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Appl Environ Microbiol, March 1998, p. 992-998, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Quinone Profiling of Bacterial Communities in Natural and
Synthetic Sewage Activated Sludge for Enhanced Phosphate
Removal
Akira
Hiraishi,1,2,*
Yoko
Ueda,2,
and
Junko
Ishihara3
Department of Ecological Engineering,
Toyohashi University of Technology, Toyohashi
441,1
Laboratory of Environmental
Biotechnology, Konishi Co., Tokyo 130,2 and
Department of Public Works, Shimane Prefecture, Matsue
690,3 Japan
Received 8 September 1997/Accepted 4 December 1997
 |
ABSTRACT |
Respiratory quinones were used as biomarkers to study bacterial
community structures in activated sludge reactors used for enhanced
biological phosphate removal (EBPR). We compared the quinone profiles
of EBPR sludges and standard sludges, of natural sewage and synthetic
sewage, and of plant scale and laboratory scale systems. Ubiquinone (Q)
and menaquinone (MK) components were detected in all sludges tested at
molar MK/Q ratios of 0.455 to 0.981. The differences in MK/Q ratios
were much larger when we compared different wastewater sludges (i.e.,
raw sewage and synthetic sewage) than when we compared sludges from the
EBPR and standard processes or plant scale and laboratory scale
systems. In all sludges tested a Q with eight isoprene units (Q-8) was the most abundant quinone. In the MK fraction, either tetrahydrogenated MK-8 or MK-7 was the predominant type, and there was also a significant proportion of MK-6 to MK-8 in most cases. A numerical cluster analysis
of the profiles showed that the sludges tested fell into two major
clusters; one included all raw sewage sludges, and the other consisted
of all synthetic sewage sludges, independent of the operational mode
and scale of the reactors and the phosphate accumulation. These data
suggested that Q-8-containing species belonging to the class
Proteobacteria (i.e., species belonging to the beta
subclass) were the major constituents of the bacterial populations in
the EBPR sludge, as well as in standard activated sludge. Members of
the class Actinobacteria (gram-positive bacteria with high
DNA G+C contents) were the second most abundant group in both types of
sludge. The bacterial community structures in activated sludge
processes may be affected more by the nature of the influent wastewater
than by the introduction of an anaerobic stage into the process or by
the scale of the reactors.
| |
INTRODUCTION |
Activated sludge processes with
changing anaerobic-oxic (AO) or anaerobic-aerobic conditions have been
used successfully for phosphate removal from wastewater. It is typical
in the enhanced biological phosphate removal (EBPR) process that the
sludge releases Pi (with concomitant uptake of wastewater
organic carbon) in the anaerobic phase and takes up Pi
rapidly in the aerobic stage. The Pi removed from
wastewater is accumulated as a form of polyphosphate (polyP) in the
sludge bacteria. Therefore, which phylogenetic and taxonomic groups of
bacteria are responsible for phosphate removal and polyP accumulation
has been and is still a subject of major concern for understanding the
EBPR mechanism and the control of this process (for reviews see
references 28 and 31).
Previous studies performed with conventional cultural methods suggested
that Acinetobacter species predominate and/or play an
important role in the EBPR process (6, 11, 12, 33, 45).
However, none of Acinetobacter isolates tested exhibited characteristics that are consistent with the biochemical model of this
process (13, 37). Some other species of bacteria were also
isolated and identified as possible phosphate removers in the
anaerobic-aerobic system (5, 18, 29, 33, 46, 47). Recently,
a new polyP-accumulating gram-positive bacterium named Microlunatus phosphovorus was isolated from an EBPR process
(36). To our knowledge, this bacterium is the first organism
to exhibit Pi release and uptake in response to changing
anaerobic-aerobic stages at the pure-culture level. Nevertheless, it is
difficult to reconstruct bacterial community structures in activated
sludge by studying only cultural information because of the well-known biases of culture-dependent methods, which may apply to only 1 to 15%
of the total population of the sludge (1) and may provide misleading information about community structure (48).
