Polyphosphate kinase genes from full-scale activated sludge plants

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. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

"Candidatus Accumulibacter" population structure in enhanced biological phosphorus removal sludges as revealed by polyphosphate kinase genes

We investigated the fine-scale population structure of the "Candidatus Accumulibacter" lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of "Candidatus Accumulibacter" 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the "Candidatus Accumulibacter" lineage. Sequences from at least five clades of "Candidatus Accumulibacter" were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using "Candidatus Accumulibacter"-specific 16S rRNA and "Candidatus Accumulibacter" clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total "Candidatus Accumulibacter" lineage and the relative distributions and abundances of the five "Candidatus Accumulibacter" clades. The qPCR-based estimation of the total "Candidatus Accumulibacter" fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined "Candidatus Accumulibacter" clades. The relative distributions of "Candidatus Accumulibacter" clades varied among different EBPR systems and also temporally within a system. Our results suggest that the "Candidatus Accumulibacter" lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.     

Widespread detection of Candidatus Accumulibacter phosphatis,a polyphosphate-accumulating organism,in sediments of the Columbia River estuary

Enhanced biological phosphorus removal (EBPR) exploits the metabolism of polyphosphate-accumulating organisms (PAOs) to remove excess phosphorus (P) from wastewater treatment. Candidatus Accumulibacter phosphatis (Accumulibacter) is the most abundant and well-studied PAO in EBPR systems. In a previous study, we detected polyphosphates throughout peripheral bay sediments, and hypothesized that an estuary is an ideal setting to evaluate PAOs in a natural system, given that estuaries are characterized by dynamic dissolved oxygen fluctuations that potentially favour PAO metabolism. We detected nucleotide sequences attributable to Accumulibacter (16S rRNA, ppk1) in sediments within three peripheral bays of the Columbia River estuary at abundances rivalling those observed in conventional wastewater treatment plants (0.01%–2.6%). Most of the sequences attributable to Accumulibacter were Type I rather than Type II, despite the fact that the estuary does not have particularly high nutrient concentrations. The highest diversity of Accumulibacter was observed in oligohaline peripheral bays, while the greatest abundances were observed at the mouth of the estuary in mesohaline sediments in the spring and summer. In addition, an approximately 70% increase in polyphosphate concentrations observed at one of the sites between dawn and dusk suggests that PAOs may play an important role in P cycling in estuary sediments.     

Diversity of nitrite reductase genes in "Candidatus Accumulibacter phosphatis"-dominated cultures enriched by flow-cytometric sorting

"Candidatus Accumulibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumulibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.     

Comparative genomics of two ‘Candidatus Accumulibacter' clades performing biological phosphorus removal

Members of the genus Candidatus Accumulibacter are important in many wastewater treatment systems performing enhanced biological phosphorus removal (EBPR). The Accumulibacter lineage can be subdivided phylogenetically into multiple clades, and previous work showed that these clades are ecologically distinct. The complete genome of Candidatus Accumulibacter phosphatis strain UW-1, a member of Clade IIA, was previously sequenced. Here, we report a draft genome sequence of Candidatus Accumulibacter spp. strain UW-2, a member of Clade IA, assembled following shotgun metagenomic sequencing of laboratory-scale bioreactor sludge. We estimate the genome to be 80–90% complete. Although the two clades share 16S rRNA sequence identity of >98.0%, we observed a remarkable lack of synteny between the two genomes. We identified 2317 genes shared between the two genomes, with an average nucleotide identity (ANI) of 78.3%, and accounting for 49% of genes in the UW-1 genome. Unlike UW-1, the UW-2 genome seemed to lack genes for nitrogen fixation and carbon fixation. Despite these differences, metabolic genes essential for denitrification and EBPR, including carbon storage polymer and polyphosphate metabolism, were conserved in both genomes. The ANI from genes associated with EBPR was statistically higher than that from genes not associated with EBPR, indicating a high selective pressure in EBPR systems. Further, we identified genomic islands of foreign origins including a near-complete lysogenic phage in the Clade IA genome. Interestingly, Clade IA appeared to be more phage susceptible based on it containing only a single Clustered Regularly Interspaced Short Palindromic Repeats locus as compared with the two found in Clade IIA. Overall, the comparative analysis provided a genetic basis to understand physiological differences and ecological niches of Accumulibacter populations, and highlights the importance of diversity in maintaining system functional resilience.     

Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation

Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were beta-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the beta-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing >/=98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the beta-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related to Rhodocyclus and Propionibacter.     

