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1 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
2 Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA
3 Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720, USA
Correspondence
Satoshi Hanada
s-hanada{at}aist.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Published online ahead of print on 24 January 2003 as DOI 10.1099/ijs.0.02520-0.
The GenBank/EMBJ/DDBJ accession number for the 16S rRNA gene sequence of strain T-27T is AB072735.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). In the past decade, culture-independent rRNA-based molecular methods have provided means of identifying the types of micro-organisms that occur in microbial communities without the need for cultivation (Amann et al., 1995; Pace et al., 1986; Hugenholtz & Pace, 1996). Consequently, the number of bacterial phyla (divisions) identified has more than tripled to over 30 due, in significant part, to culture-independent phylogenetic surveys of environmental microbial communities (Hugenholtz et al., 1998; Hugenholtz, 2002). Approximately a third to one half of these phyla are yet to be represented by cultured organisms and are characterized only by environmental 16S rDNA clone sequences (Hugenholtz, 2002).
Recently, a candidate division named the BD group was proposed by Hugenholtz et al. (2001) based on environmental sequence data. In their report, the BD group consisted of five 16S rDNA sequences from different environmental samples: two sequences from deep-sea sediments after which the group was named (Li et al., 1999), two unpublished sequences from soil samples and one sequence from an enhanced biological phosphorus removal (EBPR) sludge (Hugenholtz et al., 2001). The phylogenetic novelty of this group was recognized independently by other researchers at almost the same time and they named the lineage candidate division KS-B (Madrid et al., 2001). Additional environmental sequences, particularly from soil and marine habitats, are expanding the BD group (see below), but no cultivated representative of the group has been identified up to now.
We have isolated a bacterium, designated strain T-27T, from an anaerobic–aerobic sequential batch reactor operated under EBPR conditions as part of an ongoing survey of the diversity of this habitat. Phylogenetic analyses based on 16S rRNA gene sequences indicate that strain T-27T belongs to the BD group. In this paper, we characterize strain T-27T using a polyphasic approach and propose the name Gemmatimonas aurantiaca gen. nov., sp. nov. for the isolate. We also name the phylum that it represents Gemmatimonadetes phyl. nov.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). A 2·5 l polycarbonate cylinder (inner diameter, 10 cm; depth, 36 cm) with a working volume of 1·8 l was used as a reactor for the enrichment of EBPR sludge. The reactor was operated in a cycle with three distinct periods, an anaerobic period (60 min), an aerobic period (90 min) and a settling period (30 min). A volume of 0·9 l of treated water was replaced with fresh synthetic wastewater to give a biochemical oxygen demand (BOD) loading rate of 0·5 kg m-3. The synthetic wastewater contained 177 mg acetate, 55 mg peptone, 14·4 mg CaCl2, 95·0 mg MgSO4.7H2O, 12·7 mg (NH4)2SO4 and 21·1 mg KH2PO4 in 1 l tap water. The sludge retention time was controlled to 20 days and the sludge concentration remained at 4–5 g dry weight l-1. After 2 months of operation, the mean phosphorus content in the sludge at the end of aerobic period was about 60 mg P (g dry weight)-1.
Enrichment strategy and isolation.