In recent years, attempts have been made to describe bacterial
communities in activated sludge systems by using non-culture-dependent chemotaxonomic and molecular methods (3, 4, 7, 15-19, 23, 24, 34,
43, 48, 49), and these approaches have provided data which
contradict the previous results obtained with laboratory cultivation
methods. Immunofluorescence (7) and quinone-profiling
studies (17, 18) have indicated that the numbers of
Acinetobacter cells are low in the EBPR process, as well as
in the standard activated sludge process. Similar results were obtained
with two molecular approaches (4, 49). One of these
approaches, rRNA-targeted in situ hybridization, showed that members of
the beta subclass of the Proteobacteria and gram-positive bacteria with high DNA G+C contents (now classified as the class Actinobacteria [44]) were numerically
abundant in the EBPR system (49). The other approach
involved PCR cloning and sequencing of environmental 16S rRNA genes,
and this approach also showed that members of the beta subclass of the
Proteobacteria were the major population constituents in
this system (4).
Although rRNA approaches have become common in this area of study, the
information obtained with these methods is still uncertain and somewhat
different depending on the method used. This may be due in part to
technical problems specific to the molecular methods, including
problems with DNA retrieval, PCR bias, hybridization efficiency, and
imposed selection of the retrieved or target sequences. For example,
quinone pattern analyses have shown that partially saturated
menaquinones (MKs), which are good biomarkers of the class
Actinobacteria, usually constitute more than 20% of the total quinone content in plant scale sewage sludge (19, 23, 24), whereas 16S ribosomal DNA (rDNA) clone library approaches appear to underestimate the numbers of these bacteria (4,
43).
Previously, we used the quinone profile method to characterize
bacterial community structures in the EBPR process as noted above
(17) because of its simplicity and high reproducibility as a
culture-independent technique. However, our previous research had a
weakness; namely, a laboratory scale anaerobic-aerobic system fed with
synthetic wastewater was the only system studied. Therefore, this study
was designed to reexamine bacterial community structures in both plant
scale and laboratory scale activated sludge reactors for EBPR by using
respiratory quinone profiles. We report here that there were small
differences in quinone profiles (i.e., community structures) between
the EBPR and standard activated sludge systems. The usefulness of
quinone profiling as a non-culture-dependent tool for quantitative
evaluation of population shifts over time and space is also discussed.
| |
MATERIALS AND METHODS |
Sludge samples.
All of the activated sludge samples tested
are listed in Table 1. Four EBPR sludge
samples were collected from a main aeration basin used for the AO
process in the Shinjiko-tobu sewage treatment plant (24) in
Matsue, Japan, from July to December 1992; these samples were
designated P-AO1, P-AO2, P-AO3, and P-AO4. Plant scale standard
activated sludge, designated P-St, was collected from a sewage
treatment plant in Chiba Prefecture, Japan. All plant sludge samples
were placed in sterile polyethylene bottles, transported to the
laboratory at 
20°C, and stored at 80°C until analysis. For
comparison, activated sludge cultivated in our laboratory was used. The
laboratory system, which consisted of four jar fermentors with
temperature and dissolved oxygen controllers, was seeded with standard
sludge from the Chiba plant and was operated on a fill-and-draw basis
with a 24-h batch cycle as described previously (18). The
level of mixed liquor suspended solids (MLSS) was adjusted to 700 to
900 mg/liter every day. The reactors were fed either with raw sewage
taken from the plant or with synthetic sewage (15, 23) at a
biological oxygen demand loading rate of 220 to 300 mg/g of MLSS/day.
The synthetic sewage was composed of (per liter of tap water) 3.0 g of Polypeptone (Daigo, Tokyo, Japan), 3.0 g of meat extract
(Kyokuto, Tokyo, Japan), 3.0 g of anhydrous sodium acetate,
1.0 g of (NH4)2SO4, 1.0 g
of KH2PO4, 1.0 g of
K2HPO4, 0.2 g of MgCl2
· 6H2O, and 0.1 g of CaCl2 · 2H2O (pH 7.0); it was diluted with tap water at a given
biological oxygen demand loading rate before feeding. The four reactors
were operated as an AO system with raw sewage (L-AO/RS), an AO system with synthetic sewage (L-AO/SS), a standard system with raw sewage (L-St/RS), and a standard system with synthetic sewage (L-St/SS). After
5 weeks of acclimation, sludge samples were removed from the reactors
and analyzed.
Extraction and fractionation of quinones.
Sludge was
harvested by centrifugation (12,000 × g, 10 min),
washed with 50 mM phosphate buffer (pH 6.8) containing 1 mM
ferricyanide, and resuspended in this buffer by using a total volume of
10 ml. Quinones were extracted three times with 2.5 volumes of a
chloroform-methanol mixture (2:1, vol/vol), evaporated in a vacuum, and
reextracted three times with n-hexane-water (1:1, vol/vol).
