Environmental Science and Engineering Program and Department of Civil Engineering, National University of Singapore, Singapore 117576
Correspondence
Wen-Tso Liu
cveliuwt{at}nus.edu.sg
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences reported in this paper are AY351635AY351641 and AY351643.
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INTRODUCTION |
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Culture-dependent and culture-independent molecular techniques have been used to identify GAOs. One postulated GAO that has been described is the coccobacillus-shaped organism seen in an acetate-fed deteriorated EBPR reactor (Liu et al., 1996), and phylogenetically placed in the bacterial lineage GB consisting of at least seven phylogenetic subgroups (Nielsen et al., 1999
; Kong et al., 2002b
), or Candidatus Competibacter phosphatis' (Crocetti et al., 2002
) in the Gammaproteobacteria. This group exhibits several metabolic traits similar to those proposed for GAOs (Nielsen et al., 1999
; Crocetti et al., 2002
), and appears to be widely distributed in laboratory- and full-scale EBPR processes (Crocetti et al., 2002
; Kong et al., 2002b
).
The tetrad-forming organisms (TFOs), which occur as clusters of four or more cells, are another hypothesized GAO (Cech & Hartman, 1993; Liu et al., 1996
; Tsai & Liu, 2002
). So far, TFOs dominate mainly in laboratory-scale EBPR systems fed with synthetic carbon sources. Culture-dependent studies have obtained several bacterial isolates with morphological traits resembling TFOs observed in EBPR systems. These isolates have been shown phylogenetically to be members of the Alphaproteobacteria (Amaricoccus spp., and Defluvicoccus vanus), Betaproteobacteria (Quadracoccus sp.), Gammaproteobacteria, and Actinobacteria (Tetrasphaera spp., Micropruina glycogenica and Kineosphaera limosa) (Maszenan et al., 1997
; Kong et al., 2001
; Shintani et al., 2000
; Hanada et al., 2002
; Liu et al., 2002a
). However, except for M. glycogenica, none of those isolates have been seen in abundance in either full- or laboratory-scale EBPR processes (Kong et al., 2001
; Seviour et al., 2003
). While the absence of these TFO isolates in EBPR systems remains unexplained (Seviour et al., 2003
), molecular approaches have detected several as-yet-unidentified TFOs from different major phylogenetic lineages dominating in laboratory-scale EBPR systems (Kong et al., 2001
, 2002a
; Levantesi et al., 2002
; Tsai & Liu, 2002
). These findings suggest that using morphological traits to identify members of a physiologically functional group like the GAOs is inappropriate. There needs to be an improvement in the current understanding of the microbial diversity and metabolic functions of TFOs, and their distribution and possible role as GAOs in systems designed to perform EBPR.
Membrane bioreactors (MBRs) have emerged as important wastewater treatment technologies because of their small footprint, high mass/liquid separation efficiencies and low sludge production and operational costs (i.e. high biomass concentration) (Stephenson et al., 2000). Applying MBRs to biological phosphorus removal is also promising (Adam et al., 2002
). Here, a sequencing MBR with anaerobic, aerobic and liquid-solid separation stages was established, but it failed to perform EBPR over an operation period of 260 days. Microscopic observation revealed that the microbial community was dominated by cells with a TFO morphology. This paper describes the in situ physiological traits of these TFOs, resolves their phylogenetic affiliation and diversity, and examines their occurrence in laboratory- and full-scale EBPR and non-EBPR systems.
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METHODS |
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Microscopy.
Epifluorescence microscopy and confocal laser scanning microscopy (CLSM) were both used for microscopic observations and staining methods. The epifluorescence microscope (model BX51, Olympus) was equipped with a cooled CCD camera SPOT-RT Slider (Diagnostic Instruments), a 100 W HBO bulb, and three different fluorescence filter sets (U-MWU2, U-MWB2 and U-MF2). The CLSM model LSM 5 Pascal (Carl Zeiss) was equipped with an inverted microscope, an argon-ion laser (458514 nm), two helium/neon lasers (543 nm and 633 nm), three Zeiss filter sets (01, 09 and 15), and different objective lenses (x20, x40, x63 and x100 oil-immersion). Image processing and analysis were performed with the software package provided by Zeiss, Metamorph (Universal Imagine) and Adobe Photoshop software (Adobe).
Chemical staining.
Neisser and Sudan black B staining procedures were used to confirm the presence of intracellular polyphosphate and PHA granules, respectively (Jenkins et al., 1993). Due to equipment setup, Nile blue A staining (Ostle & Holt, 1982
) combined with FISH was used in CLSM to detect accumulated intracellular PHA in microbial cells of interest.