A sludge sample was taken from the reactor and diluted to a concentration of 1000 mg l-1 with a dilution buffer containing 20 mM sodium pyrophosphate and 20 mM EDTA (pH 7·0). After a short ultrasonic treatment (Tomy Seiko; model UR-200P) (equipped with a micro-tip; output 10 W, 15 s, constant pulsing) to disperse aggregates, the sludge suspension was centrifuged at 100 g for 5 min to remove remaining aggregates. The supernatant was then subjected to a further centrifugation at 1400 g for 5 min to pellet heavier individual cells. After washing three times with distilled water, the pelleted bacterial cells were diluted and spread onto plates of NM-1 medium [Nakamura et al., 1995; containing (l-1) 0·5 g glucose, 0·5 g polypepton, 0·5 g sodium glutamate, 0·5 g yeast extract, 0·44 g KH2PO4, 0·1 g (NH4)2SO4, 0·1 g MgSO4.7H2O, 1 ml vitamin solution and 20 g agar]. The pH was adjusted to 7·0 with the addition of NaOH. The vitamin solution contained (l-1): 1·0 g nicotinic acid, 1·0 g thiamin hydrochloride, 50 mg biotin, 0·5 g p-aminobenzoic acid, 10 mg vitamin B12, 0·5 g calcium pantothenate, 0·5 g pyridoxine hydrochloride and 0·5 g folic acid. Strain T-27T was obtained by picking single, non-transparent colonies from the agar plates after incubation at 25 °C for 2 weeks. Strain T-27T was cultivated aerobically in liquid NM-1 medium at 30 °C for physiological and morphological analyses.
Microscopy.
Cell morphology was examined under a phase-contrast microscope (Olympus AX80T). For electron microscopy of ultrathin sections, cells were embedded in Spurr medium (Kushida, 1980) after fixing with 2·5 % (v/v) glutaraldehyde and 1 % (w/v) osmium tetroxide. Photomicrographs were obtained with a Hitachi H-7000 transmission electron microscope operated at 75 kV. Scanning electron micrographs were obtained as described previously (Sekiguchi et al., 2000).
Physiological and biochemical characterization.
Gram staining was performed as described by Magee et al. (1975). Oxidase activity was determined by monitoring the oxidation of tetramethyl-p-phenylenediamine on filter paper and catalase activity was determined using a 3 % hydrogen peroxide solution (Smibert & Krieg, 1994). For determination of nitrate reduction, strain T-27T was grown in NM-1 medium supplemented with 2·0 g NaNO3 l-1 under anaerobic conditions. Fermentative growth was tested in a screw-capped tube filled with NM-1 medium in the presence of 0·2 % glucose and/or 0·2 % yeast extract. Neisser staining was performed as described by Jenkins et al. (1993). DAPI (4,6-diamidino-2-phenylindole) staining was performed by addition of 10 µl of a solution of 1 mg DAPI l-1 to 1 ml cell suspension and subsequent incubation for 5 min. DAPI-stained cells were observed under a fluorescence microscope (Olympus AX80T). Carbon source utilization was determined using a basal medium supplemented with test substrates (0·5 g l-1). The basal medium contained (l-1) 0·44 g KH2PO4, 0·1 g (NH4)2SO4, 0·1 g MgSO4.7H2O and 1 ml vitamin solution, the pH being adjusted to 7·0.
Quinone, fatty acid and cell wall analyses.
Quinones were extracted with chloroform/methanol (2 : 1, v/v). The extract was purified by a Sep-Pak Plus column (Waters) and analysed by reverse-phase HPLC (Beckman System Gold with a Hewlett Packard Zorbox ODS column) for identification (Tamaoka et al., 1983). Cellular fatty acid methyl esters were analysed by a Hitachi M7200A GC/3DQMS system equipped with a DB-5ms capillary column (30 m by 0·25 mm) coated with (5 %-phenyl)-methylpolysiloxane (J & W Scientific). The analytical procedure was described by Hanada et al. (2002a). The presence of diaminopimelic acid (DAP) isomers in the cell-wall peptidoglycan was determined by TLC with a silica gel plate (Merck; no. 5716) after hydrolysis with 6 M HCl at 100 °C for 18 h (Komagata & Suzuki, 1987).
DNA base composition.
Total DNA of strain T-27T was extracted according to the procedure of Saito & Miura (1963). Total DNA was digested with P1 nuclease using a Yamasa GC kit (Yamasa Shoyu). The G+C content was measured by HPLC (Kamagata & Mikami, 1991).
16S rRNA gene sequence and phylogenetic analysis.