Then, the crude quinone extract in n-hexane was concentrated
and applied to a Sep-Pak Plus Silica column (Waters Corp., Milford,
Mass.). MK and ubiquinone (Q) fractions were eluted with 20 ml of
n-hexane-diethyl ether (98:2, vol/vol) and then with 20 ml
of n-hexane-diethyl ether (90:10, vol/vol), respectively.
The presence of MKs and Qs in both fractions was confirmed by silica
gel thin-layer chromatography and UV light detection prior to
high-performance liquid chromatography (HPLC) assays. Detailed
information concerning the procedure used for quinone extraction and
fractionation has been given elsewhere (19, 24).
Identification and quantification of quinones.
Quinone
components were separated and identified by reverse-phase HPLC and
photodiode array detection with internal and external standard
quinones. The analytical system used has been described previously
(24). Silver ion-modified thin-layer chromatography (20) and mass spectroscopy (8) performed with a
Shimadzu model QP-2000 mass spectrometer were also used as
supplementary tools for quinone analysis. Standard Qs and phylloquinone
(K1) were obtained from Sigma Chemical Co. (St. Louis,
Mo.). MK standards were prepared from known species of bacteria
(15, 19). Below, Qs, rhodoquinones (RQs), and MKs with
n isoprene units in their side chains are designated
Q-n, RQ-n, and MK-n, respectively
(Fig. 1 shows chemical structures).
Partially hydrogenated MKs are designated MK-n(Hx), where x
indicates the number of hydrogen atoms saturating the side chain.
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FIG. 1.
Four quinone structural classes found in activated
sludge. (a) Q-n. (b) RQ-n. (c) MK-n.
(d) K1.
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|
Numerical analysis.
To indicate dissimilarities among sludge
samples on the basis of quinone profiles, a dissimilarity index
(D) was calculated by the overlap method as described
previously (19). This parameter was defined as follows:
where
xik = xjk = 100
and
xik and
xjk indicate
the percentages of quinone homolog
k in samples
i
and
j, respectively.
Another parameter, the microbial divergence index based on quinone
profiles (
MDq) (
23,
26), was also
used to show the
degree of microbial divergence of sludge. This
parameter was defined
as follows:
where
xk 
0.001, and
xk indicates the molar ratio of quinone homolog
k to the total quinone content.
Calculation of
D and
MDq values,
tabulation of
D value matrix data, and construction of a
dendrogram for clustering were performed
with a program written by us
(
27) by using the Microsoft Visual
Basic programming system.
The algorithm of the neighbor-joining
method (
41) was used
for dendrogram construction. This method
has been shown to give more
accurate topography of dendrograms
than the group average linkage
method (
23), which was used previously
for clustering
quinone patterns (
19).
Other analytical methods.
Total cell counts were determined
by epifluorescence microscopy performed with membrane filtration and
staining with 4',6-diamidino-2-phenylindole as described previously
(39, 49). The phosphorus compounds in sludge were extracted
and fractionated by the method of Langen et al. (32) with
small modifications, and the acid-hydrolyzed Pi in each
fraction was measured colorimetrically as described previously
(25). MLSS and volatile solids in MLSS (VSS) were determined
by using standard methods (2).
| |
RESULTS |
Phosphorus and quinone contents.
The phosphorus and quinone
contents of all sludges tested, together with information on the
processes from which the sludges were collected, are shown in Table 1.
All of the EBPR sludges from the plants and the laboratory except
sample P-AO4 contained high amounts of phosphorus (range, 1.35 to 1.82 µmol per mg of MLSS), whereas the phosphorus contents of the standard
sludges were 0.58 to 0.67 µmol/mg of MLSS. The phosphorus contents of the EBPR sludges corresponded to 4.6 to 6.1% of the dry weight. More
than 50% of the total phosphorus contents of the EBPR sludges was
included in the alkali- and hot acid-soluble polyP fractions (data not
shown). These data suggested that all of the EBPR sludges tested except
P-AO4 had actually worked as phosphate removal systems.
All of the sludges tested contained both Qs and MKs at MK/Q molar
ratios of 0.455 to 0.981. The plant sludges also contained
much lower
but appreciable amounts of RQs, which are derivatives
of Qs. We
observed no marked differences in quinone contents and
MK/Q ratios
between the EBPR and standard sludges. On the other
hand, there were
significant differences in MK/Q ratios between
the sludges loaded with
raw sewage and synthetic sewage.
Relationships between biomass and quinone contents.
