Caloramator Coolhaasii Sp. Nov., a Glutamate- Degrading, Moderately Thermophilic Anaerobe

International Journal of Systematic and Evolutionary Microbiology (2000), 50, 1155–1162 Printed in Great Britain

Caloramator coolhaasii sp. nov., a glutamate- degrading, moderately thermophilic anaerobe

Caroline M. Plugge,1 Erwin G. Zoetendal1,2 and Alfons J. M. Stams1

Author for correspondence: Caroline M. Plugge. Tel: j31 317 482105. Fax: j31 317 483829. e-mail: Caroline.Plugge!algemeen.micr.wau.nl

1 Laboratory of An obligately anaerobic, moderately thermophilic, glutamate-degrading Microbiology, Wageningen bacterium (strain ZT) was isolated from an enrichment culture obtained from University, H. van Suchtelenweg 4, anaerobic thermophilic granular sludge. The cells were rod-shaped to 6703 CT Wageningen, ﬁlamentous and showed no motility or spore formation. The cell wall had a The Netherlands Gram-positive structure, which was revealed by electron microscopy. Optimum 2 Wageningen Centre for growth of the strain was observed under neutrophilic conditions at 50–55 SC. Food Sciences, PO Box 557, The doubling time of strain ZT grown in rich medium was approximately 1 h at 6700 AL Wageningen, T The Netherlands optimal pH and temperature. Strain Z was able to grow on a variety of organic

compounds. Most carbon sources were converted to acetate, CO2,H2, and T traces of propionate and lactate. Strain Z oxidized glutamate to acetate, CO2, M NH4 , traces of propionate and H2. The doubling time on this substrate was 16d. The strain fermented glutamate syntrophically in co-culture with Methanobacterium thermoautotrophicum Z-245T to the same products, but the co-culture had a fourfold higher growth rate. 16S rDNA sequence analysis revealed a relationship with Thermobrachium celere, Caloramator indicus and Caloramator proteoclasticus. The GMC content was 317 mol%. Based on its morphological, phylogenetic and physiological characteristics, it is proposed that strain ZT should be classiﬁed in the genus Caloramator as a new species, Caloramator coolhaasii.

Keywords: Caloramator coolhaasii sp. nov., interspecies hydrogen transfer, glutamate degradation, methanogenic archaea, syntrophic consortia

INTRODUCTION mate in co-culture with hydrogen-scavenging methanogens (Cheng et al., 1992; Tarlera, 1997; Baena et Proteins are important substrates in anaerobic di- al., 1999a; Nanninga & Gottschal, 1985; Stams & gestion (Barker, 1981; McInerney, 1989). Amino acids, Hansen, 1984). Some of these organisms are meso- the hydrolysis products of proteins, can serve as carbon philic, but Selenomonas acidaminovorans, recently and energy sources for many types of anaerobic renamed Thermanaerovibrio acidaminovorans (Baena bacteria. Utilization of amino acids has been used as a et al., 1999b), and Caloramator proteoclasticus are classiﬁcation characteristic for clostridia (Mead, moderately thermophilic. These bacteria grow slowly 1971; Elsden & Hilton, 1979). Several anaerobic in pure culture, but more rapid growth is achieved bacteria have been described that can grow on gluta- when grown syntrophically with a partner organism mate. Most of them are members of the low-GjC which removes the hydrogen. These bacteria degrade − + Clostridium subphylum, which convert glutamate to one glutamate to two acetate, one HCO$, one NH% + − acetate, butyrate, CO#,NH% and traces of hydrogen and one H#, or to one propionate, two HCO$, one + (Buckel & Barker, 1974; Laanbroek et al., 1979). Also, NH% and two H#, where the ratio is dependent on the fermentative anaerobes are described that utilize gluta- hydrogen partial pressure. In this paper, the properties of strain ZT, a novel

...... organism isolated from anaerobic methanogenic Abbreviations: MPN, most probable number; TEM, transmission electron granular sludge, are described. This bacterium microscopy. degrades glutamate in pure culture, but much faster The GenBank accession number for the 16S rDNA sequence of strain ZT is growth is achieved in co-culture with hydrogeno- AF104215. trophic methanogens.

