J. Gen. Appl. Microbiol., 48, 125–133 (2002)

Full Paper

Isolation and characterization of a Gram-positive polyphosphate-accumulating bacterium

Shin Onda and Susumu Takii*

Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo 192–0397, Japan

(Received October 31, 2001; Accepted March 9, 2002)

A Gram-positive polyphosphate-accumulating bacterium was isolated from phosphate-removal activated sludge using pyruvate-supplemented agar plates. The isolate was oval or coccobacilli (0.4–0.70.5–1.0 mm) that occurred singly, in pairs or irregular clumps. Polyphosphate granules in the cells were observed by toluidine blue staining. The pure culture of the isolate rapidly took up phosphate (9.2 mg-P/g-dry weight) in the 3-h aerobic incubation without organic substrates, after anaerobic incubation with organic substrates containing casamino acids. When acetate was the sole carbon source in the anaerobic incubation, the isolate did not remove phosphate. These physiological features of the isolate were similar to those of Microlunatus phosphovorus. However, unlike M. phosphovorus the P-removal ability of the isolate was relatively low and was not accelerated by repeating the anaerobic/aerobic incubation cycles. Phylogenetic analysis and comparison of several characteristics showed that the isolate was identified as elongata which was recently proposed as a new polyphosphate-accumulating isolated

from activated sludge. As the isolate contained menaquinone (MK)-8(H4) as the predominant iso- prenoid ubiquinone, it may be significantly responsible for phosphate removal, because MK-

8(H4) has reportedly been found in fairly high proportions in many phosphate-removing activated sludges.

Key Words——activated sludge; menaquinone-8(H4); phosphate-removal; polyphosphate; Tetra- sphaera elongata

Introduction ganic pollutants from wastewater. Since phosphate in wastewater causes eutrophication, phosphate-removal Activated sludge systems are widely used for treat- (P-removal) is important to prevent water pollution. In ment of municipal sewage and industrial wastewater. the EBPR system, microbes called polyphosphate-ac- An enhanced biological phosphorus removal (EBPR) cumulating organisms (PAOs) are considered to be re- system is one of the activated sludge systems modi- sponsible for phosphate removal. Under anaerobic fied by attaching an anaerobic tank in front of the aera- conditions, PAOs take up carbon substrates (e.g., ac- tion tank to efficiently remove phosphate along with or- etate, propionate) and store them as a form of polyhy- droxyalkanoate (PHA) while releasing phosphate, uti- lizing polyphosphate (poly-P) in their cells as an en- * Address reprint requests to: Dr. Susumu Takii, Department of Biological Sciences, Graduate School of Science, Tokyo Met- ergy source. Under subsequent aerobic conditions, uti- ropolitan University, 1–1 Minami-ohsawa, Hachioji, Tokyo 192– lizing the carbon storage in their cells, PAOs take up 0397, Japan. more phosphate than they released under anaerobic E-mail: [email protected] conditions and accumulate it in the form of poly-P 126 ONDA and TAKII Vol. 48