The total
cell counts in the aeration basins as measured by fluorescence
microscopy ranged from 1.0 × 109 to 3.0 × 109 cells/ml. The concentrations of MLSS determined were
700 to 2,430 mg/liter, and VSS accounted for 72 to 91% of the MLSS
(data not shown). There was a positive relationship among the total
quinone concentration, the total cell count, the MLSS content, and the VSS content in the aeration basins. Examples of the relationships between the total quinone concentration and the total cell count or VSS
content are shown in Fig. 2; the
correlation coefficients (r) determined for the former and
latter relationships were 0.765 and 0.990, respectively. The regression
equations derived from these relationships indicated that 1 nmol of
quinones corresponded to 1.3 × 109 cells and 0.8 mg
of VSS. The weaker direct relationship between quinone content and
total cell count was probably due to the effect of dispersion and
dilution of sludge flocs during cell counting.
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FIG. 2.
Relationships between total quinone contents and total
cell counts (a) or VSS (b) in aeration basins. The correlation
coefficients (r) for the former and latter relationships are
0.765 and 0.990, respectively (n = 9).
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|
Quinone composition.
The quinone compositions of all sludges
tested, as determined by HPLC, are summarized in Table
2. In the Q fraction, Q-8 predominated,
Q-10 was the second most common type, and Q-9 and other homologs were
minor components in all test sludges. Also, Q-8 was the most abundant
type in the total quinone (35 to 50% of the total). Small proportions
of RQs, mainly RQ-8, as confirmed by mass spectrometry (M+
at m/z 712), were detected in all plant scale sludges. In
the MK fraction, either MK-7 or MK-8(H4) predominated and
there were significant proportions of MK-6 and MK-8 in the plant scale
sludges. In the laboratory sludges, MK-8(H4) was
predominant and MK-7 was the second most common type.
Numerical analysis.
The differences in quinone profiles
among the samples tested were evaluated quantitatively by calculating
the D and MDq values (Table
3). The D values when
the sludges were compared ranged from 5.1 to 24.2%. The D
values were relatively low (5.1 to 13.8%) for the sludges from the
same wastewater type (i.e., sludges with raw sewage or with synthetic
sewage), independent of the mode of operation (EBPR versus standard),
the scale of the reactors (plant scale versus laboratory scale), and
the phosphate accumulation. The values increased to 14.3 to 24.2%
(mostly >20%) for the raw sewage and synthetic sewage sludges. The
MDq values were similar (range, 12.4 to 13.8)
for the raw sewage sludges, whereas the MDq
values were lower (7.9 to 8.3) for the synthetic sewage sludges, regardless of the mode of operation and the scale of the reactors.
Based on the
D matrix data shown in Table
3, a dendrogram
grouping the sludges tested was constructed by using the algorithm
of
the neighbor-joining method (Fig.
3). The
sludges tested were
divided into two major clusters, one of which
consisted of all
of the EBPR and standard sludges containing raw sewage
and one
of which included all laboratory sludges fed with synthetic
sewage.
Thus, the topology of the dendrogram was independent of the
capacity
of sludge for phosphate accumulation. Within the raw sewage
cluster,
the plant and laboratory sludges overlapped each other.
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FIG. 3.
Dendrogram grouping the sludges tested based on
D value matrix data. The dendrogram was constructed by using
the algorithm of the neighbor-joining method. The two major clusters
for raw sewage sludges and synthetic sewage sludges are surrounded by
lines.
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|
When the quinone profile data reported previously for standard and EBPR
sludges fed with the synthetic sewage (
18,
23)
were
incorporated into the numerical analysis, all of these sludges
fell
into the synthetic sewage cluster described above at a dissimilarity
level of less than 13% (data not shown).
Assignment of different quinone producers to phylogenetic
taxa.
The distributions of different quinone homologs in the EBPR
sludges (based on the data in Table 1) are illustrated in Fig. 4, and these quinones were assigned to
phylogenetic taxa (bacteria that may have been present) on the basis of
the available chemotaxonomic information. Q-8 was assigned to the beta
subclass of the Proteobacteria and some members of the gamma
subclass; Q-9 was assigned members of the gamma subclass, such as
members of the genera Acinetobacter and
Pseudomonas; and Q-10 was assigned to the alpha subclass of the Proteobacteria (50). RQ-8 might have been
derived from the second quinone component of certain members of the
beta subclass, such as Brachymonas (21) and
Zoogloea (22) species. MKs with short isoprene
units (MK-6 to MK-8) were assigned to the
Flavobacterium-Cytophaga group (35, 38), the
gram-positive bacteria with low G+C contents (9), the genus
Planctomyces (42), and the delta subclass of the
Proteobacteria (9, 10), and MKs with longer side
chains or partially hydrogenated chains were assigned to the class
Actinobacteria. Partially saturated MKs are distributed in
some species of the sulfate-reducing proteobacteria (10).
