Appl Microbiol Biotechnol (1997) 48: 7379

Springer-Verlag 1997

ORIGINAL PAPER

E. J. Vainio A. Moilanen T. T. Koivula

D. H. Bamford J. Hantula

Comparison of partial 16S rRNA gene sequences obtained

from activated sludge bacteria

Received: 4 November 1996 / Received revision: 24 February 1997 / Accepted: 28 February 1997

Abstract The cultivated and uncultivated bacterial

communities of an activated sludge plant were studied.
Two samples were taken and a total of 516 bacterial
isolates were classied into groups using their whole-cell
protein patterns. The distribution of bacteria into protein-pattern groups diered signicantly between the
two samples, suggesting variation in culturable bacterial
ora. Partial 16S rRNA gene sequences were determined
for representatives of the commonest protein-pattern
groups. Most of the sequences obtained were previously
unknown, but relatively closely related to known sequences of organisms belonging to the a, b or c subclasses of the proteobacteria, the rst two subclasses
being predominant. This classication of bacteria isolated on a diluted nutrient-rich medium diered from
recent culture-dependent studies using nutrient-rich
media. The uncultivated bacterial community was
studied by analyzing ten partial 16S rRNA gene sequences cloned directly from activated sludge. None of
the cloned sequences was identical to those determined
for culturable organisms; or to those in the GenBank
database. They were, however, related to the a or b
subclasses of the proteobacteria, or to the gram-positive
bacteria with a high G+C DNA content.
E. J. Vainio J. Hantula (&)
Forest Research Institute, P.O. Box 18,
FIN-01301 Vantaa, Finland
Tel.: +358 9 8570 5682
Fax: +358 9 8572 575
e-mail: jarkko.hantula@metla.
A. Moilanen
Department of Ecology and Systematics,
Division of Population Biology, P.O. Box 17,
FIN-00014 University of Helsinki, Finland
T. T. Koivula
Department of Biosciences, Division of Microbiology,
P.O. Box 41, FIN-00014 University of Helsinki, Finland
D. H. Bamford
Institute of Biotechnology and Department of Biosciences,
Division of Genetics, P.O. Box 56,
FIN-00014 University of Helsinki, Finland

Introduction
The revolution in molecular biology during the last decades has provided novel tools for studies of microbial
ecology. The availability of culture-independent methods like polymerase-chain-reaction-assisted direct cloning (PCR cloning), sequencing of rRNA genes, and in
situ hybridization has changed our view about the diversity and species composition of natural microbial
communities (Giovannoni et al. 1990; Amann et al.
1991; Fuhrman et al. 1992; DeLong et al. 1989; Olsen
et al. 1994).
The bacterial communities inhabiting activated
sludge plants have been studied for almost a century by
analyzing culturable bacterial isolates (Russel and
Bartow 1916; Buttereld 1935; Allen 1944; Dias and
Bhat 1964; Benedict and Carlson 1971; Blaim et al. 1984;
Hantula et al. 1991a). As a result, they are known to be
very diverse and dynamic systems, where proteobacteria
predominate. A novel insight to this community was
obtained when studies based on PCR cloning and in situ
hybridization were carried out (Wagner et al. 1993;
Manz et al. 1994; Wagner et al. 1994a, b; Wallner et al.
1995; Bond et al. 1995). The most interesting observations were that microbes belonging to the c subgroup of
the proteobacteria seemed to be less abundant in activated sludge than was observed by culture-dependent
techniques, and that gram-positive bacteria with a high
G+C DNA content seemed to occur more frequently
than previously suggested. It was also observed (Wagner
et al. 1993, 1994a) that b-subclass proteobacteria were
found more commonly in hybridization studies in situ
than in culture-dependent control experiments using
nutrient-rich media (Luria-Bertani medium or trypticase/soy/agar). Bacteria belonging to this group were,
however, frequently observed in earlier studies using
more dilute media (Dias and Bhat 1964; Benedict and
Carlson 1971; Seiler and Blaim 1982; Blaim et al. 1984)
and therefore the signicance of this observation is
unclear. Thus, the relationship between the results

74

obtained by culture-dependent and culture-independent

methods is not well understood, and therefore studies on
culturable microbial populations should complement the
results obtained by culture-independent methods (for
more detailed discussion see Palleroni 1994).
In this study a large collection of bacterial cultures
was isolated from a Finnish municipal wastewater
treatment plant. The isolates were classied into clusters
(or groups) using their whole-cell protein patterns. In
addition, a number of partial 16S rRNA gene fragments
were PCR cloned directly from the same sample. Partial
16S rRNA gene sequences were determined for representatives of 15 groups based on the whole-cell protein
patterns and compared to those obtained with direct
PCR cloning.

