FEMS Microbiology Letters 246 (2005) 143–149
www.fems-microbiology.org
Microbial community dynamics in bioaugmented sequencing
batch reactors for bromoamine acid removal
Yuanyuan Qu a, Jiti Zhou a, Jing Wang
a
a,*
, Xiang Fu b, Linlin Xing
a
School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalina Linggong Road, Liaoning 116024, PR China
b
Department of Civil and Environmental Engineering, Stanford University, Stanford, CA 94305-4020, USA
Received 12 January 2005; received in revised form 3 March 2005; accepted 7 April 2005
First published online 19 April 2005
Edited by C. Edwards
Abstract
Sphingomonas xenophaga QYY with the ability to degrade bromoamine acid (BAA) was previously isolated from sludge samples.
The enhancement of BAA removal by strain QYY in sequencing batch reactors (SBRs) was investigated in this study. The results
showed that augmented SBRs exhibited stronger abilities to degrade BAA than the non-augmented control one. In order to estimate
the relationship between community dynamics and function of augmented SBRs, a combined method based on fingerprints
(ribosomal intergenic spacer analysis, RISA) and 16S rRNA gene sequencing was used. The results indicated that the microbial
community dynamics were substantially changed, and the introduced strain QYY was persistent in the augmented systems.
This study suggests that it is feasible and potentially useful to enhance BAA removal using BAA-degrading bacteria, such as
S. xenophaga QYY.
Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Bioaugmentation; Bromoamine acid; Community dynamics; Ribosomal intergenic spacer; Sphingomonas xenophaga
1. Introduction
Bromoamine acid (1-amino-4-bromoanthraquinone2-sulfonic acid, BAA), a major intermediate, is widely
used in synthesis of anthraquinone dyes. It is water-soluble and stains water bodies red resulting in serious
environment pollution [1]. Nevertheless, there is little
information on bio-treatment of colored wastewater
containing anthraquinone compounds, e.g., BAA [2].
Efficient and reliable color removal is critically important to treatment of dyes in wastewater. However, the
conventional activated sludge systems often fail to
*
Corresponding author. Tel.: +86 411 84706250; fax: +86 411
84706252.
E-mail address: [email protected] (J. Wang).
achieve high efficiency in treating such wastewater [3].
Bioaugmentation is expected as the most straightforward strategy to remediate such failure [4].
Bioaugmentation is the application of indigenous or
genetically modified organisms to bioreactors or other
polluted waste sites in order to accelerate the removal
of undesired compounds [4,5]. It has been reported to
enhance removal of specific pollutants [6–8], such as 3chloroaniline, resin acid and aromatic hydrocarbons,
etc. Unfortunately, there are few microorganisms suitable for bioaugmentation [4,6]. Nowadays, the genus
Sphingomonas has received a lot of attention and became
biotechnologically interesting microorganism for its degradation capabilities of xenobiotic compounds [9,10]. In
our laboratory, some BAA-degrading bacteria were
isolated and characterized [11], of which S. xenophaga
0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2005.04.006
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Y. Qu et al. / FEMS Microbiology Letters 246 (2005) 143–149
QYY demonstrated the strongest BAA degrading ability
[12], and the initial test showed that it was suitable for
bioaugmentation of BAA removal in laboratory based
SBRs.
Bioaugmented systems host complex microbial communities, however, very little is known about their normal dynamics. And the relationship between community
structure and system function remains unknown. Currently, DNA fingerprint technologies such as denaturing
gradient gel electrophoresis (DGGE) and ribosomal
intergenic spacer analysis (RISA) have been exploited
to investigate bacterial community dynamics [13–16].
In order to reveal the microbial community thoroughly,
combined methods such as fingerprints and 16S rDNA
sequence analysis are becoming more useful [15].
