additional 16% and when the two probes were used in
combination, 38% of the Chloroflexi could be identified. For
this reason, the two probes are recommended to be applied
together for identification of filamentous Chloroflexi in
activated sludge communities. The Chloroflexi species tar-
geted by probe EU25-1238 or probe Chl1851 was, however,
abundant in only three plants (FI4 1.5).
In 34 other WWTPs, filaments with the same morpholo-
gical appearance, but only hybridizing with the subdivision
probe (10 samples) or the phylum probe (26 samples), were
responsible for a high filament index. This emphasizes that
unidentified species are more common than these isolates in
full-scale WWTPs. It also shows that phylum probes
(CFXMIX) should be applied for screening the presence of
filamentous Chloroflexi species in WWTP. Almost all probe-
defined Chloroflexi filaments also hybridized with EUBMIX;
however, only half of the CFX109-positive filaments de-
tected in the samples used for ecophysiological studies gave a
positive EUBMIX signal, suggesting that some Chloroflexi
species did not hybridize with the EUBMIX probes. This
needs to be resolved in future studies.
The morphology of most gene probe-defined Chloroflexi
from the ecophysiological studies fell within the broadly
defined morphotype 1851. Minor differences were seen, for
example, in cell diameter and Gram staining but these
characters also differ in the two identification manuals
(Eikelboom, 2002; Jenkins et al., 2004). Morphotype 1851
filaments without epiphytic bacteria have been observed
frequently in industrial WWTP, and in this study with gene
probe-defined Chloroflexi, no epiphytic bacteria were ob-
served in three of four industrial WTP subjected to ecophy-
siological characterization. Almost all Chloroflexi sp.
examined here showed variability in Gram staining, which
might reflect an unusual cell wall, as reported in Beer et al.
(2002). This has also been observed for a close relative,
Chloroflexi aurantiacus, which also stains Gram-negative,
although the peptidoglycan composition has Gram-positive
characteristics (Garrity & Holt, 2001).
In 26% of the WWTPs investigated, probe-defined Chlor-
oflexi constituted a filament index higher than 1.5, which
could have an impact on floc structure and WWTP opera-
tion. In only a few of the cases observed here (TNO17 and
TNO25) were representatives of the Chloroflexi directly
involved in bulking. Morphology-based surveys have also
been reported, and morphotype 1851 only dominated in a
FEMS Microbiol Ecol 59 (2007) 671–682 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
679Ecophysiology of filamentous Chloroflexi species
few incidences. However, these results are difficult to com-
pare as the FISH screening in our study showed that filaments
with different morphology than that of morphotype 1851
were in some cases targeted by some of the probes applied.
Ecophysiological behaviour
The Chloroflexi sp. in the WWTP samples examined were
only active in situ under aerobic conditions, which is
different from many other filamentous bacteria in activated
sludge (Nielsen et al., 2002, Thomsen et al., 2002, Kragelund
et al., 2006). The substrates taken up were mainly sugars.
Butyrate and a few short chained fatty acids were also used
by some filamentous Chloroflexi, but not all. Butyrate is not
often taken up by activated sludge bacteria (Kragelund and
Nielsen, unpublished results). Acetate was taken up only by
filaments in one WTP targeted by the Chloroflexi phylum
probe. Whether some unknown Chloroflexi species can
consume this compound, as was shown for the new isolates
in the pure culture growth tests, or whether the observation
is due to unspecific phylum probes is so far unknown. N-
acetylglucosamine uptake for Chloroflexi was observed in
half of the WWTPs tested. This monosaccharide is a
component in lipopolysaccharides and peptidoglycan, con-
stituting the cell wall of most bacteria (Barker & Stuckey,
1999). It is not a substrate commonly used by filamentous
bacteria in activated sludge (Kragelund and Nielsen, unpub-
lished results), but would be available continuously due to
cell decay and subsequent release of N-acetylglucosamine
units. Uptake of N-acetylglucosamine has also been observed
under aerobic conditions for filamentous Chloroflexi present
in a biofilm (Kindaichi et al., 2004). The expression of exo-
enzymes also supports the degradation of polysaccharides,
e.g. chitinase, galactosidase, and glucuronidase activity. All
examined Chloroflexi filaments appeared hydrophilic, and
they were often observed in large bundles inside sludge flocs
and were not always visible using phase contrast microscopy.
Comparing the isolates EU25 and Ver9Iso2 with the
strains of Kohno et al. (2002), many identical physiological
traits were observed. We tested more substrates supporting
the specialization on sugars and some short chain fatty
acids. However, reduction of nitrate to nitrite observed by
Kohno and coworkers (two of the five strains) was not seen
for isolate EU25 or Ver9Iso2. The strains isolated by Kohno
and coworkers were able to grow anaerobically on sugars
(glucose and fructose), whereas the ability to grow anaero-
bically for strain EU25 and Ver9Iso2 was not confirmed in
our study. A small uptake of glucose using MAR was
observed under conditions where nitrate served as e-accep-
tor for isolate EU25, but growth could not be observed.
Comparing the results from the pure cultures with in situ
studies it is clear that the pure culture shows greater
versatility in physiology than the gene probe-defined fila-
ments in corresponding activated sludge samples. For in-
stance, fewer substrates could be assimilated, and no uptake
under anaerobic conditions was recorded. This phenomen-
on is in agreement with other studies (Rossetti et al., 2005)
showing that isolates tend to show their greater physiologi-
cal potential in pure culture, whereas under in situ condi-
tions they are more restricted in their physiological activity.
Possible role in sludge
Filamentous members of the Chloroflexi phylum are fre-
quently observed in activated sludge and contribute to the
overall filament index number. Most likely, the population
sizes of Chloroflexi species in sludge have been underesti-
mated by conventional microscopical identification due to
their location inside sludge flocs. Furthermore, many as yet
unidentified members are present in activated sludge sam-
ples which no gene probes target beyond phylum level.
Interestingly, acetate was not used (except possibly in one
case) by Chloroflexi filaments under in situ conditions,
although it is one of the most common substrates present
in activated sludge (Hvitved-Jacobsen et al., 1995). Chloro-
flexi filaments appear to be specialized in polysaccharide
degradation as different monosaccharides were taken up,
combined with exo-enzyme activity used for polysaccharide
degradation. The location of the investigated filaments
inside sludge flocs and preferential use of sugars suggest that
they grow on colloids and particles from the incoming
wastewater trapped in the surrounding exopolymeric ma-
trix, on exopolymers produced by other microorganisms,
and on decaying cells. Other filamentous members in
activated sludge, e.g. members of Bacteroidetes (Kindaichi
et al., 2004), have also been shown to use the same type of
substrate, and competition between these species is therefore
likely. The influence of morphotype 1851 on floc structure
has been observed, causing bridging and even open struc-
tured sludge flocs if they become more abundant. However,
this requires a large population size and is not frequently
observed. All in all, filamentous members of Chloroflexi are
commonly observed in municipal and industrial WTP, but
are only occasionally involved in bulking or foaming in-
cidences.
Acknowledgements
This study was funded by the EU program ‘Dynamics and
composition of filamentous micro-organism communities
in industrial water systems’ (DYNAFILM).
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