01148 # 2000 IUMS 1155 C. M. Plugge, E. G. Zoetendal and A. J. M. Stams

(a) (c)

(d)

(b)

...... Fig. 1. (a, b) Phase-contrast micrographs of cells of strain ZT during growth on glutamate (a) and WC broth (b); bar, 10 µm. (c, d) Electron micrographs showing (c) the single-layered structure of the cell wall (bar, 0n1 µm; cm, cytoplasmic membrane; p, peptidoglycan layer) and (d) cell division (bar, 1 µm; sf, septum formation). Electron-dense structures are probably intracellular protein precipitates (d).

1156 International Journal of Systematic and Evolutionary Microbiology 50 Caloramator coolhaasii sp. nov.

METHODS Philips EM400 TEM. Motility was determined using Gray’s T method for flagella staining (Gerhardt et al., 1981). For Organisms, cultivation and isolation procedures. Strain Z negative staining, cells were fixed for 1 h in 2n5% (v\v) was isolated from thermophilic methanogenic granular glutaraldehyde in 0n1 M sodium cacodylate buffer (pH 7n2). sludge converting glutamate as the sole source of carbon and A Formvar-coated grid (150 mesh) was placed on a drop of energy. Methanobacterium thermoautotrophicum Z-245T (l T T a dense cell suspension for 20 min. After rinsing with de- DSM 3720 ) and Thermobrachium celere DSM 8682 were ionized water, the cells were stained with 1% (w\v) uranyl obtained from the German Collection of Microorganisms acetate and the grid was air-dried. Cells were examined in a and Cell Cultures (DSMZ, Braunschweig, Germany). Calo- JEOL TEM. ramator proteoclasticus DSM 10124T was from our own culture collection. GjC content. The GjC content of the DNA was de- et al A bicarbonate-buffered anaerobic medium as described termined at the DSMZ by HPLC (Mesbah ., 1989). previously by Stams et al. (1993), supplemented with 0n02% SDS-PAGE analysis of whole-cell protein profiles. The strains yeast extract, was used for enrichment and cultivation of were grown at 55 mC in bicarbonate-buffered anaerobic strain ZT. Wilkens–Chalgren broth (WC broth; Oxoid; 16 g −" T medium supplemented with 0n2% yeast extract and 10 mM l ) was used for the isolation of strain Z . Direct dilution glucose. Samples from cells in the exponential phase (cor- series in liquid WC broth as well as agar plates supplied with responding to OD'!! values of approximately 0n25) were WC broth were used. All incubations were done at 55 mCin centrifuged for 5 min at 16000 g. The pellets were the dark, unless otherwise stated. For direct comparison of resuspended in 100 mM Tris\HCl buffer (pH 7n2). Protein strain ZT with Caloramator proteoclasticus and T. celere, −" samples were diluted in denaturing loading buffer and boiled all cultures were cultivated on: WC broth (16 g l ); for 15 min. Cell debris was removed by centrifugation bicarbonate-buffered anaerobic medium (Stams et al., 1993); (5 min at 16000 g) and samples were stored at 4 mC until bicarbonate-buffered anaerobic medium supplemented with electrophoresis. SDS-polyacrylamide running gels with 10% 0n02% yeast extract; bicarbonate-buffered anaerobic acrylamide and 4% acrylamide stacking gels were run in a medium supplemented with 0n2% yeast extract; and −" Bio-Rad Mini Protean II system at 20 mA for 2 h. Approxi- YTG medium. YTG medium contained (l ): Na#CO$, mately 10 µg protein was loaded in each lane. High- 5 3g;NaHPO .2H O, 0 356 g; KCl, 0 075 g; yeast extract n # % # n n molecular-weight markers for SDS-PAGE were from LKB- 5 g; tryptone, 10 g; cysteine.HCl, 0 2g;NaS.9H O, 0 2g; n # # n Pharmacia. The gels were stained with 0 1% (w\v) resazurin, 1 mg. Before use, the pH of the YTG medium was n Coomassie brilliant blue R250 in methanol\deionized adjusted to 10 by adding sterile 3 M NaOH. Glucose water\acetic acid (4:5:1, by vol.) overnight