Biotechnology Research Centre, La Trobe University, Bendigo, Victoria 3552, Australia
Correspondence
Robert J. Seviour
r.seviour{at}latrobe.edu.au
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ABSTRACT |
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Present address: Dept of Environmental Engineering, Aalborg University, DK-9000, Aalborg, Denmark.
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INTRODUCTION |
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EBPR systems are notoriously unreliable (Seviour et al., 2003) and their performance often deteriorates for no clear reason. Not all explanations for such events allow for any role of microbes in process failure (Schönborn et al., 2001
) and many that do usually lack any convincing supporting microbiological evidence (Filipe et al., 2001
; Whang & Park 2002
). Cech & Hartman (1993)
noticed large numbers of cocci in distinctive tetrads, a morphotype they called the G-bacteria, in a reactor showing poor EBPR capacity that was fed glucose. These cocci have since been called tetrad-forming organisms or TFO (Tsai & Liu 2002
). The original proposal, which has received support (reviewed by Seviour et al., 2000
, 2003
) was that these TFO were out-competing the PAO by assimilating substrates in the anaerobic zone, utilizing them for PHA production, but with no concomitant P release. This stored PHA supported their growth in the aerobic zone, but now with glycogen accumulation instead of polyP storage. Consequently, these populations have been referred to as the glycogen-accumulating organisms (GAO) (Liu et al., 1997
; Bond et al., 1999
; Crocetti et al., 2002
).
However, the precise identity of these GAO is still largely unknown. Whether some of the TFO morphotype, now known from cultured isolates to be phylogenetically diverse (Seviour et al., 2000), are GAO is also unclear. Isolates of TFO from a reactor showing poor EBPR capacity by Cech & Hartman (1993)
were identified as members of an
-proteobacterial genus, Amaricoccus (Maszenan et al., 1997
), which in pure culture failed to synthesize polyP aerobically. While they could synthesize glycogen aerobically, they did not assimilate either acetate or glucose anaerobically (Falvo et al., 2001
), and PHA synthesis occurred aerobically but not anaerobically. These are not physiological properties expected of a GAO (Hesselmann et al., 1999
; Crocetti et al., 2002
). However, it is possible that Amaricoccus spp. were not the organisms directly responsible for the poor EBPR seen by Cech & Hartman (1993)
.
Large coccobacilli have often been seen in reactors with low EBPR capacity, and from fluorescence in situ hybridization (FISH) these appeared to be -Proteobacteria (Bond et al., 1999
). However, considerable molecular data (Hesselmann et al., 1999
; Nielsen et al., 1999
; Liu et al., 2000
; Dabert et al., 2001
; Crocetti et al., 2002
; Kong et al., 2002b
) suggest that these coccobacilli are all phylogenetically closely related members of the
-Proteobacteria. This conflict has since been shown to arise from the original design of the probes and their target sites, which differ only by a single nucleotide for these two subdivisions of the Proteobacteria (Yeates et al., 2003
). Crocetti et al. (2002)
and Levantesi et al. (2002)
both considered that these
-proteobacterial coccobacilli were GAO, a conclusion based not on any direct demonstration of in situ glycogen storage in these populations, but on their abilities to store PHA anaerobically and not aerobically, and inabilities to store polyP either anaerobically or aerobically (Hesselmann et al., 1999
), as detected by staining after FISH probing. They called these Candidatus Competibacter phosphatis, implying that these were the populations which were competing with the PAO, and hence those responsible for EBPR deterioration (Crocetti et al., 2002
).
However, these -Proteobacteria are sometimes found in communities with high EPBR capacity too (Liu et al., 2000
) and do not always represent major populations in systems with low EBPR capacity and biomasses with high glycogen contents (Kong et al., 2001
, 2002a
). Furthermore, Levantesi et al. (2002)
and Crocetti et al. (2002)
detected large numbers of
-proteobacterial TFO in an EBPR community. They were not Amaricoccus spp. by FISH probing, but their identity and possible role as GAO were not pursued further, even though some, like the putative
-Proteobacteria GAO discussed above, stained for PHA when grown under anaerobic but not aerobic conditions.
Earlier, Kong et al. (2001) detailed the structurefunction relationships in several sequencing batch reactor (SBR) communities, showing that their community with no EBPR capacity was dominated by large
-proteobacterial TFO, as well as Gram-positive bacteria. Among these, Micropruina glycogenica (Shintani et al., 2000
) was held responsible for the high glycogen content of the biomass (Kong et al., 2001
). In another SBR biomass with very low EBPR capacity and high glycogen content (Kong et al., 2002a
), FISH data showed the community was again dominated by
-proteobacterial TFO. Very few
-Proteobacteria or Gram-positive bacteria, which dominated communities with high EBPR capacity, were seen. However, the
-proteobacterial populations could not be identified, and neither denaturing gradient gel electrophoresis (DGGE) profiles nor clone libraries constructed from these biomasses using universal 16S rRNA gene PCR primers revealed
-Proteobacteria in proportions commensurate with the FISH data (Kong et al., 2001
, 2002a
). The possibility of bias in the DNA extraction from these communities was discussed as a possible reason for this (Kong et al., 2001
).
