Tepidiphilus margaritifer gen. nov., sp. nov., isolated from a thermophilic aerobic digester

The vast majority of species belonging to the class Proteobacteria of the domain Bacteria are mesophilic; nevertheless, some slightly or extremely thermophilic genera and species have been described. Thermophilic organisms are distributed among the different subclasses of the Proteobacteria, and most of them are chemolithotrophs that can use H₂ or sulfur/sulfur compounds as electron donors. Within the β-subclass of the Proteobacteria, eight species of thermophilic organisms, included in five genera, have been described to date. The species Thiomonas thermosulfata (Shooner et al., 1996), Thermothrix azorensis (Odintsova et al., 1996), Thermothrix thiopara (Caldwell et al., 1976) and Tepidimonas ignava (Moreira et al., 2000) represent thermophilic organisms, characterized by their ability to use sulfur or sulfur compounds as an energy source. Tepidimonas ignava, isolated from a Portuguese hot spring, is the only strictly heterotrophic organism among these sulfur bacteria. The genus Hydrogenophilus currently comprises two species, Hydrogenophilus thermoluteolus (Hayashi et al., 1999) and Hydrogenophilus hirschii (Stöhr et al., 2001), both of which are hydrogen-oxidizing bacteria with the ability to fix CO₂. Caldimonas manganoxidans, a chemo-organoheterotrophic, thermophilic member of the β-Proteobacteria, was described recently (Takeda et al., 2002).

Isolation of these organisms has frequently occurred under conditions favouring the recovery of specific metabolic types, namely hydrogen- and sulfur-oxidizing bacteria; this may explain why the majority of thermophilic members of the β-Proteobacteria described to date are chemolithotrophic. Indeed, it is intriguing that, despite the abundance of chemo-organoheterotrophic organisms belonging to the β-Proteobacteria, only a single thermophilic representative of this metabolic group has been described to date.

The present study describes a moderately thermophilic, chemo-organoheterotrophic bacterium, strain N2-214^T, isolated from a thermophilic aerobic digester of water-treatment sludge. On the basis of 16S rDNA-based phylogenetic analyses and physiological and chemotaxonomic characteristics, it is proposed that isolate N2-214^T represents a new genus and species, for which the name Tepidiphilus margaritifer gen. nov., sp. nov. is proposed.

Isolation, cultivation conditions and bacterial strains.
Strain N2-214^T was isolated from a caprolactone enrichment culture obtained from a thermophilic aerobic digester of a domestic wastewater-treatment plant in northern Portugal. In this treatment process, the decanted sludge is subjected to a mesobiotic anaerobic digestion followed by a thermophilic aerobic digestion, which reaches a maximal temperature of about 60 °C. The sample was transported, without temperature control, and used as an inoculum for enrichment on polycaprolactone diol polymer (Solvay) with a molecular mass of 1000 Da. The enrichment was carried out in mineral medium (medium A) (Manaia & Moore, 2002) at 50 °C. Strain N2-214^T was purified from the mixed culture obtained in this enrichment by subculturing on LuriaBertani (LB) broth containing 20 g agar l^-1 (LB agar) (Carlton & Brown, 1981). The isolate was maintained on LB agar or cryo-preserved in LB broth containing 15 % (v/v) glycerol.

Determination of morphological, growth and biochemical characteristics.
Colony and cell morphology, Gram-staining reaction, production of spores, accumulation of poly-β-hydroxybutyrate (PHB) granules, the number and position of flagella and the temperature range for growth were examined as described by Manaia et al. (2003).

Phenotypic tests were carried out as described by Smibert & Krieg (1981), using 2-day cultures on LB agar. Unless otherwise stated, all incubations were performed at 50 °C. The pH tolerance range was examined in LB medium, using 10 mM MES (Sigma), for pH 5·06·0 or 10 mM TAPS (Sigma) for pH 6·09·0. Enzymic activities were tested using the API ZYM system by following the instructions of the manufacturer (bioMérieux). Hydrogenase activity was determined on the basis of the descriptions of Aragno & Schlegel (1992) and Stöhr et al. (2001) as described by Manaia et al. (2003). The nutritional pattern was characterized using the API 50CH system and a defined medium (medium B) as described by Manaia et al. (2003). Chemolithoautotrophic growth, the use of H₂ as an energy source and the ability to use sulfur or thiosulfate as electrons donors were tested as described by Manaia et al. (2003). The ability to grow in the absence of a source of combined nitrogen was tested using medium A without ammonium sulfate, under aerobic conditions or under an N₂ atmosphere with nitrate as the electron acceptor.