Materials and methods

Sampling
Two samples were taken from the Suomenoja municipal wastewater treatment plant (Espoo, Finland) with 230 000 population
equivalents. The plant treats mostly municipal wastewater, but 8%
of the inuent comes from industrial sources. The mean inuent
ow of the plant was 77 000 m3/day. Phosphorus removal was
enhanced by adding approximately 70 g FeSO4/m3 to the inuent
and 1.6 g Al/m3 to the aeration basins. The sludge age was 4.1 days
and the sludge load about 0.26 kg biological oxygen demand
(BOD7) (kg mixed liquid-suspended solids))1 day)1. The water
temperature during the experimental period was 1014 C. No
bulking, foaming or other disturbances were observed in the plant
operation during the time when the samples were collected.
The samples were taken from the same location in the aeration
basin at the same time of day (11 a.m.) on 26 October 1993
(sample 1) and 14 January 1994 (sample 2). Two 100-ml subsamples were taken within 1 min at both sampling times (subsamples
1a, 1b, 2a and 2b). For isolation of bacteria the ocs were partially
disrupted by sonication (Branson 2200 bath sonicator). The viability of the cells was monitored by plating, and the highest viable
counts were obtained by 30 min sonication. This sonication time
was then used for all the samples. Aliquots (100 ll) of a dilution
series of the sonicated sludge were plated on 10% TGY plates
(APHA 1985) containing 0.05% (w/v) Bacto tryptone, 0.025%
(w/v) Bacto yeast extract, 0.01% (w/v) Bacto dextrose and 1.5%
(w/v) Bacto agar (Difco; Detroit, Mich. USA), and incubated for
14 days at room temperature (approximately +20 C). All emergent colonies were picked and puried by successive single-colony
isolations until pure lineages were obtained. For DNA extraction,
an aliquot of sample 1a was stored at )20 C.
Sodium dodecyl sulfate/polyacrylamide gel electrophoresis
(SDS-PAGE) of total cellular proteins
The SDS-PAGE was carried out as described previously (Olkkonen
and Bamford 1989), except that 100 mM NaCl was added to the
anode buer. For the whole-cell protein analysis, solid cultures on
10% TGY were incubated at room temperature for 2 weeks. The
bacterial mass obtained was collected into a microcentrifuge tube,
resuspended in 10 mM TRIS/HCl buer (pH 8.0) containing 10 lg
lysozyme ml)1 and 5 U mutanolysine ml)1, and pretreated at 37 C
for 30 min before sample preparation.
For dendrogram construction the gels were scanned with a
charge-coupled device camera (Kodak megaplus, model 1.4; resolution 1024 1024 pixels 256 gray levels). The images were analyzed using BioImage image processing software and the wholecell protein pattern of Escherichia coli DH5a as a standard to de-

