Coprothermobacter Platensis Spm Now,, a New Anaerobic Proteolytic Thermophilic Bacterium Isolated from an Anaerobic Mesophilic Sludge

lnternational Journal of Systematic Bacteriology (1998), 48, 1297-1 304 Printed in Great Britain -~ _____~ ______~ -

Coprothermobacter platensis Spm now,, a new anaerobic proteolytic thermophilic bacterium isolated from an anaerobic mesophilic sludge

C. Etchebehere,' M. E. Pa~an,~J. Zorzopulos,2~3 M. Soubes' and L. Muxi'

Author for correspondence: C. Etchebehere. Fax: + 5982 924 1906. e-mail: cetchebe(o bilbo.edu.uy

1 Departamento de A new anaerobic, proteolytic, moderately thermophilic bacterium, strain 3RT, Mic r o b io I ogia, Facu Itad de was isolated from a methanogenic mesophilic reactor treating protein-rich Quimica y Facultad de Ciencias, Universidad de la wastewater. The cells were Gram-negative, non-spore-forming, non-motile Republica, C. C.1157, rods. The DNA base composition was 43 molo/o G+C. The optimum pH and Montevideo, Uruguay temperature for growth were 7.0 and 55 "C respectively. The bacterium * Departamento de fermented gelatin, casein, bovine albumin, peptone and yeast extract. Glucose, Microbiologia, Universidad fructose, sucrose, maltose and starch were poorly fermented. The major de Buenos Aires, Buenos Aires, Argentina fermentation products from glucose were acetate, CO, and H, and, from gelatin, propionate was also detected. Growth glucose was stimulated by 3 Laboratorio de on B iotecno I ogia, Fundaci 6 n thiosulfate, which was reduced to sulfide. Sulfate and nitrate were not P. Cassara, Buenos Aires, reduced. 16s rRNA gene analysis revealed that the isolated bacterial strain was Argentina phylogenetically related to Coprothermobacter proteolyticus (96.3 O/O sequence similarity), the only known species within the genus. DNA-DNA hybridization analysis demonstrated a very low level of homology, indicating that the isolated strain and C. proteolyticus were not related at species level. Therefore, it is proposed to classify the described strain in the genus Coprothermobacter as a new species, Coprothermobacter platensis. The type strain of C. platensis is strain 3RT ( = DSM 11748T).

Keywords : Coprotlzernzobactcr platcmis sp. nov., proteolytic bacteria, thermophiles, anaerobe, thiosulfate reduction

INTRODUCTION (Tarlera et al., 1997) and Coprothermobacter (Ollivier et al., 1985 ; Rainey & Stackebrandt, 1993 ; Kersters et Anaerobic digestion is increasingly used for carbon al., 1994). Among them, the genus Coprothermobacter decontainination of agroindustrial wastewaters (formerly classified as Tl?ermobacteroides)represents a (Speece, 1996). Proteins are frequently a major com- very deep-branching phylum within the domain Bac- ponent of'such wastes, and their degradation, initiated teria, with only one species, Coprothermobacter proteo- by extracellular proteases, is often incomplete (Mc- lyticus, reported so far (Rainey & Stackebrandt, 1993). Inerney, 1988). The vast majority of full-scale digestors The other genera, Anaerobranca, Thermobrachiuni and are mesophilic, however thermophilic treatment is also Cnloramator, are related to the clostridia, belonging to being explored as it may have advantages, especially the large low G+C content branch of the Gram- for effluents produced at high temperature (Letting, positive division (Rainey et nl., 1993; Collins et al., 1995). To start thermophilic reactors, mesophilic 1 994). anaerobic: sludge may be adapted by gradually in- creasing the incubation temperature (Van Lier et al., In this paper, we describe a novel species within the 1993). genus Coprotliermobacter, Coprothermobacter platensis, isolated from a methanogenic mesophilic reactor Although proteolytic activity is a common charac- treating a protein-rich wastewater. Furthermore, we teristic among mesophilic bacteria, very few anaerobic, present evidence of the ability of both species of thermopliilic proteolytic bacteria have been charac- Coprotliernzobacter to use thiosulfate as an electron terized. In the past 10 years four novel genera have acceptor. Many mesophilic facultative anaerobes and been described, namely Thermobrachium (Engle e? a/., strict anaerobes share this physiological trait. Among 1996), Aiicrerohranca (Engle et al., 1995), Caloramator the thermophiles and hyperthermophiles of the do-