Cells of strain T-27T were lysed by the method of Hiraishi (1992). A 16S rDNA fragment was amplified by PCR (Hiraishi et al., 1994) using the universal primers 27F (5'-AGAGTTTGATCATGGCTCGA-3'; positions 8–27 in the Escherichia coli numbering system) and 1492R (5'-GGCTACCTTGTTACGACTT-3'; positions 1510–1492) (Weisburg et al., 1991). The PCR product was sequenced directly on an ABI 377 DNA sequencer using a dRhodamine Dye Terminator cycle sequencing kit (Applied Biosystems). The compiled 16S rDNA sequence was aligned against an ARB dataset using the ARB program package (http://www.arb-home.de/) and refined manually based on primary and secondary structural considerations. 16S rDNA sequences related to T-27T were obtained from the public databases by similarity searches and added to the ARB database. Putative chimeric sequences were identified by partial treeing analysis as described previously (Hugenholtz & Huber, 2003). Twenty-seven Gemmatimonadetes reference sequences and a bacterial domain reference sequence dataset (Hugenholtz, 2002) representing most of the major recognized bacterial phyla were selected to generate a de novo evolutionary distance tree using the Olsen correction and neighbour joining (Fig. 3). The Lane mask (Lane 1991), available as a filter in ARB, or a Gemmatimonas-specific mask created in ARB based on 50 % minimal similarity was applied to the dataset to remove regions of ambiguous positional homology, leaving 1287 or 1358 bp, respectively, for comparative analyses. Sequence identities between 16S rRNA gene sequences were calculated from the 1287 bp dataset. Phylogenetic trees based on the 16S rDNA dataset and smaller datasets containing only the Gemmatimonadetes sequences and various outgroups were constructed by three different algorithms according to a previous report (Kim et al., 2000); the neighbour-joining method (Saito & Nei, 1987) with the MEGA2 package (Kumar et al., 2001), the maximum-parsimony method with the PAUP* 4.0 package (Swofford, 2002) and the maximum-likelihood method with the MOLPHY package (Adachi & Hasegawa, 1996). Bootstrap resampling analysis (Felsenstein, 1985) for 1000 replicates was performed with neighbour-joining and maximum-parsimony analyses to estimate the confidence of tree topologies. For maximum-likelihood analyses, local bootstrap probabilities were estimated by the RELL (resampling of estimated log-likelihood) method (Kishino et al., 1990; Hasegawa & Kishino, 1994). In addition, posterior probabilities of branching points were estimated by Bayesian inference using MRBAYES version 2.01 (Huelsenbeck & Ronquist, 2001) with the default settings and general time-reversible model (16 substitution types, nst=6) and a gamma rate correction (rates=gamma) suggested for RNA data. A total of 100 000 generations were calculated for two truncated Gemmatimonadetes datasets, from which a tree was saved every 100 generations. The likelihoods of the trees (with WS3 or marine group A sequences used as outgroups) had converged on a stable value after 20 000 generations, and consensus trees were constructed from the final 800 trees in PAUP*.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-hydroxyalkanoate and glycogen. Following enrichment, cells were plated onto a heterotrophic agar medium, NM-1, and incubated for up to 12 weeks. Pinpoint and slowly appearing colonies were preferentially selected from the isolation plates and purified through repeated isolation of single colonies on NM-1 media. Twenty isolates were obtained in this manner and checked for polyphosphate inclusions by Neisser staining. Among the Neisser-positive isolates (13/20), one strain, designated T-27T, warranted further investigation based upon its novel 16S rDNA sequence.
After 2 weeks incubation, colonies of strain T-27T were circular, smooth, faintly orange to pink and only 1–2 mm in diameter. This slow growth, resulting in pinpoint colonies, may explain why no closely related isolates have been obtained in pure culture previously. No diffusible pigment production was observed. Cells were rod-shaped, Gram-negative, motile, 0·7 µm in width and 2·5–3·2 µm in length (mean size 2·8 µm) (Fig. 1). Spore formation was not observed. Electron microscopy confirmed that cells of strain T-27T possess a Gram-negative cell envelope, with the cytoplasmic and outer membranes readily visible (Fig. 2). Electron-dense and transparent inclusion bodies are present in TEM images (Fig. 2a). Cells reproduce by binary fission and often show budding morphology, suggesting asymmetrical cell division (Figs 1 and 2b).
