Advanced Wastewater Management Centre, The University of Queensland, St Lucia 4072, Australia1
Department of Geology and Geophysics, University of Wisconsin-Madison, Madison, WI 53706, USA2
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK3
Author for correspondence: Linda L. Blackall. Tel: +61 7 3365 4645. Fax: +61 7 3365 4699. e-mail: blackall{at}biosci.uq.edu.au
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ABSTRACT |
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Keywords: GAOs, fluorescence in situ hybridization (FISH), wastewater treatment, EBPR
Abbreviations: CLSM, confocal laser scanning microscope/microscopy; COD, chemical oxygen demand; EBPR, enhanced biological phosphorus removal; FISH, fluorescence in situ hybridization; GAO, glycogen-accumulating organism; OTU, operational taxonomic unit; PAO, polyphosphate-accumulating organism; PHA, poly-ß-hydroxyalkanoate; SBR, sequencing batch reactor; VFA, volatile fatty acid
b The GenBank accession numbers for the sequences reported in this paper are given in Methods.
a Present address: Department of Environmental Science, Policy and Management, Hilgard Hall, University of California Berkeley, Berkeley CA 94720, USA.
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INTRODUCTION |
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For EBPR to occur, the sludge must be mixed with wastewater influent under anaerobic conditions. The mixture is cycled through an anaerobic zone followed by an aerobic zone and a sludge settlement zone. Polyphosphate-accumulating organisms (PAOs) release P from stored polyphosphate in the anaerobic zone and accumulate P as polyphosphate in excess of normal metabolic requirements in the aerobic zone (van Loosdrecht et al., 1997b ). Removal of a portion of the growing biomass from the aerobic zone results in net removal of P from the wastewater. The carbon substrates utilized in EBPR are typically volatile fatty acids (VFAs) that are supplied to the sludge in the influent. PAOs take up the VFAs in the anaerobic zone and convert them to intracellular poly-ß-hydroxyalkanoates (PHAs) using stored glycogen and polyphosphate for energy. In the aerobic zone, PAOs utilize their stored PHA for cellular growth, replenishment of their glycogen and uptake of P. It is thought that anaerobic uptake of influent carbon substrates like VFAs and storage as PHA and the anaerobicaerobic polyphosphate cycling are the selective advantages for PAOs over other micro-organisms (van Loosdrecht et al., 1997a
). One hypothesis for failure of EBPR is that non-PAOs successfully compete against PAOs for influent soluble carbon. The non-PAOs were called glycogen-accumulating organisms (GAOs) (Mino et al., 1995
). GAOs have been reported to dominate deteriorated EBPR processes where glycogen and PHA transformations are similar to those during good EBPR but P is not transformed as in PAOs (e.g. Fukase et al., 1985
; Satoh et al., 1994
). Very little is known of GAOs apart from their phenotype.
The study of highly PAO-enriched laboratory-scale bioreactors allowed the understanding and identification of one confirmed PAO as Candidatus Accumulibacter phosphatis, a close relative of Rhodocyclus in the ß-Proteobacteria (Crocetti et al., 2000 ; Hesselmann et al., 1999
) (henceforth called Accumulibacter). Studies into GAOs have been directed towards their identification (Dabert et al., 2001
; Nielsen et al., 1999
). However, there remain gaps in the knowledge of the identity of GAOs and there has been little attempt to directly demonstrate that putative GAOs have the GAO phenotype. In this study, we designed fluorescence in situ hybridization (FISH) probes from 16S rDNA clones from sludges demonstrating the GAO phenotype and evaluated them with laboratory-scale and full-scale sludges by FISH and post-FISH chemical staining for the intracellular polymers PHA and polyphosphate. Thus we have identified one GAO by linking its in situ identification with the GAO phenotype.