termine the apparent molecular masses of the protein bands. The

patterns were compared using the information about apparent
molecular masses of the bands, band spacing and band intensity.
The results were used to construct a UPGMA dendrogram (Sneath
and Sokal 1973). The bacterial clusters obtained in this analysis are
called protein groups hereafter.
Extraction and purication of total genomic DNA
from cultivated isolates
Total genomic DNA of cultivated isolates (AI047, AI049, AI051,
AI085, AI111, AI138, AI171, AI208, AI235, AI269, AI271, AI289,
AI301, AI303, AI312, AI313, AI346, AI353, AI356, AI359, AI361,
AI372, AI383, AI391, AI423, AI491, AI563) was puried according
to the method of Tsai and Olson (1991).
Extraction and purication of total DNA from activated sludge
Total DNA of activated sludge sample 1a was puried as follows.
A 15-ml sample was pelleted by centrifugation (Sorwall SS34-rotor,
5000 rpm, 10 min) and washed twice with buer L (0.15 mM
NaCl, 0.1 M Na2EDTA, pH 8.0). The pellet was resuspended in
5 ml of the same buer containing 10 lg lysozyme ml)1 and 10 U
mutanolysine ml)1. The suspension was incubated at 37 C for 2 h,
after which 5 ml buer L, 5 ml 20% (w/v) SDS and 1 mg proteinase K were added. The suspension was incubated at 37 C for
1 h. This was followed by three freeze ()70 C) and thaw (65 C)
cycles, and extractions with phenol/chloroform (1:1) and chloroform/isoamyl alcohol (24:1). The DNA was precipitated with ethanol, dried under vacuum, resuspended in 1 ml TRIS/EDTA buer
(10 mM TRIS, 1 mM EDTA, pH 8.0) and treated with RNase.
The DNA was nally polyethyleneglycol (PEG)-precipitated by
adding 600 ll solution containing 20% (w/v) PEG 6000 and 2.5 M
NaCl to 1 ml DNA sample. The mixture was incubated on ice for
5 min and centrifuged in a microcentrifuge for 10 min. The pellet
was washed with 70% (v/v) ethanol, dried under vacuum and resuspended in TRIS/EDTA.
Amplication, cloning, and sequencing
of partial 16S rRNA gene fragments
Partial sequences of the 16S rRNA gene, ranging in length from
343 to 500 nucleotides, were determined for 27 isolates from 15
protein groups (4 isolates from group 1, 3 from group 2, 2 from
groups 39, and 1 from groups 11, 12, 1416 and 18, see Table 2).
In cases where a protein group seemed to be divided into subclusters, representatives from each potential subcluster were selected for sequence analysis. The isolates belonging to protein
groups selected for sequence analysis contained 74% of all isolated bacteria (444 of 600 isolates) and 86% of the bacteria for
which a protein pattern had been analyzed (444 of 516 isolates).
In this study, the percentage occurrence of proteobacterial subclasses in the cultivated samples is expressed as the number of
isolates aliated with the proteobacterial subclasses divided by
the number of all isolates in the protein groups for which sequences had been determined (444). For database comparisons the
number of sequences was reduced from 27 to 16 by including only
one representative of isolates with identical sequence in the
overlapping area (and such identical sequences will be called sequence types hereafter). In addition, ten partial 16S rRNA gene
fragments were amplied by PCR and cloned without cultivation
from sample 1.
Amplication of partial 16S rRNA gene fragments from puried DNA of activated sludge sample 1a and the cultivated isolates
was carried out in 100-ll reaction mixtures. Each reaction tube
contained 10 ll Pfu polymerase reaction buer (200 mM TRIS/
HCl, pH 8.2; 100 mM KCl; 60 mM (NH4)2SO4; 15 mM MgCl2;
1% w/v Triton X-100), 75 lM each deoxynucleotide triphosphate,
2.5 U Pfu DNA polymerase (Stratagene), 50 pmol each primer and