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Strain T-27T was able to utilize a limited range of substrates as sole carbon sources: yeast extract, polypepton, succinate, acetate, gelatin and benzoate. The strain was also able to utilize the following substrates weakly: glucose, sucrose, galactose, melibiose, maltose, formate and -hydroxybutyrate. The following were not utilized: fructose, mannose, lactose, trehalose, raffinose, arabinose, xylose, rhamnose, glycerol, ethanol, 1-propanol, erythritol, mannitol, sorbitol, lactate, citrate, pyruvate, propionate, malate, butyrate, glutamate, aspartate, alanine, starch, glycogen, gentiobiose, turanose, methyl pyruvate, 2-oxoglutarate, 
-ketovalerate, serine, histidine, glycine, leucine and Casamino acids.
Chemotaxonomic characteristics
The major respiratory quinone of strain T-27T is menaquinone (MK)-9. The dominant fatty acids of strain T-27T are iso-C15 : 0 (45 % of total fatty acid methyl esters), C16 : 1 (27 %), C14 : 0 (12 %), C13 : 0 (9 %) and C16 : 0 (7 %). No DAP isomers were detected from the cell-wall peptidoglycan of strain T-27T by DAP analysis. The G+C content of genomic DNA of strain T-27T was 66·0 mol%.
Phylogenetic analysis
A nearly complete 16S rDNA sequence (1446 nucleotides) was obtained for strain T-27T. Comparative sequence analysis indicated that strain T-27T is not closely related to any known cultured micro-organisms in recognized bacterial phyla (
85 % sequence identity). Instead, it is a member of candidate division BD, a recently recognized phylum-level lineage in the domain Bacteria, to date containing only environmental clones obtained from a number of diverse habitats (Hugenholtz et al., 2001; see below for a discussion of this phylum). The environmental clones most closely related to strain T-27T (Ebpr21 and SBRH63, respectively 99 and 97 % sequence identity) were obtained from two geographically remote activated sludges operated under EBPR conditions (Hugenholtz et al., 2001; Liu et al., 2001). This suggests that strain T-27T and related organisms may be ubiquitous polyphosphate-accumulating bacteria in EBPR processes. Polyphosphate accumulation of the strain was suggested by Neisser and DAPI staining and the presence of electron-dense inclusion bodies (Fig. 2a
), although in situ polyphosphate accumulation remains to be verified.
How novel is strain T-27T?
Based on the standard set of phenotypic tests conducted in this study, strain T-27T is physiologically unremarkable. Many phylogenetically diverse bacteria are Gram-negative aerobic heterotrophs and a number of bacteria can accumulate polyphosphate, including ‘Accumulibacter phosphatis’ (a member of the Proteobacteria; Hesselmann et al., 1999), Microlunatus phosphovorus and Tetrasphaera species (members of the Actinobacteria) (Nakamura et al., 1995; Maszenan et al., 2000; Hanada et al., 2002b). Strain T-27T also has a pedestrian, if somewhat limited, carbon substrate utilization profile. Therefore, if isolates are screened using only phenotypic tests, phylogenetically novel micro-organisms may be overlooked. The chemotaxonomic and ultrastructural data obtained for strain T-27T, however, hint at a more exotic ancestry. The strain has an unusual combination of dominant fatty acids: 45 % iso-C15 : 0 (usually characteristic of Gram-positive bacteria) and 27 % C16 : 1 (more often found in Gram-negative heterotrophs). Similarly, it is unusual for Gram-negative bacteria to lack DAP in their cell-wall peptidoglycan and to grow aerobically using MK-9 as the major respiratory quinone (D. C. White, personal communication). Also, the cell envelope is unusual in that the space between the membranes (periplasmic space) is atypically wide for Gram-negative organisms (R. Webb, personal communication). More detailed molecular analyses, including genome sequencing, will confirm (or deny) the evolutionary novelty of strain T-27T indicated by its 16S rDNA sequence.