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METHODS |
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To achieve anaerobic conditions, N2 gas was bubbled through the liquid and to achieve aerobic conditions, air was bubbled. Sludge was wasted in the aerobic zone of each cycle to achieve a sludge age of 78 days. The reactors were operated at a temperature of 22±2 °C. Regular weekly cycle studies (samples withdrawn each 1015 min from the reactor in the anaerobic and aerobic zones) were conducted to confirm the sludge phenotype. Samples were analysed for intracellular glycogen and PHA, and supernatant P, COD and VFA. The sludge P content (P%) was also determined on the biomass collected at the end of the aerobic period when the stored polyphosphate would be maximal. The P% is calculated by (Pt-Pe)/MLSSx100, where Pt is the total sludge phosphate in mg l-1, Pe is the phosphate in the effluent in mg l-1, and MLSS is the mixed liquor suspended solids in mg l-1. All chemical analytical methods are reported in Bond et al. (1999a ).
Chemical staining.
Sudan black B (for lipophilic inclusions including PHAs), methylene blue (for polyanions including polyphosphate) and Gram stains (Murray et al., 1994 ) were conducted on sludges. Samples were viewed by light microscopy on either a Nikon Microphot FXA microscope or a Zeiss Axiophot microscope. Images were captured via a cooled charge-coupled device connected to a PC and prepared in Adobe PhotoShop (Adobe Systems). Additionally the Nile blue A staining method (Ostle & Holt, 1982
) was used to detect intracellular PHAs. For this stain, samples were viewed by epifluorescence microscopy on a Zeiss Axiophot microscope or by confocal laser scanning microscopy (CLSM), detailed later. Sudan black, methylene blue and Nile blue staining procedures are not quantitative but simply indicate storage polymers inside specific cells.
Clone library preparation and analysis.
Separate bacterial 16S rDNA clone libraries were prepared from genomic DNA extracted from frozen-stored Q and T sludge. Briefly, DNA was extracted and purified (Burrell et al., 1998 ), primers 27f and 1492r (Lane, 1991
) were used for PCR amplification of near-complete 16S rDNAs and amplified genes were cloned using a TA cloning kit (Invitrogen). Inserts from individual clones in each library were amplified and grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism (RFLP) analysis using methods previously described (Burrell et al., 1998
) and restriction enzymes HinPI and MspI. OTU representatives were fully sequenced (Blackall, 1994
) and 16S rDNA sequences of clones were compiled using the software package SeqEd (Applied Biosystems). Each compiled sequence was compared to those in publicly available databases by use of the Basic Local Alignment Search Tool (BLAST, Altschul et al., 1997
) to determine approximate phylogenetic affiliations and detect sequences with high identity. The compiled sequences were aligned using the ARB software package (http://www.arb-home.de) and alignments were refined manually. Phylogenetic analysis of the sequences was by methods previously reported (Hugenholtz et al., 2001b
). Distance and parsimony methods were carried out in PAUP* version 4.0b2a, with and without corrections for rate variation and GC bias. The robustness of the tree topology was tested by bootstrap analysis with a range of outgroups (Dalevi et al., 2001
).
GenBank accession numbers.
The EMBL accession numbers for the 15 sequences reported in this paper are as follows. Novel -Proteobacteria cluster: SBRQ171, AF361089; SBRQ185, AF361090; SBRQ191, AF361091; SBRQ157, AF361092; SBRQ196, AF361093; SBRQ152, AF361094; SBRH10, AF361095; SBRT185, AF361096. Candidate phylum OP10: SBRT152, AF368186; SBRT161, AF368187; SBRT197, AF368188; SBRT162, AF368189. Acidobacteria: SBRT166, AF368181.
-Proteobacteria: SBRT155, AF368183. Bacteroidetes: SBRT303, AF368190.
FISH and post-FISH chemical staining.