75
approximately 100 ng puried template DNA. The primers used in
the amplications have been reported by Lane et al. (1985). Universal primer C, 5ACGGGCGGTGTGTRC (where R = A/G),
and a primer complementary to universal primer A, 50 CAGCMGCCGCGGTAATWC (where M = A/C; W = A/T), were
used for direct amplication of partial rRNA genes from sample
1a. The amplication product covered the area 5191406 of the 16S
rRNA (primers included) according to the E. coli numbering system and its length was approximately 900 bp. The primers used for
the cultivated isolates were universal primer C and a primer
complementary to primer B, 50 AAACTYAAAKGAATTGACCC
(where Y = C/T; K = G/T). The length of the amplication
product was approximately 500 bp covering the area 9071406 of
the 16S rRNA (according to the E. coli numbering system). The
reaction mixtures were covered with mineral oil and placed in a
thermocycler. After an initial denaturation at 95 C for 5 min, 25
cycles were carried out: 95 C for 1 min, 48 C for 1 min and 72 C
for 30 s. The amplication products were puried by phenol and
chloroform/isoamyl alcohol (24:1) extractions following electrophoretic separation and excision from agarose gels.
To prepare the 16S rRNA gene library from sample 1a, the
puried PCR products were treated with T4 polynucleotide kinase
(Promega) and ligated into SmaI-digested pUC18 cloning vector
using T4 DNA ligase (Promega) according to the manufacturer's
instructions. The ligation products were transformed into
competent E. coli DH5a cells and plated on Luria-Bertani agar
plates (Sambrook et al. 1989) containing 150 lg ampicillin ml)1
and 50 lg 5-bromo-4-chloro-3-indolyl b-D-galactoside ml)1. Plasmids were extracted according to the method of Birnboim and
Doly (1979) and inserts were detected from SmaI digests by agarose
gel electrophoresis. Both strands of the plasmid inserts were sequenced manually using the Sanger dideoxy-DNA method (Sambrook et al. 1989) with Sequenase version 2.0 T7 DNA polymerase
(USB). Primer C and the primer complementary to primer B were
used in sequencing.
The puried PCR products of the cultivated isolates were sequenced manually using a CircumVent thermal cycle sequencing kit
(New England Biolabs) with approximately 3050 ng template
DNA. Both strands of the amplication products obtained were
sequenced using primer C and the primer complementary to primer
B. Reaction conditions were chosen according to the manufacturer's instructions and 20 cycles of amplication were carried out:
95 C for 20 s, 55 C for 20 s and 72 C for 20 s. The amplication
products were analyzed in 7% (w/v) acrylamide gels according to
Sambrook et al. (1989).
Data analysis
The similarity values presented in Table 2 were obtained from
GenBank comparisons. Other similarity values appearing in the
text were calculated using PC/GENE computer software (Bairoch
1992).
Nucleotide sequence accession numbers
The nucleotide sequence data reported in this paper will appear in
the GenBank nucleotide sequence database with the following accession numbers: AI047, U45667; AI049, U45687; AI051, U45691;
AI085, U45690; AI111, U46932; AI138, U45666; AI171, U45668;
AI208, U45669; AI235, U45670; AI269, U45671; AI271, U45672;
AI289, U45673; AI301, U45674; AI303, U45675; AI312, U45676;
AI313, U45677; AI346, U45678; AI353, U45679; AI356, U45680;
AI359, U45681; AI361, U45682; AI372, U45683; AI383, U45684;
AI391, U45685; AI423, U45686; AI491, U45688; AI563, U45689;
CL01, U45698; CL02, U45699; CL03, U45700; CL04, U45701;
CL05, U45692; CL06, U45693; CL07, U45694; CL08, U45695;
CL09, U45696 and CL10, U45697. For the accession numbers of
previously published GenBank sequences see Table 2.

Results
Success and reproducibility of the SDS-PAGE analysis
A total of 150 isolates were originally picked from each
subsample. In subsamples 1a, 1b, 2a and 2b the analysis
of whole-cell protein patterns separated by SDS-PAGE
was successful for 138, 139, 122 and 117 isolates respectively. The number of isolates analyzed was decreased for two reasons: (i) several isolates were lost
during the single-colony isolation procedure and (ii) no
bands were observed in some of the protein patterns,
possibly because of inecient cell disruption during
sample preparation.
The grouping of the 516 protein patterns obtained
with the UPGMA algorithm separated 53 protein
groups altogether (Table 1). Subsamples 1a and 1b as
well as subsamples 2a and 2b were collected independently to control the reproducibility of our sampling
procedure and protein pattern analysis. Comparison of
subsamples 1a and 1b showed that 6 out of the 27 protein groups were common to both subsamples. Although
this number was relatively low, a more detailed analysis
of the data indicated that all protein groups with more
than 3 isolates contained isolates from both subsamples
(Table 1). A similar analysis with subsamples 2a and 2b
showed that 22 out of 43 protein groups observed were
common to both subsamples. Again all large protein
groups (with more than 4 isolates) were observed in both
subsamples (Table 1). These analyses conrmed that our
sampling procedure was adequate for culturable organisms in terms of the reproducibility of the results, and
therefore subsamples 1a and 1b, and 2a and 2b were
combined to form samples 1 and 2 respectively.
Comparison of samples 1 and 2
The grouping of isolates from sample 1 showed that
protein groups 1 and 2 contained 204 and 29 isolates
respectively. These gures represent 73% and 10% of
all isolates. Thus, the remaining 25 protein groups
contained only about 17% of isolates in sample 1
(Table 1).
The grouping of sample 2 isolates revealed more
equal distribution (Table 1), the largest protein groups
being 3 (containing 16% of isolates), 1 (9%), 2 (9%), 4
(9%) and 5 (8%). In addition, protein groups 9 (5%), 6
(4%), 8 (4%) and 7 (4%) were composed of at least 9
isolates in sample 2 (Table 1).
The comparison of samples 1 and 2 revealed that 15
protein groups (of 53) were shared by the two samples
(Table 1). Almost all groups with 10 or more isolates
contained isolates originating from both samples, the
only exceptions being protein groups 28 and 32, which
were composed of isolates from the second sample only
(Table 1).