This paper provides in situ evidence to support the view that the putative GAO in these communities are previously undescribed members of the genus Sphingomonas in the -Proteobacteria. It also suggests that the putative
-proteobacterial GAO reported elsewhere (Crocetti et al., 2002
; Kong et al., 2002b
) are not significant there.
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METHODS |
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Community fingerprinting with DGGE profiling.
Plasmids extracted from host cells of the clone libraries and the genomic DNA originating from each SBR community were both analysed by DGGE profiling, following the experimental approach detailed by Kong et al. (2001). The aim was to preferentially amplify the populations of the
-Proteobacteria which might be present in the SBR biomasses. DGGE analyses of the cloned fragments were used to compare their migratory positions with the bands generated from the SBR community genomic DNA templates separated by DGGE, adopting basically the same approach as Kong et al. (2001)
. This method was chosen in preference to the more common procedure of excising individual DGGE bands for sequencing, which is known to be problematic (Heuer et al., 2001
). Only those cloned fragments which had identical migratory mobilities with the bands detected in DGGE profiles of genomic DNA from each community were then sequenced. A 3070 % gradient gel (8 % acrylamide) was chosen for these separations because, of the several screened, this gave the best resolution and sharpest bands with the large fragments (approx. 950 bp) generated from PCR with the ALF1b and 907r primers. This method of screening the clones was preferred over the more usual RFLP fingerprinting method (Wagner & Loy, 2002
), which does not permit such a direct elucidation of these relationships. Partial 16S rRNA gene sequences (approx. 950 bp) were obtained from most of the DNA cloned fragments with migratory properties corresponding to those genomic DNA fragments of interest from each SBR, allowing their tentative identification. No evidence was seen to suggest that any of these large fragments remained undenatured and moved towards the bottom of the gels.
FISH probe design.
The clone sequence data from identified separated DGGE DNA fragments from the SBR communities were then used to design 16S rRNA targeted oligonucleotide probes against populations of the -Proteobacteria detected in each, with programs available through BioManager (ANGIS). Candidate probe sequences were generated with the software package Prime (Genetics Computer Group, GCG) and the likely specificity of each was then assessed with BLASTN (Altschul et al., 1997
). The probe sequences for the
-Proteobacteria described here are given in Table 2
. As it was not possible to validate these probes against pure cultures of the
-Proteobacteria of interest, biomass samples corresponding to the SBR source of each clone were used instead. The strength of fluorescent signal and specificity of each of the probes, as judged from the morphology of the cells fluorescing with each, was compared over a range of formamide concentrations (from 0 to 40 % in 5 % steps) and the optimum formamide concentration was then selected (Weller et al., 2000
; Crocetti et al., 2002
). It is understood that this approach may mean that other unsequenced bacteria in different biomass samples may respond positively to the probes described here, especially since these probes were designed against only partial sequences of individual clones (i.e. only 950 bp). All probes were supplied commercially by ProOligo and labelled with either the Cy3 or Fluos fluorochrome.
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Staining of PHA and polyP in SBR biomasses.
Bacterial cells accumulating PHA in each SBR biomass were visualized with the staining protocol of Kitamura & Doi (1996). Briefly, fixed biomass samples from the end of the anaerobic phase were treated with nile blue A (100 mg l1) in absolute ethanol for 30 min. After microscopic examination, de-staining was carried out by immersion of slides in absolute ethanol for 30 min. PolyP accumulation in samples from the aerobic stages was detected with 4',6-diamidino-2-phenylyindole (DAPI) with the method of Liu et al. (2001)
. FISH analysis was carried out as previously described in this paper to identify the PHA- and polyP-accumulating populations.
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RESULTS |
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DGGE profiles of the ALF1-907r-generated DNA fragments from the communities in SBR6, 7, 8 and 9 are shown in Fig. 1. As expected from previous FISH and chemical analyses (Kong et al., 2001
, 2002a
), profiles obtained for SBR communities 7 and 8, both with very similar and high EBPR performances, are almost identical to each other, and substantially different to those from SBR communities 6 and 9, with little or no EBPR capacity. Most of the
-proteobacterial clone fragments in the SBR7 community profile were also seen in the community profile in SBR8. The SBR9-1 clone fragment was of particular interest. Although present in all communities profiled, it had a much greater fluorescent intensity on DGGE gels generated from the communities in SBR6 and 9, and especially the latter (Fig. 1
). This sequenced clone was only 92 % similar to its closest relative, an uncultured Sphingomonas sp. (AF181572). About 30 % of the clones screened from these two SBRs had identical DGGE migratory mobilities to the SBR9-1 clone.
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PHA and polyP production by the -Proteobacterial TFO
Sequential nile blue A staining and FISH showed that cells fluorescing with the SBR9-1a probe in biomass samples taken at the end of the anaerobic period from SBR6 and 9 contained PHA (Fig. 3d), while those taken under aerobic conditions contained neither PHA (Fig. 3e
) nor polyP (data not shown).