Published probes and two designed probes (Table 1) were used in FISH (Amann, 1995
; Manz et al., 1992
). Probes were commercially synthesized and 5' labelled with either the fluorochrome FITC or with one of the sulfoindocyanine dyes Cy3 and Cy5 (ThermohybaidInteractiva, Ulm, Germany). The probe design tool of the ARB software package was used to design two probes from the clone library sequences (GAOQ probes in Table 1
). The design parameters used were as described by Hugenholtz et al. (2001a
, b
). Probe sequences were subsequently confirmed for specificity using BLAST, commercially synthesized and evaluated and optimized for FISH with paraformaldehyde- and ethanol-fixed Q sludge and the pure cultures Rhodocyclus purpureus DSM168 [from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany] and Desulfobulbus propionicus DSM2032 (from DSMZ) using methods previously described (Crocetti et al., 2000
).
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In some preparations, pot-FISH chemical staining was done. FISH was carried out and representative images captured, then the coverslip was removed from the slide, the mountant washed off and the chemical staining carried out. Fields photographed in FISH were relocated and rephotographed with the chemical stain. This procedure was first reported by Crocetti et al. (2000) for FISH and methylene blue staining to identify a PAO.
One laboratory-scale anaerobicaerobic cycling SBR and four full-scale activated sludge mixed liquors (from Noosa, Loganholme, Gibson Island and Luggage Point Sewage Treatment Plants, South-East Queensland, Australia) were collected from the end of the anaerobic zone and the aerobic zone and fixed. They were FISH probed with EUBMIX and GAOQ431 (Table 1). The laboratory-scale SBR and two full-scale sludges (Noosa and Loganholme Sewage Treatment Plants) were post-FISH chemically stained for PHA and polyphosphate.
Microbial quantification.
Methods used were essentially those reported previously (Bouchez et al., 2000 ). Samples from both Q and T sludges were pipetted into thick smears on slides to achieve reasonable homogeneity of sample. FISH preparations on these samples were viewed on the CLSM. Twenty different fields of view were selected randomly in the X, Y and Z planes for each reported population measurement. Area measurements with all probes (labelled with Cy3 or Cy5; Table 1
) were reported as a proportion of the area of all Bacteria in each field according to the probe set EUBMIX (labelled with FITC). Area measurements were performed on CLSM images using Image-Pro Plus (Version 4.0 for Windows; Media Cybernetics). The area of all pixels above a manually determined minimum pixel intensity was measured from the greyscale image of each probe in a field. The upper greyscale pixel intensity value remained constant at the maximum greyscale value of 255, thus allowing for the varying fluorescent signals from different populations of cells to be measured above the highest possible background and non-specific levels of fluorescence. The proportion of Cy5-labelled EUBMIX-binding cells to SYBR Green I (Molecular Probes)-binding cells was also determined (Schmid et al., 2000
).
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RESULTS |
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The SBR from which the T sludge was obtained had been operating for 198 days, with the latter 88 days of operation showing good to excellent EBPR (Bond et al., 1999b ). Beginning on day 198 and spanning a 3 week period, the P in the influent was progressively lowered from 30 mg PO4-P l-1 to 14 mg l-1, to 4 mg l-1 and then on day 219 to 2 mg PO4-P l-1. During this reduction, the P% in the sludge dropped from 12% to 1·5%, but carbon transformations (PHA and glycogen) remained essentially the same as during good EBPR. After approximately a week of operation at 2 mg PO4-P l-1 in the influent (day 225), the T sludge sample was taken from the end of the aerobic period, a portion was fixed and an aliquot frozen. We observed a high proportion of both Gram-negative and Gram-positive cells in the T sludge. Two abundant morphotypes of Gram-negative cells were large cocci arranged in tetrads and clusters of coccobacilli (approx. 1·5 µm diameter). Cells in this aerobic period sample did not contain polyphosphate or PHA. It had been previously reported that many cell types but not the cocci in tetrads stored PHA anaerobically (Bond et al., 1999b
).