(Comeau et al., 1986; van Groenestijn et al., 1987; Ko- and Morikawa, 1999). rtstee et al., 2000; Mino et al., 1998). Acinetobacter spp. were first investigated as PAOs Materials and Methods possibly responsible for P-removal (Kortstee et al., 2000; Mino et al., 1998). Microlunatus phosphovorus Reactor and activated sludge sample. An acti- (Nakamura et al., 1995; Ubukata and Takii, 1997) and vated sludge sample was collected from the outlet of Lampropedia sp. (Stante et al., 1997) were also iso- the aeration tank of a laboratory-scale anaerobic/aero- lated as PAOs which showed a similar ability to metab- bic continuous flow reactor. The reactor consisted of olize phosphate to P-removal activated sludges. How- an anaerobic tank (5 L working volume) and an aero- ever, some of their characteristics are different from bic tank (2 L working volume). The artificial sewage the P-removal activated sludges, and culture-indepen- fed was composed of (mg/L): CH3COOH (100), dent methods showed that they did not dominate in P- C2H5COOH (75), peptone (140), yeast extract (60), removal activated sludges (Bond et al, 1999; Crocetti H3PO4 (15.5 as P), KHCO3 (65), NaHCO3 (100), et al., 2000; Hesselmann et al., 1999; Hiraishi et al., MgSO4 ·7H2O (100), CaCl2 ·5H2O (25) and NaCl (100). 1998; Kortstee et al., 2000; Mino et al., 1998). The influent was loaded at 30 L/day, and the return Culture-independent methods such as molecular sludge was 120 L/day (Maejima and Matsuo, 1997). At methods and quinone profiling have been used to in- sampling time, mixed liquor suspended solid (MLSS) vestigate bacterial communities in P-removal activated and mixed liquor volatile suspended solid (MLVSS) sludges. Recently, Hesselmann et al. (1999) and Cro- were 5,160 mg/L and 3,790 mg/L, respectively. The pH cetti et al. (2000) reported that a bacterium closely re- was 6.8 in the anaerobic tank, 7.6–8.2 in the aerobic lated to Rhodocyclus, named Candidatus Accu- tank. The concentration of PO4-P in the effluent was mulibacter phosphatis (Hesselmann et al., 1999), was 0.2 mg/L and the total phosphorus content of the responsible for P-removal by cloning of 16S rDNA and sludge was 8.4% (Onda et al., 2002). fluorescent in situ hybridization (FISH). In our previous Pure isolation and media. The 10-ml activated report (Onda et al., 2002) this bacterium was also sludge sample was diluted with 40 ml of pyrophos- shown to be the dominant PAO in a laboratory-scale phate solution (sodium tripolyphosphate 5 mg/L, NaCl anaerobic/aerobic continuous flow reactor by using de- 8.5 g/L) and homogenized (11,500 rpm) with a waring naturing gradient gel electrophoresis of PCR-amplified blender (Ace Homogenizer: Nihon Seiki Co., Ltd., 16S rDNA, quinone profiling, and FISH. However, it Tokyo, Japan) for 5 min. One milliliter of sludge sus- has never been successfully isolated, so far as we pension was diluted with 19 ml pyrophosphate solution know. Culture-independent methods also suggested and homogenized again for 10 min. The sludge sus- that having menaquinone (MK)-8(H4) pension was serially diluted with the pyrophosphate was another important member of the EBPR microbial solution and spread on agar plates. For isolation and community (Hiraishi et al., 1998). Recently, poly-P ac- growth, R2A (Reasoner and Geldreich, 1985), 1/2PYG cumulating Tetrasphaera spp. were isolated and de- supplemented with pyruvate (Sugitate and Morikawa, scribed from P-removal activated sludges (Hanada et 1999), and artificial sewage supplemented with pyru- al., 2002; Maszenan et al., 2000). However, these vate (ASP) media were used. The compositions of the strains have not been reported to show characteristics media were as follows: R2A medium (g/L); yeast ex- similar to the phosphate metabolism in relation to P-re- tract (0.5), peptone (0.5), casamino acids (Difco, De- moval activated sludges. troit, MI, USA) (0.5), glucose (0.5), soluble starch (0.5),