The aim of this paper was to evaluate the potential
utility of bioaugmentation with S. xenophaga QYY to
enhance BAA removal and monitor the dynamics of
microbial communities. This is the first report of bioaugmentation of BAA using S. xenophaga.
lized cells (ASI). Immobilized cells were established as
described previously [17]. The inoculation amounts
were 1.81%, 4.53% and 9.07% (the dry weight of the
supplemented culture to that of indigenous AS),
respectively. In addition, there were two operation conditions for both ASS and ASI systems. The partition
points for both systems were on day 16 and day 28,
respectively. In the ASS systems, the secondary inoculation (4.53%) was performed after day 16. But for the
ASI systems, the cycle time was changed to 24 h after
day 28.
2. Materials and methods
The genomic DNA was extracted from the sludge
samples by the method described previously [18].
The universal primers S926f (5 0 -CTYAAAKGAATTGACGC-3 0 ) and L189r (5 0 -TCATGAGATGYTTMARTTC-3 0 ) were used, which anneal to
positions 910–926 of the 16S rRNA gene and positions
189–207 of the 23S rRNA gene (Escherichia coli numbering), respectively. PCR amplification was conducted
as described previously with the exception of annealing
temperature (48 °C) [19]. PCR amplified fragments were
separated on native polyacrylamide (6%) gels, which
were stained with ethidium bromide and photographed
for RIS analysis.
Clone libraries of RIS-rDNA amplicons were constructed from the corresponding sample in RIS analysis
of ASS (day 15, day 28) and ASI (day 40), with inoculum of 9.07% using a TA Cloning Kit (TaKaRa, Dalian
Co., Ltd).
2.1. Microorganism, media and activated sludge samples
S. xenophaga QYY was originally isolated from
sludge samples in our laboratory, and is deposited in
the China General Microorganism Culture Center with
the number 1172.
The media used in this study were BAA-Luria-Bertani medium (BLB), which contained (g l1): 10 Bactotryptone, 5 Bactoyeastextract, 10 NaCl, 0.1 BAA, pH
7.2, and the synthetic wastewater medium (SW), which
contained (g l1): 1.3 KH2PO4, 1.5 Na2HPO4, 0.1
(NH4)2SO4, 0.01 FeCl3, 0.02 tryptone, and BAA was
of 110–520 mg l1, pH 7.0.
Samples of fresh activated sludge (AS) were taken
from Dalian Chunliuhe Wastewater Treatment Plant,
which were used as the indigenous populations.
2.2. Operation of SBRs and construction of augmented
SBRs
The AS system was simulated with 250-ml flask
containing 3.08 g l1AS and 50-ml of SW medium,
which was used as a control and was incubated at 30
°C on a shaker. The SBRs were operated on a 12 h cycle, and each cycle consisted of 10 min fill, 11 h react,
40 min settle, 10 min decant. The mean cell residue
time (sludge age) was controlled at about 10 days by
regular discharging sludge. Samples were taken once
a day for both BAA removal and molecular fingerprint
analysis.
The augmented systems were: (i) AS system with
suspended cells (ASS), and (ii) AS system plus immobi-
2.3. Analytical method
Cell concentration was measured spectrophotometrically at 660 nm. And the concentration of BAA was
determined by monitoring the changes at 485 nm using
a Jasco UV-560 spectrophotometer (Japan).
2.4. Genomic DNA extraction, PCR and construction
of clone libraries
2.5. 16S rDNA sequencing and phylogenetic analysis
The recombinant plasmids with the rDNA inserts of
different sizes from each library were selected randomly,
and the 16S rDNA regions were sequenced by TaKaRa
Biotechnology (Dalian) CO., Ltd.
The determined partial 16S rDNA sequences (ca. 500
bp) were compared using the BLAST algorithm. And
the sequence most similar to each clone was used as
the reference affiliation. Phylogenetic trees were constructed using Clustal X 1.8 by the neighbor-joining
method, and the evolutionary distances were calculated
according to the model of Juke–Cantor [20]. Bootstrap
analysis for 1000 replicates was performed to estimate
the confidence of tree topologies.