However, since these MKs are limited to MKs with short isoprene units
(less than seven units long), most of the partially hydrogenated MKs
found in the sludges could be assigned to the class
Actinobacteria.
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FIG. 4.
Distribution of different quinone homologs in EBPR
sludges and assignment of these quinones to their possible sources
(phylogenetic groups of bacteria). Symbols:  , average data for the
plant EBPR sludges with high phosphate contents (P-AO1, P-AO2, and
P-AO3);  , data for the laboratory EBPR sludge fed with synthetic
sewage (L-AO/SS). Cb, cyanobacteria; F/C,
Flavobacterium-Cytophaga cluster in the
Cytophaga-Bacteroides phylum. Pm, Planctomyces.
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| |
DISCUSSION |
Advances in microbial chemotaxonomy based on quinone patterns
(8, 9) provide a basis for the idea that quinone profiling of an environmental sample yields insight into the proportions of
species with different homolog types in the total population. One of
the major problems that complicate the assessment of in situ bacterial
populations by quinone profiling is that the quinone contents of
bacterial cells vary among taxa or in response to environmental stress.
A previous study showed that the ratio of total quinones to
biomass as measured by membrane lipid phosphate was different under
different environmental conditions (14). In this study,
however, we found that the total quinone content was highly correlated
with the biomass, as indicated by the VSS data for activated sludge
systems (Fig. 2). Another research group has also demonstrated that
there is a positive relationship between quinone content and
biomass in soil (40). Biomarker approaches, including
quinone profiling, in general suffer from another problem, namely, that only information concerning culturable
bacteria can be considered when a community structure is
interpreted, even if numerous unknown bacteria are present.
Nevertheless, this is not a problem in cluster analyses of microbial
communities based on numerically processed quinone data.
Our previous studies have shown that the quinone patterns of municipal
sewage activated sludges are similar to each other (15-19, 23,
24). The general quinone profiles of these sludges are as
follows: (i) Qs and MKs are present at MK/Q ratios of 0.6 to 1.0 in
most cases (but the MK/Q ratios are 0.3 to 0.6 for synthetic sewage
sludges); (ii) Q-8 is the most abundant type of quinone; (iii) the
proportion of Q homologs decreases in the order Q-8 > Q-10 > Q-9 > other Qs; and (iv) the predominant MK type is either MK-7, MK-8, or MK-8(H4) [in some cases, MK-6 or
MK-9(H8)]. The results of this study are consistent with
the general information noted above and also support our previous data
obtained for laboratory scale EBPR activated sludge with synthetic
sewage (17).
In view of the present data, together with the previous findings, it is
clear that most of the bacteria in both EBPR and standard activated
sludge systems are members of the Proteobacteria and that Q-8-producing species (i.e., species belonging to the beta subclass) are the most abundant species (>30% of the total
population) (Fig. 4). Some species belonging to the gamma subclass,
such as the enterobacterial species, also contain Q-8 as a major
quinone. However, the contribution of these bacteria to the
Q-8-producing population may be negligible, because
demethylmenaquinone, another indicator of the enterobacteria, did not
occur in appreciable amounts in any sludge. The low Q-9 contents in all
sludges suggest that Acinetobacter and
Pseudomonas species, which are representatives of the gamma
subclass, constitute minor populations (less than 4% of the total
population). Members of the alpha subclass of the
Proteobacteria may constitute about 10% of the total
population, as judged from the Q-10 content. These results agree well
with the results of rRNA in situ hybridization and 16S rDNA clone
library studies, all of which have indicated that members of the beta subclass of the Proteobacteria are predominant and minor
populations of Acinetobacter strains occur in EBPR and
standard systems (4, 30, 43, 48, 49). The finding that MKs
with long isoprene units (n 
10) and with partially
saturated chains were present at relatively high levels (>18%)
suggests that the Actinobacteria is the most abundant
phylogenetic group next to the beta subclass of the
Proteobacteria. A high proportion of members of the
Actinobacteria in an EBPR process has also been revealed by
rRNA-targeted oligonucleotide probing (49), whereas 16S rRNA
clone library studies failed to detect this phylogenetic group as a
major population component (4, 43). It has been suggested
that the polyP-accumulating bacterium M. phosphovorus, which
contains MK-9(H4) as its sole quinone (36),
is only a minor component of the populations in EBPR sludges if it is
present at all, in view of the low MK-9(H4) contents of
these sludges (less than 3%). A similar result was obtained by dual
staining of EBPR sludge with rRNA-targeted probes and a polyP-specific
fluorescent dye (30). The MK profiles of the sludges also
suggest that bacteria containing MK-6 to MK-8 (e.g.,
bacteria in the Cytophaga-Flavobacterium group,
Planctomyces strains, and/or gram-positive
bacteria with low G+C contents) are present at significant levels in
both EBPR and standard processes. The presence of these phylogenetic
groups in the EBPR process was demonstrated by a 16S rDNA clone library
study (4). In view of the quinone profiles of the EBPR
sludge, it is necessary to consider a number of species of at least two
phylogenetic groups, the Proteobacteria and the
Actinobacteria, as possible phosphate removers.