and destained in (10 mM) was used as the carbon source, except in the case of WC broth, where no additional carbon source was added. methanol\deionized water\acetic acid (4:5:1, by vol.) for 3h. Determination of growth parameters. The growth rate of T 16S rDNA sequence analysis. Genomic DNA was isolated strain Z on WC broth was determined by measuring the T increase in OD'!! in triplicate with time. The growth rate on from strain Z and the 16S rDNA was amplified using glutamate with and without yeast extract was determined by bacterial primers 8f and 1510r (Lane, 1991) under previously measurement of product formation with time. Growth on described conditions (Harmsen et al., 1995). PCR products different substrates was tested using a bicarbonate-buffered were purified and concentrated using a QIA Quick kit anaerobic medium as described previously by Stams et al. (Qiagen). Approximately 1 µg purified PCR product was (1993), supplemented with 0n02% yeast extract. The in- used for sequence analysis using the Sequenase T7 oculum size of strain ZT was 1%. Substrates were added sequencing kit (Amersham) according to the manufacturer’s from sterile stock solutions up to a concentration of 10 mM instructions. Infrared Dye 41 (MWG-Biotech) labelled unless stated otherwise. For syntrophic growth tests, 1% of primers 515r, 338f (Lane, 1991) and 968f (Nu$ bel et al., 1996) strain ZT was inoculated in hydrogen pregrown cultures of were used as sequencing primers. The sequences were M. thermoautotrophicum Z-245T. In all cases, growth was automatically analysed on a LI-COR DNA sequencer 4000L followed by visual examination of culture turbidity and by and corrected manually. The software of the  package product formation after 8 weeks of incubation at 55 mC. The (Strunk & Ludwig, 1991) was used to check reading errors temperature optimum was determined in WC broth at pH 7 and for phylogenetic analysis. A neighbour-joining tree was in triplicate bottles at a temperature range of 15–80 mC. The constructed with the closest relatives.  homology pH optimum was tested in WC broth adjusted with NaOH searches were done using the GenBank and EMBL data- or HCl, and triplicate bottles were incubated at 55 mCata bases. The homologies were checked with the  programs. pH range of 4–10. Both temperature and pH were determined by measuring OD after 2 weeks incubation. Nucleotide accession numbers. The EMBL database accession numbers of the strains most closely related to strain Cellular characterization. The Gram type was determined ZT are: Caloramator proteoclasticus (DSM 10124T), X90488; using Gram staining and electron microscopy. Cells from Caloramator indicus (ACM 3982T), X75788; Thermo- active cultures were stained for Gram type using 2% (w\v) brachium celere (DSM 8682T), X99238; Clostridium crystal violet and 2% safranin S (w\v) as counterstain. botulinum, X68187; Clostridium homopropionicum, X76744; Additionally, a 2% (w\v) KOH solution was used to test Clostridium acetobutylicum, X78071; and Clostridium lysis of bacteria. For transmission electron microscopy algidicarnis, X77676. (TEM), cells were fixed for 2 h in 2n5%(v\v) glutaraldehyde in 0n1 M sodium cacodylate buffer (pH 7n2) at 0 mC. After Other analyses. Substrate and fermentation product concen- rinsing in the same sodium cacodylate buffer, a post-fixation trations were measured using GC and HPLC methods as was done in 1% (w\v) OsO% and 2n5% (w\v) K#Cr#O( for described previously (Stams et al., 1993). Amino acids were 1 h at room temperature. Finally, the cells were post-stained measured as described by Kengen & Stams (1994), with an in 1% (w\v) uranyl acetate. Micrographs were taken with a extension of the derivatization time to 30 min. Sulfide was