Phylogenetic delineation and environmental distribution of the phylum Gemmatimonadetes
The phylogenetic placement of the T-27T 16S rDNA was determined by construction of a neighbour-joining tree with related environmental clones and representative sequences of recognized bacterial phyla (Fig. 3; based on the dataset of Hugenholtz, 2002). Bootstrap resampling of the dataset using evolutionary distance, maximum parsimony and maximum likelihood and calculation of posterior probabilities using Bayesian inference confirmed that T-27T reproducibly affiliates with sequences belonging to the BD group and that the group represents an independent phylum-level lineage in the bacterial domain (Fig. 3). This is consistent with previous analyses (Hugenholtz et al., 2001; Madrid et al., 2001; Hugenholtz, 2002). For this reason, we have renamed group BD the phylum Gemmatimonadetes phyl. nov., in accordance with its first cultured representative (see conclusions below).
Over 100 environmental clone sequences belonging to the Gemmatimonadetes were identified in the public databases by similarity searches and comparative analysis. Of these, the majority are partial length clones (<1000 bp; not shown), 27 are near full-length (mostly >1250 bp, shown in Fig. 3) and five are putatively chimeric as determined by partial treeing analysis (Table 1; Hugenholtz & Huber, 2003). The maximum 16S rDNA sequence divergence for the phylum is 19 %, which is greater than the divergence found in the Actinobacteria (18 %) but less than the phylum ‘Proteobacteria’ (23 %; Dojka et al., 2000). Four subdivisions of the Gemmatimonadetes could be defined in the present analysis (Fig. 3), i.e. lineages of two or more full-length 16S rDNA sequences within the phylum that are reproducibly monophyletic and unaffiliated with all other representatives of the phylum (Hugenholtz et al., 1998). Interestingly, the subdivisions appear to be distinct in terms of where their representatives are found. Subdivision 1 clones were all obtained from terrestrial habitats and clones belonging to subdivisions 2 and 4 were all obtained from marine habitats (Fig. 3). This distinction may be due to sampling artefacts or may represent a real evolutionary split according to habitat type. However, subdivision 3, currently represented by only two full-length clones, is a mixture of one terrestrial (soil) and one marine (gas hydrate) clone (Fig. 3). Terrestrial habitats include soils (Axelrood et al., 2002; Dunbar et al., 2002; Holmes et al., 2000; Valinsky et al., 2002), activated sludges (Hugenholtz et al., 2001; Liu et al., 2001) and saline cave waters (Holmes et al., 2001). Marine habitats include deep-sea sediments (Li et al., 1999), gas hydrates (Lanoil et al., 2001), arctic bacterioplankton (Bano & Hollibaugh, 2002), coastal mobile mud (Madrid et al., 2001) and marine sponge symbionts (Hentschel et al., 2002; Webster et al., 2001). An rRNA clone, SB2 (accession no. AJ404563), obtained by RT-PCR from arthritis synovial tissue (Kempsell et al., 2000), was identified as belonging to subdivision 1 of the Gemmatimonadetes, suggesting that members of this phylum may also be present in clinical niches. Note that the class Gemmatimonadetes classis nov. defined below is phylogenetically equivalent to subdivision 1 in Fig. 3.
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), so we predict that members of the Gemmatimonadetes will have a Gram-negative cell envelope that lacks DAP.
Taxonomic conclusions
The genotypic and phenotypic findings detailed above indicate that strain T-27T is a novel and distinctive bacterium. Thus, we propose a new genus and species, Gemmatimonas aurantiaca gen. nov., sp. nov., to accommodate this isolate and create a new phylum, Gemmatimonadetes phyl. nov., with T-27T as the sole cultured representative. The taxonomic ranks between phylum and genus are also proposed according to the precedent set for Chrysiogenetes (Garrity & Holt, 2001).
