Polyphosphate kinase genes from full-scale activated sludge plants
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- McMahon, K.D., Yilmaz, S., He, S. et al. Appl Microbiol Biotechnol (2007) 77: 167. doi:10.1007/s00253-007-1122-6
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The performance of enhanced biological phosphorus removal (EBPR) wastewater treatment processes depends on the presence of bacteria that accumulate large quantities of polyphosphate. One such group of bacteria has been identified and named Candidatus Accumulibacter phosphatis. Accumulibacter-like bacteria are abundant in many EBPR plants, but not much is known about their community or population ecology. In this study, we used the polyphosphate kinase gene (ppk1) as a high-resolution genetic marker to study population structure in activated sludge. Ppk1 genes were amplified from samples collected from full-scale wastewater treatment plants of different configurations. Clone libraries were constructed using primers targeting highly conserved regions of ppk1, to retrieve these genes from activated sludge plants that did, and did not, perform EBPR. Comparative sequence analysis revealed that ppk1 fragments were retrieved from organisms affiliated with the Accumulibacter cluster from EBPR plants but not from a plant that did not perform EBPR. A new set of more specific primers was designed and validated to amplify a 1,100 bp ppk1 fragment from Accumulibacter-like bacteria. Our results suggest that the Accumulibacter cluster has finer-scale architecture than previously revealed by 16S ribosomal RNA-based analyses.
KeywordsEnhanced biological phosphorus removalActivated sludgeRhodocyclusAccumulibacter phosphatisPolyphosphate kinase
The enhanced biological phosphorus removal (EBPR) activated sludge process is widely used to remove phosphorus from municipal wastewaters. The process is often studied in laboratory scale sequencing batch reactors (SBRs), fed with synthetic feed containing acetate or propionate as carbon sources. Researchers on four continents employing similar SBR operating conditions have repeatedly enriched bacteria affiliated with a phylogenetically coherent group within the β−Proteobacteria, (assessed using 16S ribosomal RNA [rRNA] analysis; Crocetti et al. 2000; Hesselmann et al. 1999; Liu et al. 2001; McMahonet al. 2002a). This group shares > 96.4% 16S rRNA sequence identity and appears to be a member of the Rhodocyclaceae family. Fluorescence in situ hybridization (FISH) was used to document their abundance and confirm their role in EBPR in lab-scale SBRs (Crocetti et al. 2000; Hesselmann et al. 1999; Liu et al. 2001; McMahon et al. 2002a), pilot-scale systems (Lee et al. 2003), and full-scale wastewater treatment plants (WWTPs; He et al. 2006, 2007; Kong et al. 2004; Zilles et al. 2002b). Hesselmann et al. (1999) proposed a genus and species name for one member of this group; Candidatus Accumulibacter phosphatis. Members of the candidate genus have not yet been cultured in isolation, and must be studied in lab-scale enrichment cultures or in full-scale WWTPs.
Recently, the metagenomes of two highly Accumulibacter-enriched EBPR sludges were sequenced, confirming the presence of two distinct Accumulibacter “species” in the two lab-scale SBRs (Garcia Martin et al. 2006). In this paper, we will discuss Accumulibacter-like bacteria using a hierarchical nomenclature of “Group-Cluster-Type-Clade” which is similar to the Linnaean “Phylum-Genus-Species-Strain,” acknowledging the open debate about bacterial species definitions (e.g.(Gevers et al. 2005)) and neglecting levels between Phylum and Genus. We consider the “Accumulibacter cluster” to constitute the organisms generally detected by commonly used PAOMIX 16S rRNA-targeted FISH probes (Crocetti et al. 2000; Hesselmann et al. 1999). Bacteria responsible for EBPR can take up large quantities of inorganic phosphate (Pi) and store it as polyphosphate (polyP). In model organisms, polyphosphate kinase 1 (ppk1) is the primary enzyme thought to be responsible for polyphosphate synthesis from ATP (Ahn and Kornberg 1990; Akiyama et al. 1992; Tzeng and Kornberg 1998). Ppk1 genes were previously retrieved from Accumulibacter-like bacteria cultivated in an acetate-fed SBR in Berkeley, CA, USA, using degenerate PCR primers (McMahon et al. 2002a). In that study, four ppk1 genotypes were discovered, two of which (Types I and II) appeared to be derived from Accumulibacter-like bacteria, sharing a maximum of 86% nucleotide identity. Unlike the 16S rRNA gene, the ppk1 gene appears to be a powerful genetic marker for revealing finer-scale population structure within co-occurring groups of Accumulibacter-like organisms.