To better understand EBPR systems, isolation of sodium pyruvate (0.3), K2HPO4 (0.3) and MgSO4 · dominant PAOs is important. In this experiment we 7H2O (0.05); 1/2PYG with pyruvate medium (g/L); pep- tried to isolate and characterize new PAOs from the tone (1), yeast extract (0.5), glucose (0.25) and laboratory-scale reactor described above (Onda et al., sodium pyruvate (0.15); ASP medium (mg/300 ml); 2002). For isolation, pyruvate-supplemented agar sodium acetate (50), sodium propionate (50), yeast ex- plates were used in this study, because it was reported tract (50), casamino acids (Difco) (100), sodium pyru- that the addition of sodium pyruvate to agar media vate (45), activated sludge extract (60 ml), MgSO4 · greatly increased the numbers of colonies on agar 7H2O (15), CaCl2 ·2H2O (5), KH2PO4 (50) and NaCl plates from some environmental samples (Sugitate (100). The media for the agar plates were supple- 2002 A polyphosphate-accumulating bacterium 127 mented with 1.5% agar. All media were adjusted to the Gram-staining and the toluidine blue staining. PHA pH 7.5. After the inoculation, the agar plates were in- granules were stained with Nile blue according to the cubated at 28°C for about three weeks. Colonies ap- protocol of Ostle and Holt (1982) and were examined pearing on the agar plates were picked up and purified under an epifluorescent microscope (BH-2) with filter by repeated streaking. set BH2-DMU. Cultivation of isolates and P-uptake experiments. Physiological and biochemical characteristics. Ni- Isolates were aerobically grown at 20°C or 30°C in trate reduction, gelatin hydrolysis, indole production, shaking flasks (500 ml) plugged with cotton plugs. The catalase and oxidase were examined according to culture media were the same in composition as the Smihert and Krieg (1981) using R medium of Martin et isolation media. To obtain sufficient cells for P-uptake al. (1997) as the basal medium. The GC content of experiments, organic substrates (casamino acids 1.2 DNA was determined by the HPLC method (Suzuki et g/L, acetate 1.2 g/L and yeast extract 0.4 g/L) were al., 1987). Menaquinones were analyzed by reverse added to the APS medium. phase HPLC (Yamada and Kuraishi, 1982). The ability of the isolates to remove phosphate was Total cell counting. Total cell numbers of the acti- tested under similar conditions to those in EBPR sys- vated sludge sample and liquid cultures were deter- tems as follows. Organic substrates (mixture of mined by acridine orange staining. The sample was di- casamino acids 1.2 g/L, acetate 1.2 g/L and yeast ex- luted with the pyrophosphate solution and dispersed tract 0.4 g/L) were first added to their cultures at the by sonication (60 s, 5 W/ml, Sonicator 5202; Ohtake stationary phase, and the gas phase was replaced Works, Tokyo, Japan). The dispersed sample was with N2 gas to create anaerobic conditions. After 5-h stained with acridine orange (final concentration: anaerobic incubation at 20°C, the cells were harvested 0.01%) for 1 min. The stained sample was trapped by by centrifugation (3,000g, 10 min) and resuspended vacuum onto a track-etched membrane filter (Nucle- in an inorganic medium composed of (mg/L): MgSO4 · pore, pore size, 0.2 mm; Nuclepore Corp., Pleasanton,

7H2O (30), CaCl2 ·2H2O (10), NaCl (100) and KH2PO4 CA, USA). An epifluorescence microscope (IMT-2:

(50, 20 or 10 as PO4-P), with pH adjusted to 6.8–7.0. Olympus Co.) with filter set IMT2-DMU was used for The cell suspension was aerobically incubated for 19 h counting cells. The cell number was calculated by av- at 20°C. The concentration of phosphate in the super- eraging the count of 10 random fields on each filter. natant was determined by the ascorbic acid method DNA sequence and phylogenetic analysis. DNA (Japanese Industrial Standard Committee, 1989). This was extracted from the isolate as described previously P-uptake test was successively repeated 4 times for (Onda et al., 2002), and 16S rDNA was amplified by each isolate. PCR with a primer set of 27f and 1492r (Lane, 1991). The effects of substrates added during anaerobic in- The DNA sequences of the PCR products were deter- cubation on the P-uptake were also examined for one mined with an automatic DNA sequencer (ABI PRISM isolate, strain ASP12. The cultured cells were har- 377: Applied Biosystems, San Jose, CA, USA). The vested by centrifugation (3,000g, 10 min, 4°C) and sequencing reactions were carried out with an ABI washed once with the inorganic medium. Acetate PRISM BigDye Terminator Cycle Sequencing Ready (1.2 g/L) only, casamino acids (1.2 g/L) only or the Reaction Kit (Perkin Elmer, Foster City, CA, USA) ac- mixed substrates described above were added to each cording to the manufacturer’s instructions. The se- cell suspension under anaerobic conditions and the P- quences were compared with available 16S rDNA se- uptake ability was examined by the procedure de- quences in the DNA Databank of Japan (DDBJ). scribed above. SSEARCH program with Smith-Waterman algorithm Morphological observation. Gram-staining of the (Smith and Waterman, 1981) was used to search the isolates was done by using Favor-G Set-F (Nissui relatives of the isolate and to calculate similarities. The Pharmaceutical Co., Ltd., Tokyo, Japan). The isolates phylogenetic tree was constructed by the neighbor- were stained with 1% toluidine blue to visualize poly-P joining algorithm (Saitou and Nei, 1987) using the granules (Doetcsh, 1981). Toluidine blue stains cells in Clustal W program. blue and poly-P in red. Light microscopes (BH-2: 16S rDNA sequence accession number. The se- Olympus Co., Tokyo, Japan; Axioplan 2: Carl Zeiss quence of the isolate, strain ASP12, has been de- Co., Ltd., Jena, Germany) were used for examining posited in DDBJ under the accession number of 128 ONDA and TAKII Vol. 48