76
Table 1 Distribution of isolates into dierent protein groups
Protein
group

Samples
1a

1b

2a

2b

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53

112
13
0
0
0
2
0
0
0
0
1
1
1
2
0
0
0
0
0
0
0
1
2
0
0
0
0
0
0
0
0
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

92
16
2
1
0
0
3
1
0
2
3
5
1
2
1
0
0
0
1
3
0
0
0
2
1
1
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

8
17
20
12
8
7
6
6
5
4
2
2
4
1
2
4
0
2
1
0
1
0
0
0
1
0
1
1
1
0
0
0
0
0
0
0
0
0
1
1
1
1
1
0
0
0
0
0
0
0
0
0
1

14
5
18
10
11
3
3
4
6
4
3
1
1
1
3
2
4
1
1
0
2
1
0
0
0
1
1
1
1
2
2
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
0

226
51
40
23
19
12
12
11
11
10
9
9
7
6
6
6
4
3
3
3
3
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

138

139

122

117

516

Similarity of partial 16S rRNA sequences

within protein groups
The 16S rRNA gene sequence analysis was limited to
343500 nucleotides in order that most of the major
protein groups could be analyzed. Despite isolates being
selected for sequence analysis from potential subclusters,

no dierences in the analyzed sequences were observed

within protein groups 2, 3, 4, 5, 6, 8 and 9. This indicates
that, in most cases, the phenotypic classication was
accurate. The sequences from protein groups 6 and 14
were identical, indicating these 2 groups to be closely
related. However, intragroup dierences were observed
in protein groups 1 and 7. In protein group 1, 3 of the 4
isolates studied shared identical sequences, but 1 isolate
diered from them by 5.0% in sequence similarity. In
protein group 7 the subclusters observed should be
considered as separate protein groups as the 2 isolates
studied shared only 80% in sequence similarity. Thus,
the clustering of the 2 isolates into protein group 7 was
incorrect. This conclusion was also conrmed by visual
comparison of the protein patterns.
Aliation of sequences to taxonomic groups
The sequences were aliated with proteobacterial subclasses according to 16S rRNA signature nucleotides of
bacterial groups reported by Woese (1987) and also according to the phylogenetic status of the most similar
sequence in the GenBank database (Burks et al. 1992).
The similarity values presented in Table 2 were obtained
from GenBank comparisons. In all cases the same afliation was obtained with both criteria used. It should
be noted that the sequence type from protein group 5
was aliated with the b subclass of the proteobacteria
although it shared a high similarity with ``unidentied a
proteobacterium 34-P''. This aliation was based on the
16S rRNA signature nucleotides and on the fact that the
sequence type also shared a high similarity (over 98%)
with Variovorax paradoxus, a representative of the b
subclass. It should also be noted that, in the original
report (Gosink and Staley 1995), this isolate (34-P) was
clustered among b-subclass proteobacteria although the
authors had submitted the sequence to GenBank as a
representative of the a subclass. No phylogenetic conclusions were drawn above the subclass level because of
the limited amount of sequence data.
Distribution of proteobacterial subclasses
in samples 1 and 2
The major sequence groups consisted of representatives
of the a and b subclasses of the proteobacteria. The
protein groups related to the a and b subclasses contained 65% and 30% of isolates respectively. However,
these two subclasses were unevenly represented in the
two samples. Thus, 82% of all isolates related to the a
subclass were isolated from the rst sample and 92% of
isolates related to the b subclass from the second
sample. The sequences related to the c subclass of the
proteobacteria accounted for protein groups containing
only 5% of isolates and were evenly represented in
both samples (48% and 52% in samples 1 and 2
respectively).

77
Table 2 Comparison of partial 16S rRNA gene sequences to those
of the GenBank database. PCR polymerase chain reaction; isolates
or clones with identical sequences are indicated by /. The percenProtein
group

tage sequence similarity is given for the longest sequence in cases

where two or more identical (in the overlapping area) sequences
were determined. a, b, c proteobacteria of the a, b and c subclasses

Isolate or
PCR clone

Length of
sequences
(bp)

Most similar GenBank sequences

(accession code)

Taxonomic
aliation

Percentage
sequence
similarity

AI049/AI051/AI313
AI359

384/500/411
377

Bradyrhizobium sp. strain ``129'' (D14508)

Bradyrhizobium japonicum IAM 12608 (D12781)