International Journal of Systematic and Evolutionary Microbiology 50 1157 C. M. Plugge, E. G. Zoetendal and A. J. M. Stams analysed as described by Tru$ per & Schlegel (1964). Anions when 0n02% yeast extract was added. The doubling were analysed as described by Scholten & Stams (1995). times of strain ZT in glutamate media without and supplemented with 0n02% yeast extract were approximately 3n5 and 1n6 d, respectively. The doubling time RESULTS T T of strain Z in WC broth was about 1 h. Enrichment and isolation of strain Z T T Strain Z grew with complex substrates like yeast Strain Z was enriched from anaerobic methanogenic extract, Casamino acids, gelatin, casitone, peptone (all granular sludge fed with glutamate as the sole carbon at 2%, w\v) and WC broth. and energy source. Strain ZT was the predominant The following single substrates supported growth of glutamate-degrading organism in most probable T number (MPN) determinations of diluted biomass. strain Z : glutamate, aspartate, alanine, arginine, Methanogens were present as the only microscopically methionine, glucose, galactose, fructose, mannose, visible impurity. The enrichment obtained from the maltose, ribose, starch (0n5%,w\v), xylose, cellobiose MPN determinations was serially diluted in the pres- (5 mM) and pyruvate. Growth on glutamate, aspartate, alanine, arginine, methionine, mannose, ence of 5 mM bromoethane sulphonate to inhibit the T methanogens. In the absence of methane production, ribose and pyruvate was enhanced when strain Z was T co-cultivated with M. thermoautotrophicum Z-245T.It growth of strain Z on the mineral salts medium was −" poor. Strain ZT was therefore serially diluted and produced acetate (13n9 mM), H# (10n1 mmol l me- transferred to anaerobic bottles containing WC broth dium) and traces of propionate (1n3 mM) from " (16 g l− ). The growth yield and growth rate of strain glutamate (9n8 mM). The carbon and electron ZT were considerably higher in this medium. To recoveries were 97% and 84%, respectively, assuming remove traces of methanogens, strain ZT was plated on that glutamate conversion to two acetate yields one WC broth agar. This resulted in the development of CO#, whereas two CO# are formed when glutamate is circular, brownish, shiny colonies. Four colonies were converted to propionate. From the glutamate orig- picked from the highest-diluted plate with growth and inally added, 60–70% was degraded. Glutamate metabolism was inﬂuenced by the presence of M. thermo- transferred to a dilution series in WC broth. The T autotrophicum Z-245 ; glutamate (18n6 mM) was con- highest dilution with growth was used to inoculate WC −" verted to acetate (30n7 mM), CH% (8n6 mmol l me- broth agar plates again, resulting in the development −" of the type of colonies as described above. This dium; equivalent to 34n4 mmol H# l medium) and procedure was repeated once more and resulted in a traces of propionate (1 mM). The carbon and electron pure culture of strain ZT when checked by light and recoveries were 102% and 109%, respectively, and electron microscopy. glutamate was completely converted. The growth rate was about fourfold higher when M. thermoautotrophicum Z-245T was present. When strain ZT was Morphology and cell structure grown on glutamate under H#\CO# (80\20) only Cells of strain ZT growing in WC broth were rod- 7–10% of the glutamate was converted. Under these shaped to ﬁlamentous, with a size of 2–40 µmby conditions, it was not possible to obtain a closed 0n5–0n7 µm as measured by light microscopy (Fig. 1a, electron balance. Glucose was fermented to acetate, b). During exponential growth, the cells were very lactate, CO# and H#. When the amount of yeast extract long. Rapid lysis was observed as soon as the cells in the medium was increased from 0n02% to 0n2%, entered the stationary phase. However, in mineral salts formate and traces of ethanol could be detected as medium with glutamate, cells were smaller and did not reduced end products as well as the usual products. lyse in the stationary phase. Spores were never The following components did not support growth of strain ZT either in the absence or presence of M. observed and no growth occurred after pasteurization T of the culture (20 min, 90 mC). Cells stained Gram- thermoautotrophicum Z-245 : glycine, leucine, cys- negative, but the cell wall ultrastructure resembled that teine, arabinose, mannitol, melobiose, rhamnose, of Gram-positive anaerobic bacteria (Fig. 1c). casein (0n5%,w\v), cellulose (0n5%,w\v), citrate, iso- citrate, 2-oxoglutarate, fumarate, malate, benzoate, Physiological characteristics and metabolism methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol or acetoin. A Stickland reaction was not T Strain Z was a strictly anaerobic bacterium. No observed with H#, alanine or leucine as electron donor growth occurred when traces of oxygen were present in and glycine, arginine and proline as electron acceptor. the medium, as indicated by the pink colour of the The combination of alanine and arginine resulted in a medium in which resazurin was present. Its optimal higher production of hydrogen as compared to alanine growth temperature was 50–55 mC with lower and alone, indicating an oxidative deamination of arginine. upper limits of 37 and 65 mC, respectively. The pH Yeast extract stimulated growth on glutamate, but was optimum was 7n0–7n5. Below pH 6n0 and above pH 8n5, not required. Sulphide-reduced media were required no measurable growth was observed. Growth of strain for growth. Oxygen, nitrate, sulphate, thiosulphate ZT was possible in a mineral salts medium supple- and fumarate could not serve as electron acceptors for mented with vitamins and glutamate as the sole source growth on glutamate; no measurable decrease in these of carbon and energy. However, growth was enhanced compounds could be found after incubation.