Fig. 1. Time courses of colony formation on agar plates. A. Comparison of the colony formation on artificial sewage with pyruvate (ASP, square) medium and medium without pyruvate (cir- cle). B. Comparison of the colony formation on three different media containing pyruvate: ASP (square), R2A (circle) and 1/2PYG with pyruvate (triangle).

AB051430.

Results and Discussion

Effect of pyruvate addition to agar media on colony for- mation Figure 1 shows the time courses of colony formation on the enumeration plates. The artificial sewage medium amended with pyruvate (ASP medium) recov- ered more than 2-fold the number of colonies grown on the medium without pyruvate (Fig. 1A). Of three isolation media, the ASP medium yielded the highest count of colonies (Fig. 1B). More than 30% of the total Fig. 2. Phosphate release in anaerobic incubation and sub- cell count formed colonies on the APS agar plate, sequent phosphate uptake in aerobic incubation by strain whereas the other media yielded colonies less than ASP12. 20% of total cells. Total cell count was 1.8109 cells ml1 at the start of the aer- obic incubation. Strain isolation and P-uptake experiments

A total of 49 colonies on the ASP agar plates and 32 PO4-P/cell) in the 5-h anaerobic incubation, and took colonies on the other media were picked up at random up 9.2 mg PO4-P/g-dry weight (5.6 fg PO4-P/cell) in the and isolated by repeated streaking. Each isolate was 3-h aerobic incubation. About 73% of initial phosphate sorted into groups based on their colony and cell mor- was taken up within 1 h. This physiological characteris- phology. Some isolates from each group were used for tic was similar to that of P-removal activated sludge the P-uptake experiments. (Kortstee et al., 2000; Mino et al., 1998). However, P- Only one isolate, strain ASP12, took up phosphate uptake amount by the isolate was less than that by the in the aerobic incubation without organic matter and original P-removal activated sludge sample which took released it under the anaerobic condition with organic up 51 mg PO4-P/g-dry weight (73 fg- PO4-P/cell) in 1- substrates (the mixture of acetate, casamino acids and day incubation (Onda et al., 2002). The cell number yeast extract). Figure 2 shows the time course of did not change during the anaerobic incubation for phosphate concentration in the ASP12 culture. The 10 h, but it increased from 6.8108 to 1.0109/ml dur- isolate released 7.1 mg PO4-P/g-dry weight (4.3 fg ing the aerobic incubation without organic substrate for 2002 A polyphosphate-accumulating bacterium 129

Table 1. Effect of repeating anaerobic/aerobic incubation cycles on aerobic phosphate removal by ASP12.

Anaerobic/aerobic incubation cycle 1st 2nd 3rd 4th

Phosphate removed in aerobic incubation (mg PO4-P/L) 7.3 8 6.3 5.9

Initial cell density, 1.3109 cells ml1. 5-h anaerobic incubation followed by 19-h aerobic incubation at 20°C.

Table 2. Effect of organic substrates on anaerobic phosphate release and aerobic phosphate removal by ASP12.

Anaerobic release of phosphateb Aerobic uptake of phosphatec (mg PO -P/L) (mg PO -P/L) Substrate 4 4

1st 2nd 1st 2nd

Mixed substratesa 5.6 9.5 9.3 11.6 Casamino acids 9.4 9.5 8.1 11.9 Acetate 0.6 1.5 2.7 0.8

a Mixture of acetate, casamino acids and yeast extract. b 5-h incubation. c 19-h incubation. Initial cell density, 1.8109 cells ml1; incubation temperature, 20°C.