Treatment plant characteristics
Configuration at time of sampling
Oro Loma Sanitary District (OL)
San Leandro, CA
High rate anaerobic selector (anaerobic/aerobic)
Contra Costa County Sanitary District (CC)
High rate anaerobic selector (anaerobic/aerobic)
San Jose / Santa Clara Water Pollution Plant (SJ)
Four-stage anoxic/aerobic with step feed
City of Las Vegas Water Pollution Control Facility (LV)
Las Vegas, NV
Madison Metropolitan Sewerage District Nine Springs Plant (NS)
Hampton Roads Sanitary District Nansemond Plant (NAN)
Hampton Roads Sanitary District Virginia Initiative Plant (VIP)
Durham Advanced Wastewater Treatment Facility (DUR)
East Bay Municipal Utilities District (EB)
Conventional activated sludge with pure oxygen aeration
City of Oshkosh Wastewater Treatment Plant (OSK)
Conventional activated sludge
Materials and methods
Full scale activated sludges
Activated sludge samples were taken from the mixed liquor channels of each WWTP. For WWTPs sampled in California, aliquots were immediately centrifuged (3,000 g for 10 min) and the biomass pellets were frozen on dry ice for transport to the laboratory, where they were stored at −80°C. Additional aliquots were immediately filtered through glass fiber filters (Whatman GF/B) for the soluble Pi assay. The suspended solids (total and volatile) and phosphorus content of the sludge were assayed within 2 h of sampling after transport to the laboratory at room temperature. All other WWTPs were sampled as described elsewhere (He et al. 2007). Total suspended solids, volatile suspended solids, soluble reactive phosphorus, and total phosphorus were by Standard Methods 2540B 2540E, 4500-P C, and 4500-P B.5, respectively (APHA 1995).
Lab-scale sequencing batch reactors
Two sequencing batch reactors were operated as previously described (Garcia Martin et al. 2006; McMahon et al. 2002a, b; Schuler and Jenkins 2003). Reactor “UCB” was operated at the University of California at Berkeley for 2 years and was originally inoculated with activated sludge from the Southeast Water Pollution Control Plant in San Francisco, CA. Reactor “UWM” was operated for 2 years at the University of Wisconsin–Madison and was originally inoculated with activated sludge from the Nine Springs Wastewater Treatment Plant in Madison, WI.
Genomic DNA extraction
Bulk genomic DNA was extracted from sludge samples using a series of enzymatic digestions, followed by phenol-chloroform extraction, as described previously (Purkhold et al. 2000) with minor modifications (Garcia Martin et al. 2006). Some aliquots were carried through the same extraction procedure described by Purkhold et al. (2000), while some were subjected to bead beating after the second enzyme digestion, as described (McMahon et al. 2002a).
PCR amplification of ppk1 fragments using degenerate primers
PCR conditions were optimized for each batch of genomic DNA. The following conditions were fixed for all samples. Amplification was carried out in 50 μl reactions containing 1X PCR buffer II (Applied Biosystems, Foster City, CA), 3.5 mM MgCl2, 200 μM of each dNTP, 400 nM of each forward and reverse primer (NLDE-F and TGNY-R, Table 2), 5% dimethyl sulfoxide, 200 ng bovine serum albumin, and 2.5 U AmpliTaq Gold (Applied Biosystems) on an MJ Research DNA Engine thermal cycler. A touch-down PCR program was used: an initial 12 min denaturing step at 94°C, followed by ten cycles of 94°C for 45 s, an optimized annealing temperature for 45 s (decreasing 0.5°C per cycle), and 72°C for 2 min. An additional 25 cycles were carried out with the same denaturing and extension conditions, but with 45°C annealing for 45 s, followed by a final 12 min extension at 72°C. The initial annealing temperature for each sample was varied using the gradient feature of the thermal cycler. The optimum was between 48 and 50°C, touching down to 43 and 45°C, respectively. The template concentration was titrated during optimization; the optimum amount was generally between 20 and 200 ng of extracted DNA per reaction.
ppk1 clone library construction
PCR products from several replicate reactions (usually 3–4) were purified by extraction from agarose gels using spin columns (Qiagen, Valencia, CA) and were cloned into pCR4 using the TOPO TA Cloning Kit for Sequencing (Invitrogen, Carlsbad, CA). Approximately 90 clones from each library were screened using restriction fragment length polymorphism (RFLP) analysis with MspI and Alu I (Boehringer Mannheim, Germany), in separate digests (Dojka et al. 1998). Unique representatives were chosen for sequencing.