Description of Gemmatimonadetes phyl. nov.
Gemmatimonadetes (Gem.ma'ti.mo.na.det'es. N.L. fem. pl. n. Gemmatimonas type genus of the type order of the phylum; N.L. fem. pl. n. Gemmatimonadetes phylum of the genus Gemmatimonas).
The phylum Gemmatimonadetes is defined on a phylogenetic basis by comparative 16S rDNA sequence analysis of one isolated strain and uncultured representatives from multiple terrestrial and aquatic habitats. Gram-negative bacteria lacking DAP in their cell envelopes.
Type order: Gemmatimonadales ord. nov.
Description of Gemmatimonadetes classis nov.
The description is the same as for the phylum Gemmatimonadetes.
Type order: Gemmatimonadales ord. nov.
Description of Gemmatimonadales ord. nov.
Gemmatimonadales (Gem.ma'ti.mo.na.dal'es. N.L. fem. pl. n. Gemmatimonas type genus of the order; -ales ending to donate an order; N.L. fem. pl. n. Gemmatimonadales the order of the genus Gemmatimonas).
The description is the same as for the genus Gemmatimonas.
Type genus: Gemmatimonas gen. nov.
Description of Gemmatimonadaceae fam. nov.
Gemmatimonadaceae (Gem.ma'ti.mo.na.da'ce.ae. N.L. fem. n. Gemmatimonas type genus of the family; -aceae ending to donate a family; N.L. fem. n. Gemmatimonadaceae family of the genus Gemmatimonas).
The description is the same as for the genus Gemmatimonas.
Type genus: Gemmatimonas gen. nov.
Description of Gemmatimonas gen. nov.
Gemmatimonas (Gem.ma'ti.mo'nas. L. adj. gemmatus provided with buds; L. n. monas a unit; N.L. fem. n. Gemmatimonas a budding unit).
Gram-negative. Cells are motile, rod-shaped. Spores are not formed. Cells divide by binary fission but sometimes show budding forms. Mesophilic. Cells grow under aerobic conditions. The major respiratory quinone is MK-9. The main fatty acids are iso-C15 : 0, C16 : 1 and C14 : 0. The cell wall contains no DAP isomers. The G+C content of genomic DNA of the type species is 66·0 mol%. The type species is Gemmatimonas aurantiaca.
Description of Gemmatimonas aurantiaca sp. nov.
Gemmatimonas aurantiaca (au.ran.ti.a'ca. M.L. fem. adj. aurantiaca orange-coloured).
Cells are rod-shaped (0·7 µm in width and 2·5–3·2 µm in length). Growth occurs between 25 and 35 °C with the optimum at 30 °C. The pH range is 6·5–9·5. Optimum growth occurs at pH 7·0. The optimum doubling time of growth is 12 h. Catalase and oxidase are produced. Aerobe. No fermentative growth is observed. Nitrate reduction is negative. Neisser staining is positive. Cells are stained yellow with DAPI. The following can be utilized as sole carbon sources: yeast extract, polypepton, succinate, acetate, gelatin and benzoate. The following can be utilized weakly as sole carbon sources: glucose, sucrose, galactose, melibiose, maltose, formate and hydroxybutyrate. The following carbon sources are not utilized: fructose, mannose, lactose, trehalose, raffinose, arabinose, xylose, rhamnose, glycerol, ethanol, propanol, erythritol, mannitol, sorbitol, lactate, citrate, pyruvate, propionate, malate, butyrate, glutamate, aspartate, alanine, starch, glycogen, gentiobiose, turanose, methyl pyruvate, 2-oxoglutarate, -ketovalerate, serine, histidine, glycine, leucine and Casamino acids.
The type strain, T-27T (=JCM 11422T=DSM 14586T), was isolated from a laboratory-scale anaerobic–aerobic sequential batch reactor operated under EBPR conditions.
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ACKNOWLEDGEMENTS |
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