DNA extraction and construction of the 16S rRNA gene clone library.
DNA from biomass samples was obtained by the protocol of Liu et al. (1997). Bacterial primers ALF1b, targeting the Alphaproteobacteria (Manz et al., 1992
), and EUB1512R, for the domain Bacteria (Kane et al., 1993
), were used to selectively amplify the 16S rRNA gene of the Alphaproteobacteria in the community DNA. PCR was performed with a Hybaid thermal cycler (Hybaid) as follows: an initial denaturation at 94 °C for 5 min; 30 cycles of denaturation (45 s at 94 °C), annealing (45 s at 55 °C) and extension (1 min at 72 °C); and a final extension at 72 °C for 5 min. After confirmation with electrophoresis on a 0·8 % (w/v) agarose gel, PCR products were used in the construction of the 16S rRNA gene clone libraries as reported previously (Liu et al., 2002b
). 16S rRNA gene sequences were analysed using an ABI model 377 automated sequencer (Applied Biosystems) and the Taq Dye-Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems).
Phylogenetic analysis of 16S rRNA gene clones.
The 16S rRNA gene sequences obtained were compared to GenBank using the NCBI BLAST program (Altschul et al., 1990), and also checked for chimeric artifacts with the CHECK_CHIMERA tool in the Ribosomal Database Project (RDP) (Maidak et al., 2001
). For phylogenetic analysis, sequences of those selected clones and closely related bacterial species were aligned with the CLUSTAL_W version 1.4 program (Thompson et al., 1994
) available in the BioEdit software package (Hall, 1999
). For phylogenetic analysis of cloned nucleotide sequences, a neighbour-joining tree (Saitou & Nei, 1987
) with the JukesCantor method was constructed (1000 replicate bootstraps) with the MEGA2 program (Kumar et al., 2001
).
FISH.
Fresh sludge samples were fixed in both paraformaldehyde and ethanol solutions for Gram-negative bacteria and Gram-positive bacteria, respectively, then washed with PBS, and stored in PBS/ethanol solution at 20 °C prior to further analysis (Manz et al., 1992; Roller et al., 1994
). Probes were commercially synthesized and 5'-labelled with FITC, Cy3 or Cy5 (MWG Biotech). Sludge samples were initially hybridized with the probe NON338 labelled with Cy3 to exclude nonspecific-probe-binding (Wallner et al., 1993
), and then analysed with the domain- and group-specific oligonucleotide probes listed in Table 1
to provide information on microbial community structure.
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Design and optimization of oligonucleotide probes.
16S rRNA-targeted oligonucleotide probes were designed using the probe design function in ARB (Ludwig et al., 2004). The specificity of these probes was confirmed against the Check Probe program in the Ribosomal Database Project (Maidak et al., 2001
), and optimized for FISH by determining the experimental dissociation temperature or the optimal formamide concentration against a reference strain (i.e. D. vanus strain NCIMB 13612) as described by Manz et al. (1992)
.
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RESULTS AND DISCUSSION |
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Figure 2(d) shows the CLSM-FISH image of the bacterial cells simultaneously hybridized by probes EUBmix (FITC-labelled, green) and ALF968 (Cy3-labelled, red). The bacterial cells that appear yellow (due to superimposition of red and green) represent members of the Alphaproteobacteria. From the Alphaproteobacteria at least two morphotypes of TFO by size were observed. The first, which resembled the one in Fig. 2(a)
(circle 4), contained large coccoid cells in clusters of 16 or more. The second, which resembled the TFO observed in Fig. 2(a)
(circle 2), appeared after FISH as flower buds, and differed from the first in cell size and in FISH staining response. Since the Alphaproteobacteria appeared to dominate the community, a 16S rRNA gene clone library was constructed for this subdivision to phylogenetically place the TFOs.