Data for the stoichiometry of the anaerobic carbon and P transformations in both the Q and T sludges presented in Table 2 confirm the dominant GAO phenotype in these reactors due to their high similarity with model data for non-EBPR. Data for EBPR are presented for comparison.
Clone library analysis
By RFLP analysis, 50 clones from the Q sludge fell into four OTUs, and 53 T sludge clones produced eight OTUs.
Consistent with the morphological uniformness in the Q sludge micro-organisms, the clones in the library fell into only four OTUs, which all had very similar RFLP profiles. When representatives of these OTUs were sequenced (six near-complete inserts), the sequences were 99·7% identical and according to BLAST, their closest organismal match (88% identity) in GenBank was Nitrosococcus halophilus from the -Proteobacteria subphylum. Also highlighted by BLAST were sequences from three previously reported studies of wastewater treatment reactors (Dabert et al., 2001
; Liu et al., 2000
; Nielsen et al., 1999
). Sequences reported in two studies (Liu et al., 2000
; Nielsen et al., 1999
) were very short due to the method of obtaining them, and although of limited value from a phylogenetic standpoint, were useful for discussion due to their relatively high percentage identity with our Q sludge sequences (approx. 9395%). One sequence (PHOS-HE54, GenBank accession no. AF314424) from Dabert et al. (2001)
was 1433 nt long and 94·4% identical to the Q sludge sequences, and was used in phylogenetic analysis. One further sequence from an unpublished sludge clone study (SBRH10) was 94·5% identical to the Q sludge clone sequences and was fully sequenced (GenBank accession no. AF361095) so that it could be included in the analysis. Fig. 1
shows the phylogenetic tree from the analysis of the Q sludge clone sequences. These sequences, along with SBRH10, PHOS-HE54, and a clone from the T sludge clone library (SBRT185) (detailed later) form one cluster with a mean of 96·7% identity (Fig. 1
). In the phylogenetic analysis, we used many different outgroups (Dalevi et al., 2001
) but could never confidently affiliate the Q sludge clone cluster with either the ß-Proteobacteria or the well-known
-Proteobacteria and this cluster always fell as an outlier of the
-Proteobacteria. Thus, we concluded the Q sludge cluster was a monophyletic group well supported by bootstrap analysis and in the
-Proteobacteria radiation (Fig. 1
). The short sequences highlighted by BLAST were added to the tree using the parsimony insertion tool of ARB and are indicated with dashed lines.
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Only the sequences from Q and T sludge in the -Proteobacteria radiation were considered further in this paper.
Probe design and use with Q and T sludge
Two probes called GAOQ431 and GAOQ989 were designed to target the eight sequences from Q and T sludges, and SBRH10 and PHOS-HE54 in the -Proteobacteria cluster (Table 1
, Fig. 1
). Conditions for their use were optimized with Q sludge as a positive control. For GAOQ431, Rhodocyclus purpureus (2-base mismatch) was used as a negative control, while for GAOQ989, Desulfobulbus propionicus (2-base mismatch) was the negative control. For GAOQ431, there are strains with a 1-base mismatch and therefore the specificity of this probe might not be perfect. Results with Q sludge for both probes were identical and quantification was only done with GAOQ431. GAOQ431 specifically bound 92% of the EUBMIX-positive cells in the Q sludge (Table 3
) and they were large coccobacillus-shaped bacteria. Some 88% of these GAOQ431-binding cells also bound the BET42a probe (for ß-Proteobacteria) and 11% bound the GAM42a probe (for
-Proteobacteria) (Fig. 2a
). Other results for FISH with the Q sludge are presented in Table 3
.
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FISH with GAOQ431 and post-FISH chemical staining for PHA (Sudan black B) and polyphosphate (methylene blue) demonstrated that GAOQ431-binding coccobacilli in Q and T aerobic sludges did not contain PHA or polyphosphate.