PCR amplification of ppk1 fragments using Accumulibacter-specific primers
Primers used for PCR
Most ppk1 homologsb
A dataset of known and putative ppk1 gene sequences was constructed by searching available public databases using the BLAST network service on the NCBI website (Altschul et al. 1990). The sequence fragments were translated, and the amino acid sequences were aligned against the dataset using the Seqlab program in the GCG software package version 10.0 (Genetics Computer Group, WI). Translated sequences were inspected for characteristic motifs to confirm their homology to known ppk1s (e.g., amino acid sequence ARFDE; Tzeng and Kornberg 1998). Alignments were masked manually to exclude positions with gaps in more than 15% of sequences, and the remaining aligned positions were exported for analysis. Bayesian inference of phylogeny was carried out with MrBayes version 3.1.1 using default priors (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). A mixed amino acid substitution model was used for amino acid-derived phylogenies. For nucleic acid-based phylogenies, the general time reversible model was used with rates varying according to codon positions. Trees were visualized and printed using the program PAUP* version 4.0b10 (Sinauer Associates, Sunderland, MA).
Nucleotide sequence accession numbers
The GenBank accession numbers for the nucleotide sequences determined in this study are AY963820-AY963838, DQ466618-DQ466723, DQ630733-DQ630737, DQ868996-DQ869001, and DQ883814.
Physical and chemical characteristics of sludges used for ppk1 library construction
Activated sludge sourcea
Percent Pns-content [100% x mg Pns (mg suspended solids)−1]
Aerobic zone soluble Pi
(mg P l−1)
Sludge ppk1 diversity captured in clone libraries
Accumulibacter-like ppk1s were associated with Group I–a highly supported lineage containing ppk1s from β-Proteobacteria and γ-Proteobacteria. Notably, Group I contains the previously described “Type I” and “Type II” ppk1s retrieved from lab scale EBPR reactors (McMahon et al. 2002a). Group II was also a distinct lineage, containing ppk1s from only α-Proteobacteria (Fig. S1). Groups III and IV contained only Actinobacteria and Cyanobacteria ppk1s, respectively. Group V contained ppk1s from Escherichia coli, Vibrio cholerae, and Pseudomonas aeruginosa. The partitioning of γ-Proteobacteria between Groups I and V is noteworthy, because it means that ppk1 protein- and 16S rRNA gene-based phylogenies are incongruent. This implies that ppk1 may have been horizontally transferred.
Accumulibacter-like ppk1 genes detected in EBPR sludge
Novel groups of ppk1 found in all sludges
Several novel lineages with no cultured representatives were identified during phylogenetic analyses of the newly expanded ppk1 dataset (Figs. 1 and S1). A separate tree was constructed to better illustrate the fine-scale topology of Group V, which could be confidently divided into three subgroups (Fig. S2). Subgroups Vc and Vb included Types III and IV, respectively, which were previously obtained from a lab scale SBR (McMahon et al. 2002a). All four full-scale sludges contributed members to each of the three subgroups, suggesting that the organisms contributing these genes are ubiquitous in full-scale plants, regardless of configuration. The ecological or engineering significance of organisms possessing Group V ppk1 remains to be determined.
Accumulibacter-ppk1 specific primer design and validation
The previously described degenerate primers targeting ppk1 in most bacteria can be used to retrieve novel ppk1 fragments from uncultured organisms (McMahon et al. 2002a). However, amplification with these primers is often non-specific and highly inefficient, probably because of the high level of degeneracy. Therefore, we designed new PCR primers to specifically target the Accumulibacter cluster. The primers amplified Accumulibacter-ppk1 fragments from positive controls (Fig. S3a) and sludge genomic DNA known to contain Accumulibacter-ppk1 (Fig. S3b). They are also highly specific: no amplification products were detected when ppk1 fragments not affiliated with the Accumulibacter cluster or non-EBPR sludge DNA were used as template (Fig. S3a and b, respectively).