FISH probing of putative -proteobacterial GAO in SBR communities
When the FISH probes previously designed against the putative -proteobacterial GAO were applied to these SBR communities, barely detectable numbers of cells (<4 % of total cell area) fluoresced with any of the
-proteobacterial GAO probes (Table 2
) of Crocetti et al. (2002)
and Kong et al. (2002b)
in any of the communities. Those few cells that did were large (>1·5 µm diam.) coccobacilli, often in pairs or loose clusters, consistent with their reported appearance in other activated sludge biomass samples (Crocetti et al., 2000
, 2002
; Kong et al., 2002b
). These data suggest that these putative
-proteobacterial GAO were minor populations there and so were probably not mainly responsible for the marked levels of glycogen synthesis detected, especially in the SBR6 and 9 communities.
Occurrence of putative GAO in full-scale EBPR plants
When the FISH SBR9-1a probe was applied to biomass samples taken from 10 full-scale EBPR plants from eastern states of Australia, very few contained this population, as might be expected of the communities in plants with high EBPR capacity, as these were (M. Beer, unpublished). The one exception was the Bendigo plant, Victoria, where >5±3 % of the total cell area responding to the EUBmix probe fluoresced with it. Again the cells appeared as typical TFO but scattered throughout the flocs (Fig. 3f) and none stained positively for polyP with DAPI (Liu et al., 2001
). This plant often shows erratic EBPR performance, although its biomass glycogen levels were not estimated here. Only small numbers of cells (<1 % of total cell area) responding to the
-proteobacterial GAO probes were detected by FISH in biomasses from these plants, as might be expected, and in most none were seen (data not shown).
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DISCUSSION |
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Most attempts, including ours, to grow any hypothesized GAO have been unsuccessful (Kong et al., 2001; Crocetti et al., 2002
). With the possible exception of M. glycogenica (Shintani et al., 2000
), other likely candidates obtainable in pure culture, like Amaricoccus spp., appear to behave differently in terms of their PHA formation to that expected of GAO (Falvo et al., 2001
). Putative GAO have more often been selected initially on identifying the cell morphotype which dominates a biomass showing deteriorating EBPR capacity and/or a high glycogen content (Kong et al., 2001, 2002b
; Crocetti et al., 2002
). Subsequent culture-independent 16S rRNA-based methods have then been used to try to relate the identity of these dominating morphotypes to their abilities to synthesize PHA and polyP anaerobically and aerobically.
This is essentially the method adopted in this study. The in situ physiological evidence presented here is as convincing as that from other similar studies, suggesting that the probable main candidates for GAO in the SBR9 community at least are likely to be members of the -Proteobacteria, most closely related to Sphingomonas spp (i.e. clone SBR9-1). These are phylogenetically different to the
-proteobacterial Amaricoccus spp. once considered as possible GAO candidates (Seviour et al., 2000
). Sphingomonas spp. can be abundant populations in activated sludge communities and cells appear to form large aggregates within the flocs. However, tetrad cell arrangements have not yet been reported in activated sludge for members of this genus (Neef et al., 1999
; Snaidr et al., 1997
).
No convincing evidence was found to support an important role here in glycogen accumulation for the -Proteobacteria proposed by Crocetti et al. (2002)
as putative GAO in their communities. Although the percentage of the total cell area of
-Proteobacteria increased markedly to approximately 8 % in the SBR9 biomass with a glycogen content of about 17 % (w/w) dry weight, very few cells were detected by FISH in this study with the
-proteobacterial GAO probes of either Crocetti et al. (2002)
or Kong et al. (2002b)
.
In summary, the TFO responding to the SBR9-1a and SBR9-1b probes seems a highly likely candidate as a GAO, dominating the SBR9 biomass characterized by a high glycogen and low P content (Kong et al., 2002a). These cells were also numerically significant in the SBR6 community where M. glycogenica had been suggested previously as the major possible GAO (Kong et al., 2001
). It seems unlikely that all the GAO are representatives of a single population and, like the PAO now appear to be, are probably phylogenetically very diverse (Crocetti et al., 2002
; Eschenhagen et al., 2003
). The evidence presented here supports this. The availability of the FISH probe sequences for the SBR9-1 clone should now provide the opportunity to determine how widespread these
-Proteobacteria are, especially in full-scale plants where this study has shown they may exist, and whether, for example, they are the same or similar populations to the
-Proteobacteria seen in high numbers by Crocetti et al. (2002)
in their community. However, it may be that these GAO do not always store glycogen under all conditions, which now seems to be the case with polyP and the Rhodocyclus-related PAO (Wagner & Loy, 2002
; Zilles et al., 2002
). If so, this will further complicate attempts to relate numerical dominance of particular morphotypes to their functional roles in biomasses displaying deteriorated EBPR.
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ACKNOWLEDGEMENTS |
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Received 10 October 2003;
revised 4 March 2004;
accepted 16 April 2004.
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