Determination of genotypic characteristics.
The DNA G+C content was determined as described by Manaia et al. (2003).

16S rDNA sequence analysis.
The 16S rRNA gene sequence was determined after PCR amplification as described by Manaia et al. (2003). The nucleotide sequence was compared with reference 16S rDNA sequences in the EMBL database using the FASTA program (Pearson & Lipman, 1988) and subsequently aligned with reference sequences included in the ARB package (). Evolutionary distances derived from sequence-pair dissimilarities (Jukes & Cantor, 1969) were calculated using the PHYLIP package (Felsenstein, 1989). Calculations were done by using a 50 % conservation filter for the β-Proteobacteria implemented in the ARB package and by using a second filter that excluded only the most variable regions in the 16S rRNA molecule, which corresponded to inserts of different lengths amongst the reference sequences used in the analysis. The tree shown later is the one obtained using the second type of filter, since the topology of the tree for the most closely related genera did not vary regardless of which filter was used, and the bootstrap values were slightly higher. Ambiguously determined nucleotide positions or positions for which no sequence data were available in any of the reference sequences were excluded from the calculations.

Determination of chemotaxonomic characteristics.
Polar lipids, methylated fatty acids and respiratory quinones were extracted and separated as described previously (Manaia et al., 2003).

Cultivation at 50 °C in mineral medium supplemented with a polycaprolactone diol was used to enrich moderate thermophiles capable of using synthetic polymers as the sole source of carbon and energy. The inoculum for this enrichment was taken from a thermophilic aerobic digester of sludge from a domestic wastewater-treatment plant. The enrichment procedure resulted in a mixed culture, predominantly composed by Gram-positive rods, which, on the basis of phenotypic characterization, were identified as members of the genus Bacillus. Isolate N2-214^T was obtained through purification of cultivable organisms present in the mixed culture. In pure culture, this isolate was unable to grow in mineral medium supplemented with caprolactones, although good growth was observed when acetate was the only carbon source. At the optimal temperature for growth (50 °C), the doubling time in LB medium was approximately 1 h. When cultured on LB agar, strain N2-214^T produced brilliant non-pigmented colonies, 12 mm in diameter, after 3648 h growth. Under a strong light source, these colonies had a nacre-like appearance.

Cells of strain N2-214^T were Gram-negative, non-spore-forming rods, 2·0 µm long and 0·7 µm wide, with a single polar flagellum. The isolate tested positive for oxidase and catalase and for the presence of intracellular PHB granules. Strain N2-214^T reduced nitrate to a compound more reduced than nitrite, since nitrite was not detected in the supernatant of cultures. The isolate could grow under anaerobic conditions (N₂-saturated atmosphere) using nitrate as an electron acceptor, both in complex media and in mineral medium supplemented with acetate, ethanol or benzoate. Isolate N2-214^T could reduce triphenyltetrazolium in the presence of hydrogen but not in its absence, suggesting the presence of hydrogenase activity. However, since this organism could grow in a N₂-saturated atmosphere in the absence of H₂ gas, it is possible to conclude that this organism is not a strictly hydrogen-oxidizing chemolithoheterotroph. The optimal temperature for growth of strain N2-214^T in LB medium was around 50 °C, with a maximal growth temperature of 61 °C. The nutritional pattern of strain N2-214^T revealed an inability to use the sugars that were tested and a preference for organic acids, e.g. malate, acetate or benzoate, as carbon sources; other substrates, such as ethanol and the amino acids glutamic acid and asparagine, could also be used as single carbon sources (Table 1). The strain could not grow autotrophically in mineral medium supplemented with sodium hydrogen carbonate in the presence of hydrogen, S⁰ or thiosulfate; in the presence of acetate as a carbon source, these potential electron donors did not enhance growth.