35 h. Thus, the cell division of strain ASP12 occurred only in the aerobic phase as was true of the case with M. phosphovorus (Ubukata, 1994). The anaerobic and aerobic incubation cycles were successively repeated, but the P-uptake did not in- crease (Table 1). Under the experimental conditions, the P-uptake ability was stable during at least 4 cycles. Ubukata and Takii (1994) reported that the enzyme system of excess phosphate accumulation in M. phos- phovorus was induced after at least two anaerobic/aer- obic cycles. The enzyme system of the P-uptake may be constitutive in strain ASP12. Table 2 shows the effect of the substrates added on P-release in the anaerobic incubation and subsequent P-removal in the aerobic incubation. The anaerobic in- cubation with mixed substrates or casamino acids in- duced the release of phosphate followed by the uptake of phosphate in the aerobic incubation. On the other Fig. 3. Phase-contrast micrographs of strain ASP12. hand, the isolate neither released phosphate anaerobi- Bar, 5 mm. cally nor took it up aerobically, when only acetate was added in the anaerobic incubation. This physiological characteristic was similar to M. phosphovorus, but dif- Characterization of strain ASP12 fered from that of P-removal activated sludge, because Strain ASP12, was Gram-positive, oval or coc- acetate addition in the anaerobic phase is effective in cobacilli (0.4–0.70.5–1.0 mm), occurred singly, in the release and aerobic removal of phosphate by P-re- pairs or irregular clumps, and was non-motile (Fig. 3). moval activated sludges (Kortstee et al., 2000; Mino et The isolate contained poly-P granules, but no PHA al., 1998). granules. The isolate reduced nitrate to nitrite, did not 130 ONDA and TAKII Vol. 48 produce indole, hydrolyse gelatin, and produced cata- and strain ASP12 might predominate therein. Liu et al. lase but not oxidase. Two isoprenoid quinones MK- (2001) also showed by using FISH that Tetrasphaera

8(H4) and MK-7(H4) with a composition ratio of 88 : 5 japonica and related groups occupied about 17% of were observed in the isolate. The genomic DNA GC cells hybridized with eubacteria-targeted probe (EUB content was 68 mol%. 338) in the same EBPR system used here. The acti- About 30% of the isolate that appeared on the ASP vated sludges in EBPR systems have been reported to medium was morphologically similar to strain ASP12. contain actinobacteria (Bond et al., 1999; Kawahara- Since 34% of total bacterial cells formed colonies on saki et al., 1999; Wagner et al., 1994) and also MK- the medium, this bacterial type was estimated to ac- 8(H4) (Hiraishi et al., 1998) in fairly high proportions. count for about 10% of the bacterial population in Therefore, such as strain ASP12 may be dis- the original activated sludge sample. This estimation tributed more abundantly than M. phosphovorus which seemed to be reasonable, because the activated has MK-9(H4) as the main menaquinone, and may sludge contained MK-8(H4) at 11 mol% of total iso- sometimes be important PAOs in EBPR systems. prenoid quinone (Onda et al., 2002). Culture-indepen- dent methods showed that the dominant PAO in this Phylogenetic analysis activated sludge sample was related to Candidatus 16S rDNA of strain ASP12 was sequenced (1,390 Accumulibacter phosphatis, a large coccus (1.5–2.0 bp) and compared to available 16S rDNA sequences mm) (Onda et al., 2002). That bacterium was not found in the database (DDBJ). Phylogenetic analysis showed in more than 80 isolates from the isolation media. An- that strain ASP12 was a close relative of ‘Candidatus other activated sludge sample obtained from the same Nostocoida limicola’ (97.1% similarity; Blackall et al., system after 6 months contained MK-8(H4) as the 2000), Tetrasphaera (T. australiensis, 96.8% similarity; most abundant quinone (33 mol%, Onda et al., 2002), Maszenan et al., 2000) and Janibacter (J. limosus,