1158 International Journal of Systematic and Evolutionary Microbiology 50 Caloramator coolhaasii sp. nov.

Table 1. Growth and glucose fermentation products of strain ZT, Caloramator proteoclasticus and T. celere on different media ...... k, No growth; j, growth. acet, Acetate; lact, lactate; form, formate; etoh, ethanol. Products given in parentheses represent minor products.

Medium Strain ZT Caloramator T. celere proteoclasticus

WC broth jjj Bicarbonate-buffered glucose medium acet, lact, H# kk Bicarbonate-buffered glucose acet, lact, H# acet, etoh, form, lact, H# k mediumj0n02% yeast extract Bicarbonate-buffered glucose acet, (etoh), form, lact, H# acet, etoh, form, lact, H# acet, (etoh), form, H# mediumj0n2% yeast extract YTG medium, pH 10 kkacet, (etoh), form, H#

1234 Phylogeny The nucleotide sequence (1454 bp) of a 16S rRNA gene of strain ZT was analysed and revealed that this organism belongs to the subphylum of Gram-positive clostridia with low GjC content. Sequence analysis showed that strain ZT is closely related to T. celere, Caloramator indicus and Caloramator proteoclasticus (Engle et al., 1996; Christostomos et al., 1996; Tarlera et al., 1997) with levels of similarity of 97n8, 97n2 and 96n2%, respectively. A phylogenetic tree showing the relationship of strain ZT and other related species is depicted in Fig. 3. T The GjC content of strain Z was 31n7p0n4mol%.

DISCUSSION ...... T T Fig. 2. SDS-PAGE of whole-cell proteins of strain Z and closely Phylogenetically, strain Z belongs to the low-GjC- related strains. Lanes: 1, set of marker proteins with their containing clostridial group and is closely related to T. molecular masses; 2, Caloramator proteoclasticus; 3, strain ZT ; and 4, Thermobrachium celere. celere, Caloramator indicus and Caloramator proteoclasticus (Engle et al., 1996; Christostomos et al., 1996; Tarlera et al., 1997). Within this group, all members are closely related, but have been described Direct comparison of strain ZT with Caloramator and approved as new species. Based on 16S rDNA proteoclasticus and T. celere analysis, Caloramator indicus and T. celere seem to be Strain ZT, Caloramator proteoclasticus and T. celere almost 100% related. Despite the similarity of the 16S rDNA with these mentioned species, there are several grew well in WC broth and anaerobic bicarbonate- T buffered medium supplemented with 0 2% yeast ex- differences between strain Z and other members n Caloramator et al tract and 10 mM glucose. Strain ZT and Caloramator within the genus (Collins ., 1994). proteoclasticus grew well in a bicarbonate-buffered These include the absence of endospores, immobility, optimum growth temperatu