Accumulibacter-like bacteria are thought to be responsible for phosphorus accumulation in most volatile fatty acid-fed laboratory scale EBPR systems (Crocetti et al. 2000; Hesselmann et al. 1999; Levantesi et al. 2002; Liu et al. 2001; Oehmen et al. 2005; Pijuan et al. 2004) as well as many full-scale WWTPs (Beer et al. 2006; He et al. 2006, 2007; Kong et al. 2004; Zilles et al. 2002a, b). In this paper, we describe a first effort to characterize the population structure of Accumulibacter-like bacteria using a genetic locus that provides more phylogenetic resolution than the commonly used 16S rRNA gene. The four full-scale WWTPs were chosen to represent a variety of activated sludge configurations.
Several new sequences retrieved from EBPR systems formed a distinct cluster in phylogenetic trees with other previously sequenced Accumulibacter-like ppk1s. We named the phylogenetically coherent lineage that contained this cluster “Group I” to provide a nomenclature that allows discussion of the Accumulibacter clades within a more broadly defined group. The new Accumulibacter-like sequences retrieved from the full-scale sludges clearly affiliated with the previously named Type I ppk1 (McMahon et al. 2002a). Clone SJ2-65 was the most closely affiliated with Type II, sharing 99% DNA sequence identity with the UCB clones. Notably, a clone from the SBR at the University of Wisconsin–Madison (UWMH-E5) also shared 99% DNA sequence identity with both SJ2-65 and the Type II UCB clones, suggesting that the same strain of Accumulibacter was present in the Madison SBR. This SBR also contained Type I ppk1s. The fact that no Type I ppk1s were recovered from the full scale plants is intriguing as it implies that Type I ppk1s are comparatively rare at full-scale. A quantitative survey of a larger selection of WWTPs (including those used to inoculate the SBRs) is required to confirm this observation.
The retrieval of sequences associated with several distinct clades of Accumulibacter-like ppk1 fragments is consistent with the hypothesis that several phylogenetically distinct groups of these organisms exist in WWTPs. Those clustering with the Type I and II SBR sequences are likely from Accumulibacter-like organisms. We propose that sequences branching outside the designated Accumulibacter cluster, but within Group 1, are derived from organisms qualifying as members of a different genus. It is not yet possible to correlate evolutionary distance based on 16S rRNA with ppk1 DNA or amino acid sequences. Thus, we cannot determine whether these sequences originated from the Accumulibacter-related “Dechloromonas subgroup” defined by Zilles et al. (2002b). However, because 16S rRNA sequences belonging to this subgroup were obtained by Zilles et al. (2002b) from a conventional activated sludge plant not performing EBPR, our hypothesis is consistent with the fact that several EB clones (also from a conventional non-EBPR plant) are part of a group that is distinctly separate from the Accumulibacter-ppk1 clade.
We emphasize that it is not possible to draw quantitative conclusions about ppk1 diversity, distribution, or abundance based on the number of unique RFLP types and sequences obtained using PCR-based clone libraries because many sources of bias, including different efficiencies of DNA extraction, PCR amplification, and cloning contribute to non-quantitative results (Wintzingerode et al. 1997). Degenerate primers are probably even more likely to produce amplification bias, as some sequences will have more perfect matches than others.
The newly designed and validated Accumulibacter-specific ppk1 primers will be useful for the harvesting of additional Accumulibacter-ppk1 sequences from full scale WWTPs, for further studies of Accumulibacter cluster diversity and structure and as a diagnostic tool to determine whether Accumulibacter-like organisms are present. The ppk1 locus is much more divergent than the 16S rRNA locus and can distinguish between the different Accumulibacter sub-clusters, making it a promising candidate for a reliable genetic marker for Accumulibacter-mediated EBPR. Remaining to be elucidated are the relationships between the fine-scale population structure within the Accumulibacter cluster and how it might affect EBPR process performance.
The authors wish to thank Philip Hugenholtz, Hector Garcia Martin, Victor Kunin, Jason Flowers, and Daniel Noguera for helpful discussions. This research was supported by National Science Foundation Grants BES-9912472 and BES-0332181 to DJ and JDK, and by BES-0332136 to KDM.