Table 1. Phenotypic characteristics of isolate N2-214T Strain N2-214Twas unable to use the following compounds as sole sources of carbon: glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl β-xyloside, galactose, D-glucose, D-fructose, D-mannose, L-sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl α-D-mannoside, methyl α-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, D-raffinose, starch, glycogen, xylitol, β-gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate, 5-ketogluconate, hydroxyproline, L-alanine, L-serine, L-glycine, L-histidine, DL-methionine, L-arginine, toluene and polycaprolactone (molecular mass 1000 and above). Strain N2-214T was unable to grow autotrophically with H2, S0 or , did not fix nitrogen and did not grow in the presence of ampicillin (10 µg), penicillin G (10 U) or 3 % NaCl or at pH 5 or 9. Strain N2-214T did not require growth factors (vitamins or amino acids). The following enzymes were absent from isolate N2-214T: cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, N-acetyl∼-β-glucosaminidase, α-fucosidase, α-mannosidase, tweenase (Tween 80), amylase and gelatinase.

The inability of isolate N2-214^T to fix nitrogen or assimilate nitrate was evidenced by the fact that this organism could not grow in mineral medium without combined nitrogen under aerobic conditions, or in the same medium supplemented with nitrate under a N₂ atmosphere.

The G+C content of the genomic DNA of strain N2-214^T was 64·8 mol%.

Nearly the complete 16S rDNA sequence of strain N2-214^T was determined (1456 nucleotides). Phylogenetic analysis of the 16S rDNA sequence of strain N2-214^T showed its affiliation to the β-Proteobacteria, a result that is in agreement with the polar lipid and respiratory quinone composition (see below). The 16S rDNA sequence was then compared with all species with validly published names of the most closely related genera, Azoarcus, Thauera and Hydrogenophilus (Springer et al., 1998; Hayashi et al., 1999; Song et al., 1998, 1999, 2000, 2001; Reinhold-Hurek & Hurek, 2000; Stöhr et al., 2001; Mechichi et al., 2002), and several other genera in the β-Proteobacteria. The 16S rDNA sequence of strain N2-214^T was not closely related to those of other genera within this subclass, the highest similarities (approx. 9293 %) being to the sequences of Azoarcus buckelii (Mechichi et al., 2002), Thauera aromatica (Anders et al., 1995) and H. thermoluteolus (Hayashi et al., 1999). Comparative evolutionary distance analyses of the 16S rDNA sequence of strain N2-214^T demonstrated that it branched from the lineage leading to the genus Hydrogenophilus (Fig. 1), a result that was also supported by parsimony, maximum-likelihood and bootstrapping methods. The polar lipid pattern of strain N2-214^T, obtained by TLC, revealed the presence of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) as the major phospholipids. A similar polar lipid pattern, characterized by the predominance of PE and PG, is described for other members of the β-Proteobacteria (Wilkinson, 1988). The only respiratory quinone detected for isolate N2-214^T was ubiquinone 8, which is also characteristic of micro-organisms belonging to the β-Proteobacteria (Suzuki et al., 1993).

(39K): Fig. 1. Phylogenetic relationships based on 16S rDNA sequences between strain N2-214T and representatives of related genera within the β-Proteobacteria. The dendrogram was generated from evolutionary distances, calculated from pairwise dissimilarities, using the FITCH program from the PHYLIP package. Accession numbers are given in parentheses. Bootstrap values for branches leading to the sequence of N2-214T and its closest relatives, obtained after 1000 resamplings, are shown in the tree. The sequence of Methylophaga thalassica ATCC 33146T was used as outgroup.

The fatty acid composition of strain N2-214^T was analysed using LB agar cultures grown at 50 °C. Under these conditions, the methyl esters of fatty acids C_{16 : 0} and cyclo-C_{19 : 0} were predominant, followed by the unsaturated fatty acid C_{18 : 1}. These components constituted more than 75 % of the total fatty acids (Table 2). A lower growth temperature (30 °C) promoted a dramatic shift between the content of the unsaturated fatty acids C_{18 : 1} and C_{16 : 1}, which doubled, and that of cyclo-C_{19 : 0}, which, under these conditions, represented only 3 % of total fatty acids (results not shown). The hydroxy fatty acid 3-OH C_{10 : 0} was also detected, and its level was not temperature-dependent. Characteristics that distinguish isolate N2-214^T from the type strains of the type species of genera with the closest 16S rRNA similarity to the proposed new genus are shown in Table 3.