Y. Qu et al. / FEMS Microbiology Letters 246 (2005) 143–149
(a)
1
600
Concentration of BAA (mg l-1)
Concentration of BAA (mg l-1)
(b)
2
1
250
200
150
100
50
0
145
2
500
400
300
200
100
0
0
4
8
12
16
20
24
28
Time (d)
32
0
4
8
12 16 20 24 28 32 36 40 44 48 52
Time (d)
Fig. 1. BAA removal in augmented SBRs in ASS systems (a) and ASI systems (b): BAA concentration of influent (—), 1.81% inoculum (j), 4.53%
inoculum (m), 9.07% inoculum (d).
2.6. Nucleotide sequence accession numbers
The sequences used here have been deposited in the
GenBank under the accession numbers AY611716,
AY751303 and AY755362 to AY755412.
3. Results and discussion
3.1. Performance of bioaugmented SBRs
The control system did not remove BAA under the
same conditions (data not shown), presumably due to
the absence of BAA-degrading indigenous populations.
It was reported that appropriate inoculum is a key
factor for successful bioaugmentation [21]. Thus, three
different inocula were studied in both augmented systems. The BAA removal by the ASS systems under the
two operation conditions (1 and 2) marked with arrows
was shown in Fig. 1(a). During the first BAA shock
loading, BAA removal was relatively stable. After 12
days operation, the removal efficiency of three reactors
became reduced. When BAA was further increased to
220 mg l1, the ASS systems became perturbed and in
order to remedy these suspended cells were inoculated
once more on day 16. Although all systems were restored, the performance (only 70% BAA removal) was
not as good as expected.
Fig. 2. RISA fingerprints of microbial communities (a) and cluster analysis based on percent similarity of fingerprints (b). Lane designations: M: 200
bp ladder; Q: Strain QYY; AS: activated sludge alone; ASS: AS with suspended cells of strain QYY; ASI: AS with immobilized cells of strain QYY.
The numbers 0, 15, 20, and 40, etc. indicate the sampling time (days). Bar indicates 10% similarity.
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Y. Qu et al. / FEMS Microbiology Letters 246 (2005) 143–149
In ASI systems there were also two operation conditions (1 and 2) marked with arrows (Fig. 1(b)). Results
showed that inoculum size substantially affected the removal of BAA. In addition, the ASI system could cope
with a higher BAA shock loading than the ASS ones.
The ASI system with inocula 9.07% could successfully
deal with BAA up to 520 mg l1. In order to maintain
high BAA removal, the cycle time was regulated at 24
h during condition 2 (Fig. 1(b)). The ASI systems
improved the buffering capability and prevented the
(a)
7-3 (AY755364)
99 Sphingomonas xenophaga QYY (AY6117 16)
7-10 (AY755385)
99
7-8 (AY755383)
7-15 (AY755398)
62
Sphingomonas stygia (AB025013)
99
α — Proteobacteria
7-4 (AY755365)
98
99 Brevundimonas diminuta (AB167225)
49
7-5 (AY755366)
7-1 (AY755362)
99
Alpha proteobacterium (AF236002)
99
7-7 (AY755382)
7-6 (AY755367)
99
99
Acidovorax sp. (ASP130765)
β — Proteobacteria
7-9 (AY755384)
78
Derxia gummosa (AB089482)
99
7-2 (AY755363)
99
Leucobacter k omagatae (AJ746337)
99
7-12 (AY755387)
Actinobacteria
Uncultured actinobacterium (AY500111)
99