Probing of laboratory-scale and full-scale activated sludges
The sludge from a laboratory-scale SBR operating in consistent deteriorated EBPR mode contained numerous GAOQ431-binding cells with intracellular PHA at the end of the anaerobic zone (Fig. 2c, d
) but lacking PHA at the end of the aerobic zone (data not shown). Of the four full-scale plants, Loganholme and Noosa Sewage Treatment Plants were operating for EBPR. All four plants contained GAOQ431-binding cells, and in both EBPR plants, post-FISH chemical staining for PHA demonstrated the EBPR anaerobicaerobic cycling of this polymer in GAOQ431-binding cells. FISH (Fig. 2e
) and post-FISH (Fig. 2f
) Nile blue A staining results for anaerobic Noosa sludge demonstrate this.
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DISCUSSION |
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GAOs are identified in laboratory-scale and full-scale sludges
The Q and T sludges were chosen to generate 16S rDNA sequences because they demonstrated the GAO phenotype and were therefore a good source of GAO sequences from which specific FISH oligonucleotides could be designed. Bond et al. (1999a ) previously described the highly abundant clusters of Gram-negative coccobacilli anaerobically storing PHA granules but not polyphosphate in the Q sludge. The abundance of GAOQ431-binding cells in the Q sludge (92%) was combined with the conspicuous GAO phenotype of this reactor (Table 2
and Bond et al., 1999a
) to draw a correlation between the GAOQ431-binding cells and the GAO phenotype. GAO phenotype was demonstrated in the T sludge (Table 2
and Bond et al., 1999b
), where the biodiversity by microbial morphology and FISH (Table 3
) was greater than that in the Q sludge. GAOQ431-binding cells were present at 28% in the T sludge; their morphology was similar to the GAOQ431-binding cells in the Q sludge and they demonstrated some of the GAO phenotype (no PHA or polyphosphate at the end of the aerobic phase). Therefore, we suggest they were probably one of the GAOs in this sludge, but others could have been GAOs.
A laboratory-scale SBR operating in deteriorated EBPR and two full-scale EBPR processes (Loganholme and Noosa) were confirmed to contain GAOQ431-binding cells which stored PHA anaerobically and utilized it aerobically. However, in these sludges, GAOQ431-binding cells did not accumulate polyphosphate aerobically. Thus GAOQ431-binding cells were strongly suggested to be GAOs. Other organisms in the sludges also contained PHA (Fig. 2cf) and possibly transformed it in accordance with EBPR (anaerobic production and aerobic utilization) but we only demonstrated this feature in GAOQ431-binding cells.
Relationship between our results and those of other GAO studies
One near-complete (Dabert et al., 2001 ) and five partial (Liu et al., 2000
; Nielsen et al., 1999
) 16S rDNA sequences from other sludge studies were found to be highly identical to sequences in our
-Proteobacteria cluster shown in Fig. 1
. Two of the studies (Dabert et al., 2001
; Nielsen et al., 1999
) aligned the organisms with poor EBPR and in all three studies (Dabert et al., 2001
; Liu et al., 2000
; Nielsen et al., 1999
), sequences with high identity to our
-Proteobacteria sequences were found in deteriorated EBPR reactors. Although there is corroboration between all these studies, ours markedly extends the knowledge by generating eight near-complete 16S rDNA sequences, adding to the one currently available, near-complete sequence (Dabert et al., 2001
), thus allowing precise phylogenetic analysis (Fig. 1
) and extensive probe design capacity.
Nielsen et al. (1999) designed FISH probes (GAM1019 and GAM1278) to be specific for the novel
-Proteobacteria cluster from sequences only 420 nt in length. These probes have one or more mismatches to most of the near-complete sequences in this cluster shown in Fig. 1
. Probe GAOQ989 targets with no mismatches all the near-complete sequences and all but one (AF093780) of the partial sequences in the novel
-Proteobacteria cluster (Fig. 1
). Probe GAOQ431 targets with no mismatches all the near-complete sequences but none of the partial sequences, due to the fact that none of them extend to this part of the 16S rDNA. However, GAM1019 and GAM1278 are likely to be of use to target different populations within the novel
-Proteobacteria cluster and their abundance would also be relevant in the study of GAOs.