Polyphosphate kinase from activated sludge performing enhanced biological phosphorus removal

A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like beta-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg(2+), and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.     

Enrichment, phylogenetic analysis and detection of a bacterium that performs enhanced biological phosphate removal in activated sludge.

Activated sludge communities which performed enhanced biological phosphate removal (EBPR) were phylogenetically analyzed by 16S rRNA-targeted molecular methods. Two anaerobic-aerobic sequencing batch reactors were operated with two different carbon sources (acetate vs. a complex mixture) for three years and showed anaerobic-aerobic cycles of polyhydroxybutyrate- (PHB) and phosphate-accumulation characteristic for EBPR-systems. In situ hybridization showed that the reactor fed with the acetate medium was dominated by bacteria phylogenetically related to the Rhodocyclus-group within the beta-Proteobacteria (81% of DAPI-stained cells). The reactor with the complex medium was also predominated by this phylogenetic group albeit at a lesser extent (23% of DAPI-stained cells). More detailed taxonomic information on the dominant bacteria in the acetate-reactor was obtained by constructing clone libraries of 16S rDNA fragments. Two different types of Rhodocyclus-like clones (R1 and R6) were retrieved. Type-specific in situ hybridization and direct rRNA-sequencing revealed that R6 was the type of the dominant bacteria. Staining of intracellular polyphosphate- and PHB-granules confirmed that the R6-type bacterium accumulates PHB and polyphosphate just as predicted by the metabolic models for EBPR. High similarities to 16S rDNA fragments from other EBPR-sludges suggest that R6-type organisms were present and may play an important role in EBPR in general. Although the R6-type bacterium is closely related to the genus Rhodocyclus, it did not grow phototrophically. Therefore, we propose a provisional new genus and species Candidatus Accumulibacter phosphatis.     

In situ identification of polyphosphate- and polyhydroxyalkanoate-accumulating traits for microbial populations in a biological phosphorus removal process

Polyphosphate- and polyhydroxyalkanoate (PHA)-accumulating traits of predominant microorganisms in an efficient enhanced biological phosphorus removal (EBPR) process were investigated systematically using a suite of non-culture-dependent methods. Results of 16S rDNA clone library and fluorescence in situ hybridization (FISH) with rRNA-targeted, group-specific oligonucleotide probes indicated that the microbial community consisted mostly of the alpha- (9.5% of total cells), beta- (41.3%) and gamma- (6.8%) subclasses of the class Proteobacteria, Flexibacter-Cytophaga (4.5%) and the Gram-positive high G+C (HGC) group (17.9%). With individual phylogenetic groups or subgroups, members of Candidatus Accumulibacter phosphatis in the beta-2 subclass, a novel HGC group closely related to Tetrasphaera spp., and a novel gamma-proteobacterial group were the predominant populations. Furthermore, electron microscopy with energy-dispersive X-ray analysis was used to validate the staining specificity of 4,6-diamino-2-phenylindole (DAPI) for intracellular polyphosphate and revealed the composition of polyphosphate granules accumulated in predominant bacteria as mostly P, Ca and Na. As a result, DAPI and PHA staining procedures could be combined with FISH to identify directly the polyphosphate- and PHA-accumulating traits of different phylogenetic groups. Members of Accumulibacter phosphatis and the novel gamma-proteobacterial group were observed to accumulate both polyphosphate and PHA. In addition, one novel rod-shaped group, closely related to coccus-shaped Tetrasphaera, and one filamentous group resembling Candidatus Nostocoidia limicola in the HGC group were found to accumulate polyphosphate but not PHA. No cellular inclusions were detected in most members of the alpha-Proteobacteria and the Cytophaga-Flavobacterium group. The diversified functional traits observed suggested that different substrate metabolisms were used by predominant phylogenetic groups in EBPR processes.     

Net P-removal deterioration in enriched PAO sludge subjected to permanent aerobic conditions

Recently, some research in the field of enhanced biological phosphorus removal (EBPR) has been focused on studying systems where the electron donor (substrate) and the electron acceptor (nitrate or oxygen) are present simultaneously. This can occur, for example, in a full scale wastewater treatment plant during heavy rainfall periods when the anaerobic hydraulic retention time is temporarily shortened. To study this situation that could induce EBPR failure, the operation of a sequencing batch reactor (SBR) working under alternating anaerobic-aerobic conditions with an enriched EBPR population (50% Candidatus Accumulibacter phosphatis and less than 1% Candidatus Competibacter phosphatis) was shifted to strict aerobic operation. Seven cycle studies were performed during the 11 days of aerobic operation. Net P-removal was observed in this aerobic SBR during the first 4 days of operation but the system could not achieve net-P removal after this period, although the microbial composition, in terms of percentage of Accumulibacter and Competibacter, did not change significantly. The observed changes in the different compounds analysed (phosphorus, acetate, glycogen and PHB) as well as in the OUR profile indicate that metabolic changes are produced for the adaptation of PAO to aerobic conditions.     