7-11 (AY755386)
99
Kaistella k oreensis (AF344179)
Flavobacterium columnare (AB078047 )
99
Flavobacteria
7-13 (AY755410)
99
99 7-14 (AY755397)
0.02
99 9-1(AY755374)
(b)
99
9-19 (AY755404)
Uncultured bacterium (AF513106)
99
Flavobacterium columnare (AB078047)
64
9-8 (AY755381)
99
Flavobacteria
99 9-12 (AY755405)
9-15 (AY755408)
81
99
Unidentified eubacterium (U81641)
99
9-10 (AY755395)
Clostridium sp. (AY238334)
Clostridia
9-20 (AY755412)
99
73
Dermatophilus chelonae (AJ243919)
22
9-7 (AY755380)
99
97
Uncultured actinobacterium (AY500111)
9-16 (AY755409)
99
Gamma proteobacterium (AB174846)
99
99
Actinobacteria
9-5 (AY755378)
99
ν — Proteobacteria
9-2 (AY755375)
Acidovorax sp. (AY093698)
β — Proteobacteria
9-17 (AY755402)
99
9-13 (AY755406)
99
95 Variovorax paradoxus (AF532868)
9-9 (AY755394)
52
99
Uncultured Verrucomicrobia (AY395425)
Verrucomicrobiae
9-14 (AY755407)
99 9-18 (AY755403)
Uncultured bacterium (AY424826)
76 9-6 (AY755379)
99 9-3 (AY755376)
99
Sphingomonas sp. QYY (AY611716)
α — Proteobacteria
Sphingomonas adhaesiva (D13722)
99
99
9-4 (AY755377)
95 9-11 (AY755396)
0.05
Fig. 3. Phylogenetic tree (unrooted) showing the affiliations of the partial 16S rDNA sequences (E. coli positions 926–1500) determined from ASS-15
sample (library-7) (a), ASS-28 sample (library-9) (b) and ASI-40 sample (library-8) (c). The most similar sequence was included as a reference for each
sequence. The sequences GenBank accession numbers of both clone and the reference strains are shown in parentheses. The scale bar corresponded to
an estimated 0.1 mutation per nucleotide position. Bootstrap values are shown for branches with 1000 bootstrap support.
Y. Qu et al. / FEMS Microbiology Letters 246 (2005) 143–149
(c)
147
8-8 (AY755889)
57
62
8-5 (AY755372)
91
Rhodoferax ferrireducens (AF435948)
99
Acidovorax sp. (AF235013)
99
Proteobacteria
8-10 (AY755391)
Uncultured Green Bay ferromanganous(AF2930 03)
92
8-12 (AY755393)
99
99 8-16 (AY755401)
Pseudomonas putida (AY623928)
Alcanivorax sp. (AB053125)
99
91
91
Proteobacteria
8-1 (AY755368)
99
8-15 (AY755400)
Chelatococcus asaccharovorans (AJ294349)
70 8-2 (AY755369)
52 8-3 (AY755370)
99
8-4 (AY755371)
99
Proteobacteria
8-11 (AY755392)
Sphingomonas sp. QYY (AY611716)
88 8-9 (AY755390)
8-14 (AY755399)
91 8-6 (AY755373)
8-13 AY755411
Verrucomicrobiae
Opitutus sp. (X99392)
99
8-7 (AY755388)
99
Uncultured bacterium (AF255626)
0.02
Fig. 3 (continued)
inocula from being grazed or washed out. Therefore,
bioaugmentation with immobilization of strain QYY
was potentially useful in practical wastewater
treatments.
3.2. Effects of bioaugmentation on microbial community
dynamics
RISA fingerprints were examined for the effects of
bioaugmentation. Generally, the 16S and 23S rRNA
genes are in one transcriptional unit, but are separated
by RIS, which varies in length and sequence among different bacterial species. The PCR products contain the
complete RIS and parts of the flanking rDNA–RIS.
By means of moderate electrophoresis techniques, such
difference can be distinguished and is therefore useful
for studying characteristics of complex microbial
communities.