In our study, we have directly linked cells with aspects of the GAO phenotype (PHA cycling and not polyphosphate cycling) with their phylotype, and have therefore been able to determine the in situ morphology of GAOs. Previous studies also attempted this but they were less convincing due to the use of quite narrow-specificity probes for the -Proteobacteria cluster (Liu et al., 2001
; Nielsen et al., 1999
). Additionally, we used FISH and post-FISH chemical staining to demonstrate that GAOQ431-binding cells were GAOs in one laboratory-scale deteriorated EBPR SBR and two full-scale EBPR plants, establishing the utility of GAOQ431 probe in understanding deterioration of EBPR. The determination of GAO competition for VFAs with PAOs should now be determined in full-scale processes.
The phylogeny of the -Proteobacteria GAOs (Fig. 1
) and the FISH results with GAOQ431 warrant further comment. The GAOQ431 and GAOQ989 probes were designed from sequences forming a highly supported monophyletic lineage (Fig. 1
), but in the Q sludge 88% of the GAOQ431-binding cells bound BET42a (for ß-Proteobacteria) and 11% bound GAM42a (for
-Proteobacteria) (Fig. 2a
). The BET42a and GAM42a probe targets are in the 23S rRNA and only differ from each other by one central nucleotide (Table 1
). There are no pure cultures of the
-Proteobacteria GAOs and there is no information on their 23S rDNA sequences. Other researchers (Liu et al., 2001
; Nielsen et al., 1999
) also reported probing inconsistencies where only a portion of the
-Proteobacteria GAOs also bound the GAM42a probe. However, BET42a was not simultaneously used in these studies. Thus, elucidation of the reason why the Q sludge contains mostly ß-Proteobacteria GAOs and fewer
-Proteobacteria GAOs awaits information on the BET42aGAM42a target region of the 23S rDNA for these organisms.
Factors favouring GAOs and PAOs
The complex interactions that lead to the selection of different micro-organisms in a wastewater treatment system are not well understood. When we used conditions that should select for PAOs (Q sludge operation), GAOs predominated. However, Accumulibacter was present at very low levels (2% in Q sludge; Table 3) and although this organism has the ability to cycle P and carbon according to EBPR, it was out-competed in this experiment by the novel
-Proteobacteria functioning as GAOs. It could be that competition for carbon was the deciding factor in which organism would predominate, as proposed previously (Mino et al., 1998
). We found the novel
-Proteobacteria in all four full-scale activated sludges examined, and in two EBPR plants demonstrated they had aspects of the GAO phenotype. We have also found that Accumulibacter are common in full-scale activated sludges and in EBPR processes they are PAOs (data not shown). We hypothesize that most activated sludges will contain both the novel
-Proteobacteria and Accumulibacter but the reason why one or the other predominates in EBPR conditions (anaerobicaerobic cycling) must now be determined.
Proposal for Candidatus Competibacter phosphatis
On several occasions we have attempted isolation to pure culture of the novel -Proteobacteria reported in this paper but have not been successful (results not shown). Thus, we propose that the organisms from which the novel
-Proteobacteria cluster sequences originated be named Candidatus Competibacter phosphatis (see Fig. 1
for sequences encompassed), due to their ability to compete with polyphosphate-accumulating organisms in EBPR processes.
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ACKNOWLEDGEMENTS |
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Some of the work was funded by the CRC for Waste Management and Pollution Control Ltd, a centre established and supported under the Australian Governments Cooperative Research Centres Program. Greg Crocetti was supported by a University of Queensland Postgraduate Research Scholarship and a CRC-WMPC scholarship.
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Received 20 May 2002;
revised 14 July 2002;
accepted 18 July 2002.