98.4
95.8

AI085/AI138/AI171

343/366/351

AI271
Methanotrophic sp. strain ``B-3060'' (L20845)

96.2
93.4

AI208/AI235

365/366

Variovorax paradoxus IAM 12373 (D30793)

99.5

AI269/AI312

380/390

Variovorax paradoxus IAM 12373 (D30793)

98.7

AI346/AI372

364/346

Unidentied a proteobacterium ``34-P'' (U14585)

Variovorax paradoxus IAM 12373 (D30793)

98.9
98.4

AI423/AI563

379/346

Unidentied activated sludge bacterium 7087 (Z46235)

100.0

AI271

364

AI353

371

AI171, AI085, AI138

Zoogloea ramigera ATCC 19623 (X74915)
Acidiphilium cryptum ``B-Het4''(X75265)

96.2
91.3
90.6

AI301/AI303

358/398

CL08
Leptothrix discophora ATCC 51168 (L33974)

99.5
96.0

AI383/AI391

366/365

Unidentied activated sludge bacterium 7087 (Z46235)

98.9

11

AI111

349

Unidentied c proteobacterial strain ``S13'' (U31492)

Serratia marcescens ATCC 13880 (M59160)

98.0
97.7

12

AI289

371

Aeromonas eucrenophila ATCC 7966T(X60411)

100.0

14

AI047

367

Unidentied activated sludge bacterium 7087 (Z46235)

100.0

15

AI361

361

Variovorax paradoxus IAM 12373 (D30793)

98.9

16

AI356

369

Zoogloea ramigera ATCC 19544 (X74913)

96.7

18

AI491

364

CL01
CL02
CL03

361
368
373

98.9
97.8
97.2
95.7
95.7

CL04
CL05
CL06

355
392
402

CL07
CL08

408
366

CL09
CL10

383
423

Bacterial sp. ``clone group A29mn'' (X91452)

Pseudomonas uorescens MS 1650 (L24788)
Leptothrix discophora ATCC 43182 (L33975)
Ruminococcus obeum (X85101)
Gram-negative coccus strain ``Fredericksen'' (X73223)
Bordetella bronchiseptica ``strain S-1'' (X57026)
Sphingomonas sp. ``B712'' (U20775)
Ideonella dechloratans (X72724)
CL10
Rhodocyclus tenuis strain ``DSM109'' (D16208)
Variovorax paradoxus IAM 12373 (D30793)
AI303, AI301
Leptothrix discophora ATCC 51168 (L33974)
Acidiphilium facilis ATCC 35904 (D30774)
CL06
Zoogloea ramigera ATCC 19544T (X74913)

b
High G+C a
b
a
b
b
b
b
a
b

96.9
99.0
99.3
96.5
98.3
99.5
95.9
87.8
99.3
97.2

Gram-positive bacteria with a high G+C DNA content

Nine of the ten directly cloned sequences were related

to proteobacterial subclasses (see Table 2). The proportions of the proteobacterial subclasses in the uncultivated group are only suggestive, as reliable
quantication should be based on in situ techniques (see
Amann et al. 1995). Only two PCR clones were representatives of the a subclass proteobacterial group, which
accounted for approximately 90% of the cultivated
isolates from the same sample (sample 1). In contrast,
seven cloned sequences were representatives of the b
subclass, which accounted for only 4% of the cultivated
isolates in sample 1. Only one of the PCR-cloned sequences was not related to a proteobacterial subclass,

but was most similar to that of Ruminococcus obeus, a

representative of the gram-positive bacteria with a high
G+C DNA content.
Variation within taxonomic groups
Three of the sequence types related to the a subclass
were moderately (96%98%) similar to GenBank sequences (Table 2). However, four a-subclass sequence
types diered considerably (6%12%) from the most
similar sequences in GenBank, and interestingly, two of
these sequences (those of protein group 2 and AI271 of