Monitoring the impact of bioaugmentation on the start up of biological phosphorus removal in a laboratory scale activated sludge ecosystem

The acclimatisation of activated sludge to enhanced biological phosphorus removal (EBPR) conditions requires a period of about 40–100 days but its output remains hazardous. The impact of bioaugmentation on the start-up of a laboratory scale EBPR sequencing batch reactor was evaluated by process parameters measurement and microbial community dynamics monitoring using 16S rDNA targeted polymerase chain reaction-single strand conformation polymorphism electrophoresis (PCR-SSCP). Bioaugmentation: (1) speeded up the installation of good and stable EBPR in the bioaugmented reactor by about 15 days; (2) correlated with the transient enrichment of the sludge in the added microbial populations; and (3) favoured the long-term enrichment of the sludge in the phosphorus-accumulating organism (PAO) Candidatus Accumulibacter phosphatis. However, despite a lag time period, the control non-bioaugmented reactor ended up with comparable reactor parameters and microbial community evolution, suggesting that the same PAO populations were already present from the beginning in the original non-P-accumulating seed sludge. The potential of a true installation of the added microbial populations within the bioaugmented reactor compared to their substitution by indigenous similar populations is discussed. Competition between PAOs and the antagonistic glycogen accumulating organism Candidatus Competibacter phosphatis is also highlighted during EBPR start-up.     

Detection of denitrification genes by in situ rolling circle amplification‐fluorescence in situ hybridization to link metabolic potential with identity inside bacterial cells

A target‐primed in situ rolling circle amplification (in situ RCA) protocol was developed for detection of single‐copy genes inside bacterial cells and optimized with Pseudomonas stutzeri, targeting nitrite and nitrous oxide reductase genes (nirS and nosZ). Two padlock probes were designed per gene to target both DNA strands; the target DNA was cut by a restriction endonuclease close to the probe binding sites, which subsequently were made accessible by 5′‐3′ exonucleolysis. After hybridization, the padlock probe was circularized by ligation and served as template for in situ RCA, primed by the probe target site. Finally, the RCA product inside the cells was detected by standard fluorescence in situ hybridization (FISH). The optimized protocol showed high specificity and signal‐to‐noise ratio but low detection frequency (up to 15% for single‐copy genes and up to 43% for the multi‐copy 16S rRNA gene). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA‐FISH was demonstrated on activated sludge by the differential detection of two types of nirS‐defined denitrifiers; one of them was identified as Candidatus Accumulibacter phosphatis by combining in situ RCA‐FISH with 16S rRNA‐targeted FISH. While not suitable for quantification because of its low detection frequency, in situ RCA‐FISH will allow to link metabolic potential with 16S rRNA (gene)‐based identification of single microbial cells.     

Competition between polyphosphate and glycogen accumulating organisms in enhanced biological phosphorus removal systems with acetate and propionate as carbon sources

Enhanced biological phosphorus removal (EBPR) is a widely used process for achieving phosphorus removal from wastewater. A potential reason for EBPR failure is the undesirable growth of glycogen accumulating organisms (GAOs), which can compete for carbon sources with the bacterial group responsible for phosphorus removal from wastewater: the polyphosphate accumulating organisms (PAOs). This study investigates the impact of carbon source on EBPR performance and the competition between PAOs and GAOs. Two sequencing batch reactors (SBRs) were operated during a 4-6 month period and fed with a media containing acetate or propionate, respectively, as the sole carbon source. It was found that the acetate fed SBR rarely achieved a high level of phosphorus removal, and that a large portion of the microbial community was comprised of "Candidatus Competibacter phosphatis", a known GAO. The propionate fed SBR, however, achieved stable phosphorus removal throughout the study, apart from one brief disturbance. The bacterial community of the propionate fed SBR was dominated by "Candidatus Accumulibacter phosphatis", a known PAO, and did not contain Competibacter. In a separate experiment, another SBR was seeded with a mixture of PAOs and a group of alphaproteobacterial GAOs, both enriched with propionate as the sole carbon source. Stable EBPR was achieved and the PAO population increased while the GAOs appeared to be out-competed. The results of this paper suggest that propionate may provide PAOs with a selective advantage over GAOs in the PAO-GAO competition, particularly through the minimisation of Competibacter. Propionate may be a more suitable substrate than acetate for enhancing phosphorus removal in EBPR systems.     

Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal

A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like β-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg²⁺, and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.     

Phylogeny and in situ identification of a novel gammaproteobacterium in activated sludge

Failure of a continuously aerated sequencing batch reactor (SBR) pilot plant-enhanced biological phosphorus removal (EBPR) process, designed to remove phosphorus from the clarified effluent from a conventional non-EBPR wastewater treatment plant, was associated with the dominance ( c . 50% of the biovolume) of gammaproteobacterial coccobacilli. Flow cytometry and subsequent clone library generation from an enriched population of these Gammaproteobacteria showed that their 16S rRNA genes were most similar to partial clone sequences obtained from an actively denitrifying SBR community, and from anaerobic : aerobic EBPR communities. Under the SBR operating conditions used here, these cells stained for poly-β-hydroxyalkanoates, but never polyphosphate. Applying FISH probes designed against them in combination with microautoradiography showed that they could also assimilate acetate 'aerobically'. FISH analyses of biomass samples from the full-scale treatment plant providing the pilot plant feed showed that they were present there in high numbers. However, they were not detected by FISH in laboratory-scale communities of the same aerated laboratory-scale EBPR process even when EBPR had failed, or from several full-scale EBPR plants or other activated sludge processes.     

Metagenomic analysis of two enhanced biological phosphorus removal (EBPR) sludge communities

Enhanced biological phosphorus removal (EBPR) is one of the best-studied microbially mediated industrial processes because of its ecological and economic relevance. Despite this, it is not well understood at the metabolic level. Here we present a metagenomic analysis of two lab-scale EBPR sludges dominated by the uncultured bacterium, "Candidatus Accumulibacter phosphatis." The analysis sheds light on several controversies in EBPR metabolic models and provides hypotheses explaining the dominance of A. phosphatis in this habitat, its lifestyle outside EBPR and probable cultivation requirements. Comparison of the same species from different EBPR sludges highlights recent evolutionary dynamics in the A. phosphatis genome that could be linked to mechanisms for environmental adaptation. In spite of an apparent lack of phylogenetic overlap in the flanking communities of the two sludges studied, common functional themes were found, at least one of them complementary to the inferred metabolism of the dominant organism. The present study provides a much needed blueprint for a systems-level understanding of EBPR and illustrates that metagenomics enables detailed, often novel, insights into even well-studied biological systems.     

Analysis of microbial communities using culture-dependent and culture-independent approaches in an anaerobic/aerobic SBR reactor

Comparative analysis of microbial communities in a sequencing batch reactor which performed enhanced biological phosphorus removal (EBPR) was carried out using a cultivation-based technique and 16S rRNA gene clone libraries. A standard PCR protocol and a modified PCR protocol with low PCR cycle was applied to the two clone libraries of the 16S rRNA gene sequences obtained from EBPR sludge, respectively, and the resulting 424 clones were analyzed using restriction fragment length polymorphisms (RFLPs) on 16S rRNA gene inserts. Comparison of two clone libraries showed that the modified PCR protocol decreased the incidence of distinct fragment patterns from about 63% (137 of 217) in the standard PCR method to about 34% (70 of 207) under the modified protocol, suggesting that just a low level of PCR cycling (5 cycles after 15 cycles) can significantly reduce the formation of chimeric DNA in the final PCR products. Phylogenetic analysis of 81 groups with distinct RFLP patterns that were obtained using the modified PCR method revealed that the clones were affiliated with at least 11 phyla or classes of the domain Bacteria. However, the analyses of 327 colonies, which were grouped into just 41 distinct types by RFLP analysis, showed that they could be classified into five major bacterial lineages: alpha, beta, gamma- Proteobacteria, Actinobacteria, and the phylum Bacteroidetes, which indicated that the microbial community yielded from the cultivation-based method was still much simpler than that yielded from the PCR-based molecular method. In this study, the discrepancy observed between the communities obtained from PCR-based and cultivation-based methods seems to result from low culturabilities of bacteria or PCR bias even though modified culture and PCR methods were used. Therefore, continuous development of PCR protocol and cultivation techniques is needed to reduce this discrepancy.