Fig. 4. Neighbor-joining tree of strain ASP12 and its close relatives in actinobacterial division based on a comparative analysis of 1,390 nucleotides of 16S rDNA sequences. The outgroup was Escherichia coli (X80732). Numbers on the branches refer to bootstrap values for 1,000 times. Accession num- bers appear in the parenthesis. 2002 A polyphosphate-accumulating bacterium 131 c ) m) 4 s and in m N.D. MK-8(H Janibacter c ) m) (0.2–1.2 4 m MK-8(H japonica c ) m) (0.6–1.4 4 m australiensis b N.D. V N.D. MK-8(H N.D. plants filaments clusters clusters Candidatus Tetrasphaera Tetrasphaera ‘ Nostocoida limicola’ m) (0.4–1.1 m a ) 4 1.0–1.8 N.D. N.D. N.D. N.D. N.D. N.D. N.D. MK-8(H (strain Lp2) etrasphaera elongata T m) (0.7–1.0 m able 3. Comparative phenotypic properties of the phylogenetical relatives ASP12. T ) 4 0.5–1.0 sludge sludge sewage treatment in Australia biomass in Japan irregular clumps irregular twisting arrangement and arrangement and irregular clumps variable according to strains. Single, in pairs and or irregular clumps clumps of cocci, Single, in pairs, tetrad Single, in pairs, tetrad Single, in pair (0.4–0.7 not determined; V C mol% 68 69.6 N.D. 68, 70 71 70 requirement Aerobic Aerobic Aerobic Aerobic Aerobic Aerobic aerobic conditions anaerobic conditions and P-uptake under 2 Data from Hanada et al. (2002). Data from Blackall et al. (2000). Data from Maszenan et al. (2000). Phenotypic property Strain ASP12 Catalase Oxydase G Nitrate reduction Production of indole O HabitatMotilityMajor menaquinone P-removal activated P-removal activated MK-8(H Activated sludge, Sewage treatment plant Non-motile Activated sludge Sludge, sewage waste Non-motile Non-motile Non-motile Non-motile Non-motile Cell morphology Ovals or coccobacilli. Ovoids to short rods Short filaments, Cocci. Cocci. Cocci or rods. Gelatin hydrolysis Poly-P granule P-release under N.D. a b c 132 ONDA and TAKII Vol. 48