Numerical analyses of quinone data indicated that there were small
differences in the profiles of EBPR and standard activated sludges fed
with the same type of wastewater (Table 3). The levels of dissimilarity
between the two processes, as indicated by the D values,
were 5.1 to 13.8%. Previously, it has been shown that seasonal
variations in the D values in a sewage activated sludge treatment plant are less than 20%, if the plant is operated under normal conditions (24). Similar variations in D
values have also been found among sewage sludges in different plants
which are operating under good conditions (23). Therefore,
the dissimilarities between the EBPR and standard processes for the
same wastewater type may correspond to (or be smaller than) the
dissimilarities found seasonally in the same plant or the
dissimilarities among different sewage treatment plants. In contrast,
the levels of dissimilarity between the sludges loaded with different
types of wastewater (i.e., natural sewage versus synthetic sewage) were higher (mostly >20%), independent of the operational mode, the scale
of the reactors, and the phosphate accumulation. The neighbor-joining dendrogram based on the D value matrix data indicated that
the sludges tested fell into two major clusters depending on the type of wastewater (i.e., there was a raw sewage cluster and a synthetic sewage cluster). Within the raw sewage cluster, the EBPR and standard sludges or the plant scale and laboratory scale sludges overlapped each
other. These findings suggest that the introduction of an anaerobic
stage into the aerobic process results in no more significant population shift than changes in the quality of wastewater. Also, the
scale of reactors may have no or little effect on the community structure as long as the quality and loading rate of wastewater are
constant.
The natural and synthetic sewage EBPR sludges were almost identical in
their qualitative quinone patterns (Fig. 4). However, the quantitative
quinone profiles of the two groups of sludge differed significantly, as
indicated by the D and MDq values. This may be explained by the possibility that similar genera or species
of phosphate-accumulating bacteria play the major role in both EBPR
systems but the proportions of the individual populations in the total
population differ in the two systems. Alternatively, it is possible to
speculate that the same quinone-containing species but different
species of phosphate-accumulating bacteria are present in the two EBPR
systems; this would affect the whole community structure
differentially, resulting in different levels of microbial divergence in the two systems. Although phosphate removal can be
effectively achieved by the laboratory scale synthetic sewage EBPR process, as well as by the plant scale system, it is our view that
the former system should not be considered a model of the latter with
respect to bacterial population structure.
Quinone profiling has gained general acceptance as a biomarker
approach for characterizing in situ bacterial communities but has
received less attention as a culture-independent tool than molecular
techniques that now enjoy widespread use in wastewater microbiology and
microbial ecology. Certainly, the quinone profile method is inferior to
the rRNA approaches for resolving phylogenetic taxa. However, since
this biomarker method is a direct chemical analysis method for
environmental lipids, it provides higher reproducibility and
reliability without any bias based on extraction and identification of
the molecules. It also provides quantitative data useful for grouping
whole microbial populations in situ. A neighbor-joining dendrogram
inferred from quinone-based D value matrix data is useful
for quantitative evaluation of microbial population shifts over time
and space, as reported here. A combination of the quinone profile
method with molecular and ecophysiological techniques should
provide better understanding of the EBPR process.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Ecological Engineering, Toyohashi University of Technology,
Tenpaku-cho, Toyohashi 441, Japan. Phone: 81-532-44-6913. Fax:
81-532-44-6929. E-mail: hiraishi{at}eco.tut.ac.jp.
Present address: Tama Laboratory, Japan Food Research Laboratories,
Tama 206, Japan.
| |
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Abstract
Introduction