As expected, the samples of AS day 10 and day 15
had similar fingerprints (Fig. 2(a)) with 80% similarity
(Fig. 2(b)), which was somewhat different from AS day
0, probably due to BAA addition. A band of approximately 1.5 kb, attributed to strain QYY, was evident
in the ASS and ASI systems as denoted by arrows
(Fig. 2(a)). It was concluded that bioaugmentation had
a great impact on community fingerprints. Several
bands entirely disappear or appear, suggesting large decrease or increase of certain species which is different
from other studies [7]. The ASS day 15 fingerprints appeared anomalous, which showed extensively low simi-
larity to day 0 and day 28 (Fig. 2(b)). Notably, the
double bands near 2.4 kb emerged, which did not exist
in other systems. Accordingly, such irregular changes
would result in the loss or decrease of function.
It is interesting that the immobilized cells persist in
the ASI systems (Fig. 2(a)). Possible explanation is that
S. xenophaga possesses the ability to produce unique
exopolysaccharide [10], which can incorporate itself into
sludge floc, therefore, the settle ability of strain QYY
was improved.
3.3. Phylogenetic analysis of RIS-rDNA sequences
Although RISA permits meaningful comparisons of
community similarity, it does not estimate the accurate
community structure [22]. In this study, three clone libraries designated as ASS-15 (library 7), ASS-28 (library
9) and ASI-40 (library 8) were constructed for sequence
analysis. The rDNA–RIS fragments have about 600 bp
of 16S rDNA, which is sufficient for phylogenetic identification at approximately the genus level.
Nearly 50 clones with different insert sizes from three
libraries were sequenced. The phylogenetic trees were
shown in Fig. 3 and representatives of five different bacterial divisions were found. The majority of the sequenced clones (64.7%) were affiliated with the
Proteobacteria (a, b, c), which suggests that Proteobacteria are predominant in AS systems as described previously [23]. The sequences from each library revealed
different richness of higher taxa (Fig. 3(a)–(c)).
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Y. Qu et al. / FEMS Microbiology Letters 246 (2005) 143–149
The main aims of this study were to reveal community dynamics and the fate of strain QYY, therefore,
only the clones related to QYY were correlated to their
bands in the fingerprints. According to statistical analysis, the percentage of S. sp. was 26%, 20% and 43.75%
for library 7, 9 and 8, respectively. Some clones such
as 7–3, 7–10, 7–8, 9–4, 9–11 and seven clones from library 8 were closely related and identified as S. xenophaga QYY, with 99–100% identity to strain QYY 16S
rDNA sequence. These clones were isolated from each
library locating on the bands approximately 1.5 kb in
the fingerprint patterns as denoted by the arrows (Fig.
2(a)). Such results strongly suggested that strain QYY
became predominant and played an important role in
BAA degradation by a less complex community (Fig.
3(c)) after the ASI system treatment.
Judging from Fig. 3(a) and (b), S. styagia and S. adhaesiva appeared in the ASS-15 and ASS-28, respectively,
which was reported to possess the ability to degrade
polycyclic aromatic compounds [10,24]. Possible explanation was that removal of BAA by the suspended cells
might stimulate the metabolic activity of such microorganisms to some extent. Thus, the biomass of these
microorganisms would increase and become detectable.
Therefore, the form of inoculation played an important
role in microbial community structure. There was also a
minority of sequences closely affiliated with Actinobacteria, Flavobacteria, Clostridia, etc. These bacteria should
cooperate well with others, and contribute to removing
BAA in the same community.
The genus Sphingomonas is recognized as becoming
more efficient in degrading xenobiotic compounds.
Therefore, bioaugmentation with bacteria of this genus
(e.g., strain QYY) may have a bright application future.
Acknowledgments
We thank Post Dr. Dengdi An at the Institute of
Microbiology, Chinese academy of Science in Beijing,
P.R. China and Xiaodong Yuan at TaKaRa Biotechnology, Dalian Co., Ltd for their skillful technical
assistance.
This work was supported by Cross-Century Talent
Grants from the National Education Committee of
China.
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