96.0% similarity; Martin et al., 1997) within the family (1999) Identification of some of the major groups of bacte- (Maszenan et al., 2000), which be- ria in efficient and nonefficient biological phosphorus re- long to the Gram-positive bacteria with a high GC moval activated sludge systems. Appl. Environ. Microbiol., DNA content (Actinobacteria). The neighbor-joining 65, 4077–4084. Comeau, Y., Hall, K. J., Hancock, R. E. W., and Oldham, W. K. tree of the group (Fig. 4) shows that strain ASP12 is (1986) Biochemical model for enhanced biological phos- within the cluster of ‘Candidatus Nostocoida limicola’ phorus removal. Water Res., 20, 1511–1521. and genus Tetrasphaera. The features of this bacterial Crocetti, G. R., Hugenholts, P., Bond, P. L., Schuler, A., Keller, group are shown in Table 3. Quite recently, Hanada et J., Jenkins, D., and Blackall, L. L. (2000) Identification of al. (2002) proposed a new species of Tetrasphaera, as polyphosphate-accumulating organisms and design of 16S T. elongata which was isolated as a poly-P-accumulat- rRNA-directed probes for their detection and quantitation. ing bacterium from an activated sludge. Its 16S rDNA Appl. Environ. Microbiol., 66, 1175–1182. similarity to ASP12 was 99.6%. The morphological Doetcsh, R. N. (1981) Determinative methods of light mi- croscopy. In Manual of Methods for General Microbiology, feature was also very similar to ASP12. Therefore, ed. by Gerhardt, P., Murray, R. G. E., Costilow, R. N., ASP12 should be identified as T. elongata. Nester, E. W., Wood, W. A., Krieg, N. R., and Phillips, G. None of the relatives of strain ASP12 have been re- B., American Society for Microbiology, Washington DC, pp. ported to show the following characteristics: P-uptake 21–33. under aerobic conditions in the absence of organic van Groenestijn, J. W., Deinema, M. H., and Zehnder, A. J. B. matter, and P-release under anaerobic conditions. (1987) ATP production from polyphosphate in Acinetobac- In conclusion, a Gram-positive PAO belonging to the ter strain 210A. Arch. Microbiol., 148, 14–19. Hanada, S., Liu, W.-T., Shintani, T., Kamagata, Y., and Naka- Intrasporangiaceae and having MK-8(H4) as a major quinone was isolated from P-removal activated sludge mura, K. (2002) sp. nov., a polyphosphate accumulating bacterium isolated from acti- by using the isolation media supplemented with pyru- vated sludge. Int. J. Syst. Evol. Microbiol., 52, 883–887. vate and was identified as T. elongata which was Hesselmann, R. P. X., Werlen, C., Hahn, D., van der Meer, J. newly proposed by Hanada et al. (2002). The isolate R., and Zehnder, A. J. B. (1999) Enrichment, phylogenetic rapidly took up phosphate aerobically in the absence analysis and detection of a bacterium that performs en- of organic substrates, after anaerobic incubation with hanced biological phosphate removal in activated sludge. organic substrates concomitant with releasing phos- Syst. Appl. Microbiol., 22, 454–465. phate. This physiological feature was similar to that in Hiraishi, A., Ueda, Y., and Ishihara, J. (1998) Quinone profiling P-removal activated sludges except for the inability to of bacterial communities in natural and synthetic sewage activated sludge for enhanced phosphate removal. Appl. take up acetate anaerobically and to store polyalka- Environ. Microbiol., 64, 992–998. noates. These physiological features were rather Japanese Industrial Standards Committee (1989) Phosphorus similar to that of M. phosphovorus, but its main compounds and total phosphorus (JIS K0102). In Testing menaquinone (MK-8(H4)) is more widely distributed Methods for Industrial Wastewater, Japanese Industrial than that of M. phosphovorus (MK-9(H4)) in EBPR sys- Standard Association, Tokyo, pp. 834–841 (in Japanese). tems. The isolate may well be a candidate for Gram- Kawaharasaki, M., Tanaka, H., Kanagawa, T., and Nakamura, positive PAOs frequently observed in such systems. K. (1999) In situ identification of polyphosphate-accumulat- ing bacteria in activated sludge by dual staining with rRNA- targeted oligonucleotide probes and 4,6-diamidino-2- Acknowledgments phenylindol (DAPI) at a polyphosphate-probing concentra- tion. Water Res., 33, 257–265. We are grateful to Professor Y. Matsuo of Chuo University for Kortstee, G. J. J., Appeldoorn, K. J., Bonting, C. F. C., van Niel, his kind provision of an activated sludge sample from his labo- W. J., and van Veen, H. J. (2000) Ecological aspects of bio- ratory system and its operation data. logical phosphorus removal in activated sludge systems. Adv. Microb. Ecol., 16, 169–199. References Lane, D. J. (1991) 16S/18S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, ed. by Stackebrandt, Blackall, L. L., Seviour, E. M., Bradford, D., Rossetti, S., Tandoi, E. and Goodfellow, M., Wiley, New York, pp. 115–175. V., and Seviour, R. J. (2000) ‘Candidatus Nostocoida limi- Liu, W.-T., Nielsen, A. T., Wu, J.-H., Tsai, C.-S., Matsuo, Y., and cola,’ a filamentous bacterium from activated sludge. Int. J. Molin, S. (2001) In situ identification of polyphosphate- and Syst. Evol. Microbiol., 50, 703–709. polyhydroxyalkanoate-accumulating traits for microbial Bond, P. L., Erhart, R., Wagner, M., Keller, J., and Blackall, L. L. populations in a biological phosphorus removal process. 2002 A polyphosphate-accumulating bacterium 133