Table 2. Mean fatty acid composition of strain N2-214T Values are percentages of total fatty acids. Cells were grown on LB agar at 50 °C. Components that made up less than 1 % of the total are summed as Other and included iso-C11 : 0 3-OH, C12 : 0, C15 : 0, C17 : 0, C19 : 1ω12t and C20 : 1ω9t.

Table 3. Characteristics of isolate N2-214T and related type strains Strains: 1, Thauera selenatis AXT (data from Macy et al., 1993; Song et al., 2000); 2, Azoarcus indigens VB32T (Reinhold-Hurek et al., 1993); 3, Hydrogenophilus thermoluteolus TH-1T (Goto et al., 1978; Hayashi et al., 1999; Stöhr et al., 2001); 4, N2-214T. NA, Data not available. All strains utilize acetate.

To date, few thermophilic organisms have been described in the β-Proteobacteria. However, this may be due to the difficulty in cultivating these organisms under laboratory conditions, since bacteria belonging to the β-Proteobacteria have been detected in studies employing culture-independent molecular methods to describe the biological diversity of high-temperature natural (e.g. Ward et al., 1998; Hugenholtz et al., 1998) and man-made environments (e.g. LaPara et al., 2000). The work of LaPara et al. (2000), describing the phylogenetic diversity of bacterial communities in thermophilic and mesophilic bioreactors used to treat pharmaceutical wastewater, refers to the presence of clones that have the closest phylogenetic affiliation to the β-proteobacterium HMD 444 (accession number AB015328) in both meso- and thermophilic bioreactors. In this respect, is interesting to note that strains HMD 444 and N2-214^T share 16S rRNA sequence identity above 99 % (according to FASTA analysis), suggesting that they might eventually represent the same species. However, such a conclusion would only be possible on the basis of a DNADNA hybridization study (Stackebrandt & Goebel, 1994).

Description of Tepidiphilus gen. nov.
Tepidiphilus (Te.pi.di'phi.lus. L. adj. tepidus lukewarm; Gr. adj. philos friendly to; N.L. masc. n. Tepidiphilus liker of lukewarm conditions).

Forms rod-shaped cells that stain Gram-negative. Endospores are not formed. Slightly thermophilic. PHB granules are accumulated. Grows anaerobically in the presence of nitrate. Oxidase- and catalase-positive. Major phospholipids are PE and PG; ubiquinone 8 is the major respiratory quinone. Major fatty acids are C_{16 : 0} and cyclo-C_{19 : 0}. Chemo-organotrophic. Organic acids and amino acids, but no sugars, are used as single carbon sources. The type species is Tepidiphilus margaritifer.

Description of Tepidiphilus margaritifer sp. nov.
Tepidiphilus margaritifer (mar.ga.ri'ti.fer. L. n. margarita pearl; L. masc. suffix -fer carrying; N.L. masc. adj. margaritifer pearl-carrying, referring to the nacre-like appearance of the colonies).

Shows the following properties in addition to those in the genus description. Forms rod-shaped cells, 2·0 µm long and 0·7 µm wide. Cells are motile by a single polar flagellum. Colonies grown on LB agar are nacre-like and 12 mm in diameter after 3648 h growth. Growth occurs above 25 °C and below 61 °C; the optimal growth temperature is approximately 50 °C. Growth occurs between pH 6 and 8. Hydrogenase-positive. The major fatty acids at the optimal temperature for growth are C_{16 : 0}, C_{18 : 1} and cyclo-C_{19 : 0}; C_{10 : 0} 3-OH is also present. The DNA G+C content of the type strain is 64·8 mol%.

The type strain, strain N2-214^T (=DSM 15129^T=LMG 21637^T), was isolated from a thermophilic digester of wastewater-treatment sludge.

C. M. M. thanks Nuno Cerca and Nuno Marcos (Escola Superior de Biotecnologia) for their collaboration on the preparation of the enrichment cultures and Fernanda Nobre (Departamento de Zoologia da Universidade de Coimbra) for fatty acid methyl ester analyses. The authors thank Solvay for the caprolactones and ETAR de Parada for the sampling.