Phylogenetic diversity of TFOs in the Alphaproteobacteria
A total of 51 clones were selected from the clone library, screened, and classified into 11 different operational taxonomic units (OTUs) after full sequencing (1400 bp). Phylogeny analysis (Fig. 3
) showed that the majority of these clones (eight OTUs, 67 % of the total clones) were related to the Alphaproteobacteria. Five of those eight OTUs (61 % of the total clones) formed a lineage within the family Rhodospirillales of the Alphaproteobacteria. Of these five OTUs, four formed a tight cluster with Defluvicoccus vanus, isolated from a full-scale EBPR process (Kong et al., 2002a
, b
), with a sequence similarity of 92·697·2 %. Within this Defluvicoccus cluster, OTU TFOa28 (47 % of the total clones) represented the most dominant OTU or population in the reactor. The remaining fifth OTU TFOa27, with low clone abundance in the lineage, was related to three environmental clones previously retrieved from a laboratory-scale EBPR reactor (McMaholm et al., 2002
). The other three Alphaproteobacteria-related OTUs not within this lineage were related to Paracoccus and Rhodobacter species, and Hyphomonas species (Fig. 3
). Finally, of the non-Alphaproteobacteria-related OTUs, one was identified as a chimeric sequence and the other two were related to the genus Prosthecobacter from the phylum Verrucomicrobia. It should be noted that the primer ALF1b used in clone construction is not very specific for Alphaproteobacteria, and the successful construction of the clones was attributed to the high abundance of the Alphaproteobacteria organisms as revealed through FISH analysis.
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Phylogenetic confirmation and PHA-accumulating traits of the Defluvicoccus-related TFOs
The CLSM-FISH results indicated that probes TFO_DF218 and TFO_618 could bind to approximately 86·893·2 % and 64·178·6 %, respectively, of all alphaproteobacterial cells in three biomass samples taken each 23 weeks over a period of two months. This suggests that the probe TFO_DF218 has a broader specificity than probe TFO_DF618 toward these TFOs. Probe TFO_DF862 gave no hybridization signals to any cells in these biomass samples, suggesting that D. vanus was not present in the reactor.
Fig. 4(a) shows a FISH image of a biomass sample taken at the end of anaerobic phase hybridized simultaneously with probes TFO_DF218 and TFO_DF618. At least two different FISH-positive TFOs were observed. One, in clusters of 46 cells, fluoresced with both probe TFO_DF618 (red) and probe TFO_DF218 (green) as yellow-coloured cells. The other type, which fluoresced only with probe TFO_DF218 (green) and usually formed clusters of more than 16 cells, represented about 820 % of the bacterial cells in the reactor community. These differences in probe responses suggested the existence of other as-yet-uncultured populations within this Defluvicoccus cluster. Fig. 4(b)
shows the corresponding Nile blue A-stained image of the FISH-positive cells as shown in Fig. 4(a)
. All the Defluvicoccus-related TFOs reacted positively to Nile blue A stain but with different levels of fluorescence intensity, for example, among the TFO_DF218-positive, TFO_DF618-negative cells (indicated by arrows in Fig. 4b
). These observations suggested variation among the TFOs in their ability to accumulate PHA.
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Occurrence of Defluvicoccus-related TFOs in activated sludge treatment processes
The occurrence of these Defluvicoccus-related TFOs in biomass samples from the laboratory-scale and full-scale systems with or without EBPR activity that were studied in Kong et al. (2002b) was examined using FISH and the probes designed here. With the exception of the biomass sample taken from the bioreactor operated in this study, none of the other biomass samples contained a high percentage of their total cells as Defluvicoccus-related TFOs. Unlike the gammaproteobacterial lineage GB (Kong et al., 2002b
), this survey suggested that Defluvicoccus-related TFOs were not the dominant populations in laboratory- and full-scale EBPR systems or in conventional activated sludge processes. This difference could be due to the MBR system used here, where the biomass concentration and organic loading were much higher and lower, respectively, than other conventional gravity settling systems.
In fact, dominance of TFOs in EBPR processes has been reported mainly in laboratory-scale systems. Using light microscopy, Cech & Hartman (1993) and Liu et al. (1994)
observed the proliferation of TFOs in deteriorated laboratory-scale EBPR processes. Kong et al. (2001
, 2002a)
reported that diverse unidentified alphaproteobacterial TFOs represented more than 50 % of the total bacterial cells in a deteriorated EBPR reactor fed with acetate or a mixture of acetate and glucose. They did not detect any TFOs related to the genera Defluvicoccus and Amaricoccus in their systems, and suspected the existence of other novel TFOs in the Alphaproteobacteria. Likewise, Levantesi et al. (2002)
reported that TFOs other than Amaricoccus species from the Alphaproteobacteria were dominating (25 % of total bacterial cells) in an acetate-fed EBPR reactor, but whether the reported TFOs were related to the Defluvicoccus cluster revealed here remains to be validated. Thus, the role of the Defluvicoccus-related TFOs in the deterioration of full-scale EBPR processes was less clear than the role for members of the gammaproteobacterial lineage GB reported previously (Kong et al., 2002b
).
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Received 30 April 2004;
revised 12 July 2004;
accepted 13 July 2004.
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