78

protein group 7) were moderately similar (96%) to each

other.
The sequences related to b-subclass proteobacteria
formed groups that consisted of highly similar sequence
types. These groups were composed of (i) protein groups
3, 4, 5, 15 (Table 2) and the PCR clone CL07, (ii) protein
groups 6, 9 and 14, and (iii) protein group 8 and the
PCR clones CL08 and CL01. The closest relatives
of these groups were (i) Variovorax paradoxus (see
Table 2), (ii) an activated-sludge bacterium from another wastewater-treatment plant (Hantula et al. 1991a),
and (iii) Leptothrix discophora. The rst two groups were
also moderately related to each other and probably belonged to the Comamonadaceae family.
The sequences related to c-subclass proteobacteria all
shared a high similarity with sequences in GenBank
(Table 2), but a relatively low similarity (<90%) to each
other.
In general, the main dierence between the representatives of proteobacterial subclasses was that the
a-subclass sequences were more distantly related to sequences in GenBank than were those of the b and c
subclasses. In the a, b and c subclasses the sequences
most distantly related to GenBank sequences shared
88%, 96% and 98% similarities respectively with the
most similar sequences. The most unique sequence was
found among the PCR clones. As it shared less than
90% similarity with the most similar sequence in GenBank, it may represent a bacterium belonging to an
unidentied bacterial family (Wayne et al. 1987). Another indication of a previously unknown higher-level
taxon is the sequence of isolate AI353 from protein
group 7, with only 91% similarity to the most similar
sequence in GenBank.

Discussion
In this study two bacterial culture collections were obtained from activated sludge. The culturable population
was dierent at dierent sampling times, although no
apparent changes in the plant operation were observed
during the study. The rst sample was dominated by one
culturable (in TGY medium) protein group. In contrast,
in the second sample the distribution of isolates among
protein groups was more equal and no major protein
groups were observed. The latter case has also been
observed in samples analyzed previously from another
wastewater treatment plant (Hantula et al. 1991a). The
question emerging is whether these observations reect
true dierences in diversity, or result from biases caused
by the culture-dependent techniques used.
According to partial 16S rRNA gene sequence comparisons, all the cultivated isolates were related to recognized phyla of the domain bacteria, that is, the a, b
and c subclasses of the proteobacteria. This supports the
results of in situ hybridization studies, which have revealed that proteobacteria account for about 80% of all

active bacteria found in activated sludge (Wagner et al.

1993). However, our observation that most of the culturable organisms belong to the a or b subclasses of the
proteobacteria is in contrast to results of previous cultivation-based studies (Wagner et al. 1993, 1994a) where
activated-sludge samples have been dominated by csubclass proteobacteria. This contradiction may be due
to the two rich cultivation media used in the studies, as
they favor the growth of c-subclass and select against bsubclass proteobacteria (Wagner et al. 1993) whereas
dilute media, similar to the TGY used in this study, are
known to support growth of a wider range of bacteria
(Prakasam and Dondero 1967). The low number of PCR
clones does not allow the estimation of the relative frequences of these sequences in activated sludge, which
was therefore excluded.
Most of the sequences in this study were highly or
moderately similar to previously published sequences.
Sequences determined for protein groups 6, 14 and 12
were identical to sequences in GenBank (Table 2), the
rst 2 sequences being identical to a sequence we have
determined previously (Koivula and Hantula, 1997) for
isolate 7087 from another activated sludge plant (Hantula et al. 1991a). This suggests that this undescribed
species would commonly inhabit wastewater treatment
plants. This is also supported by the presence of bacteriophages infecting this species in activated sludge
(Hantula et al. 1991b). The grouping of isolates of this
species in two clusters is not surprising, as we have
previously shown that there are size polymorphisms in
the major protein bands of the isolates (Hantula et al.
1991a). Despite some sequences being identical to previously determined ones, most of our sequences diered
from those. This indicates that neither the culturable nor
the unculturable bacteria of activated sludge are yet well
understood at the 16S rRNA sequence level.
To compare views of the activated-sludge bacterial
ora obtained by culture-dependent methods and direct
cloning, we used partial 16S rRNA sequence analysis of
PCR clones and cultured organisms. None of the directly cloned sequences was identical to the sequences
determined from the cultured isolates or to sequences in
the GenBank database. Thus, although the data are
restricted, especially those for the PCR cloned sequences, they support the idea that sequences obtained
by PCR cloning or by methods based on cultivation
present a very dierent view of the studied bacterial
population at species level. However, at higher taxonomic levels, the PCR clones belonged mostly to the
same taxons as the cultivated isolates. If both sets of
data are compared to previously published results, the
activated sludge can be considered as a very dynamic
ecosystem with only a few ubiquitous species.
Acknowledgements Ms. Marja-Leena Perala is acknowledged for
skillful technical assistance. This study was supported by research
grants from the Maj and Tor Nessling Foundation and the Finnish
Academy of Sciences.

79

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