Environ. Microbiol., 3, 110–122. Smihert, R. M. and Krieg, N. R. (1981) General characteriza- Maejima, H. and Matsuo, Y. (1997) Behavior of PHA in en- tion. In Manual of Methods for General Microbiology, ed. by hanced biological phosphorus removal activated sludge. Gerhardt, P., Murray, R. G. E., Costilow, R. N., Nester, E. Environ. Eng. Res., 34, 183–190 (in Japanese with English W., Wood, W. A., Krieg, N. R., and Phillips, G. B., American abstract). Society for Microbiology, Washington DC, pp. 409–443. Martin, K., Schumann, P., Rainey, F. A., Schuetze, B., and Smith, T. F. and Waterman, M. S. (1981) Identification of com- Groth, I. (1997) Janibacter limosus gen. nov., sp. nov., a mon molecular subsequences. J. Mol. Biol., 147, 195–197. new actinomycete with meso-diaminopimelic acid in the Stante, L., Cellamare, C. M., Malaspina, F., Bortone, G., and cell wall. Int. J. Syst. Bacteriol., 47, 529–534. Tilche, A. (1997) Biological phosphorus removal by pure Maszenan, A. M., Sevioour, R. J., Patel, B. K., Schumann, P., culture of Lampropedia spp. Water Res., 31, 1317–1324. Burghardt, J., Tokiwa, Y., and Stratton, H. M. (2000) Three Sugitate, T. and Morikawa, K. (1999) Increase in colony counts isolates of novel polyphosphate-accumulating Gram-posi- of river bacterial community by supplementing with sodium tive cocci, obtained from activated sludge, belong to a new pyruvate, Microb. Environ., 14, 85–87 (in Japanese). genus, Tetrasphaera gen. nov., and description of two new Suzuki, K., Ezaki, T., and Park, Y.-H. (1987) Analysis on nucleic species, sp. nov. and Tetrasphaera acids. In Bacterial Identification in Accordance with Recent australiensis sp. nov. Int. J. Syst. Evol. Microbiol., 50, , ed. by Education Committee, Japanese Society 593–603. of Bacteriology, Saikon Publishing, Tokyo, pp. 88–123 (in Mino, T., van Loosdrecht, M. C. M., and Heijnen, J. J. (1998) Japanese). Microbiology and biochemistry of the enhanced biological Ubukata, Y. (1994) Some physiological characteristics of a phosphate removal process. Water Res., 32, 3193–3207. phosphate removing bacterium isolated from anaerobic/ Nakamura, K., Hiraishi, A., Yoshimi, Y., Kawaharasaki, M., Masu- aerobic activated sludge. Water Sci. Technol., 30, da, K., and Kamagata, Y. (1995) Microlunatus phospho- 229–235. vorus gen. nov., sp. nov., a new Gram-positive phosphate- Ubukata, Y. and Takii, S. (1994) Induction ability of excess accumulating bacterium isolated from activated sludge. Int. phosphate accumulation for phosphate removing bacteria. J. Syst. Bacteriol., 45, 17–22. Water Res., 28, 247–249. Onda, S., Hiraishi, A., Matsuo, Y., and Takii, S. (2002) Polypha- Ubukata, Y. and Takii, S. (1997) Some physiological character- sic approaches to the identification of predominant istics of a phosphate-removing bacterium, Microlunatus polyphosphate-accumulating organisms in a laboratory- phosphovorus, and a simplified isolation and identification scale anaerobic/aerobic activated sludge system. J. Gen. method for phosphate-removing bacteria. Water Sci.Tech- Appl. Microbiol., 48, 43–54. nol., 38, 149–157. Ostle, A. G. and Holt, J. G. (1982) Nile blue A as a fluorescent Wagner, M., Erhalt, R., Manz, W., Amann, R., Lemmer, H., stain for poly-b-hydroxybutyrate. Appl. Environ. Microbiol., Wedi, D., and Schleifer, K.-H. (1994) Development of a 44, 238–241. rRNA-targeted oligonucleotide probe specific for the genus Reasoner, D. J. and Geldreich, E. E. (1985) A new medium for Acinetobacter and its application for in situ monitoring in the enumeration and subculture of bacteria from potable activated sludge. Appl. Environ. Microbiol., 60, 792–800. water. Appl. Environ. Microbiol., 49, 1–7. Yamada, Y. and Kuraishi, H. (1982) Ubiquinone and Saitou, N. and Nei, M. (1987) The Neighbor-joining method: A menaquinone. In Experimental Methods for Chemotaxon- new method for reconstructing phylogenetic trees. Mol. omy of Microorganisms, ed. by Komagata, K., Gakkai Biol. Evol., 4, 406–425. Shuppan Center, Tokyo, pp. 143–155 (in Japanese).