~ ~~

00755 0 1998 IUMS 1297 C. Etchebehere and others main Bacteria, it has been reported for the genera (Alltech Associates) column. The mobile phase was H,SO, Thermoanaerobucter and Thermounaerobacterium (Lee (0.005 M), the flow rate was 0-8 ml min-' and the tem- et al., 1993) and more recently for Thermotogales perature was 35 "C. Hydrogen was measured by gas chroma- (Fardeau et al., 1997; Ravot et al., 1995, 1996). tography using a 14A gas chromatograph with a thermal conductivity detector (Shimadzu), equipped with a Carbo- sieve S I1 column and argon as the gas carrier. The flow rate METHODS was 30 ml min-' and the initial temperature was 35 "C, with an increase of 32 "C min-l up to 225 "C. Anions (nitrate, Strains. Coprothermohacter proteolyticus BTT (T = type nitrite, sulfate) were meseaured by HPLC using a UV strain) (DSM 5265T)was obtained from DSMZ (Deutsche detector (Shimadzu) and an ICPAK ANION (Waters Sammlung von Mikroorganismen und Zellkulturen, Braun- Millipore) column, with a flow rate of 1.2 ml min-l and a schweig, Germany). temperature of 42 "C. The mobile phase was phosphate Enrichment and isolation. A sample (5 ml) taken from the buffer (0.01 M, pH 6.8). Sulfide was detected by the meth- sludge of a mesophilic anaerobic digester treating waste- ylene blue method (Rand et al., 1975). Gelatin was measured water from baker's yeast production in Montevideo, Uru- as described by Bradford (1976). guay, was anaerobically transferred to 50 ml of BCYT Growth conditions. Optimum pH, temperature and NaCl medium - a basal medium containing yeast extract (Difco ; concentration ranges for growth were determined in PY 1 g 1-I) and triptone (Difco; 1 g 1-') (Touzel & Albagnac, medium, using a 5 % (v/v) inoculum. The optimum pH was 1983) ~ supplemented with glucose (5 g lF') and gelatin determined by incubating cultures at 55 "C at initial pH (Sigma; 5 g 1-I). Gelatin and glucose were used to enrich the values from 4.3 to 9.1 adjusted by adding NaOH or HC1. The medium because many thermophilic glycolytic anaerobes optimum concentration of NaCl was determined at pH 7 exhibit protease activity (Wiegel, 1992). This medium was and 55 "C. prepared as previously described (Muxi et al., 1992), sparged with NJCO, (80:20, v/v), autoclaved for 15 min at 121 "C Susceptibility tests. Antibiotic resistance was determined in and reduced with filter-sterilized sulfide-cysteine solution liquid PY medium at pH 7 and 55 "C. Antibiotics were filter (Touzel & Albagnac, 1983). Enrichments were incubated at sterilized and the following concentrations were tested : 55 "C and examined for growth with a phase-contrast vancomycin (2.5 and 5.0 mg lF1), neomycin (0.15 g lF1), microscope (Axioplan; Zeiss). Positive cultures were trans- polymyxin B (20 and 40 mg 1-I), penicillin G (20 U ml-I), ferred periodically to fresh medium by using 10% (v/v) kanamycin (300 and 600 ng ml-I), sodium azide (0.5 and inocula. Isolation was performed in plates with the same 1.0 g 1-l). medium solidified with agar (1.8%, w/v) and incubated at Protease assays. Protease activity was assayed using the 55 "C in an anaerobic chamber (Coy Laboratory Products) azocasein method under anaerobic conditions (N, atmos- with a gas atmosphere containing N,/H,/CO, (80: 10 : 10, by phere) as described by Brock et al. (1982). The specific vol.). After 10 d, a colony was picked and reisolated twice in activity (in units) was expressed as pg azocasein hydrolysed the same medium. Purity was checked microscopically and h-' (mg bacterial protein)-'. Protein was determined as by growth in the same medium incubated anaerobically and described by Bradford (1 976). aerobically at 55 "C. A purified strain (3RT) was grown under N, atmosphere, in pre-reduced PY broth medium - a Electron microscopy. For electron microscopic studies, the basal medium containing peptone (Difco; 10 g 1-I) and yeast culture was centrifuged for 5 min at 3000 r.p.m. The super- extract (Difco; 10 g IF') (Smibert & Krieg, 1994) - and natant was discarded and the pellet fixed in glutaraldehyde stored at room temperature. (2%) in sodium cacodylate buffer (0.1 M) at pH 7.4 for 30 min. The process was repeated, the pellet was then washed Substrate utilization and end product formation. Substrate in phosphate buffer (pH 7.4) and post-fixed with osmium utilization was determined in pre-reduced BC medium tetroxide (1 "0). Specimens were dehydrated in an ascending (BCYT medium without tryptone and with yeast extract gradient of ethanol (50, 70, 80, 90 and 95%) and then 0-2 g 1-'). Stock solutions of soluble substrates (100 g 1-') impregnated in propylene oxide. Finally, they were em- were anaerobically prepared, filter sterilized and anaero- bedded in Poly/Bed 8 12 resin (Polysciences 18976-2590). bically dispensed into tubes containing pre-reduced BC Ultrathin sections were cut with an ultratome Super Nova medium (10 ml) with a N, atmosphere. Insoluble substrates (Reichter-Jung) and mounted in a copper grid, stained with were weighed and transferred to culture tubes. Then, BC uranyl acetate and lead citrate and examined in a JEM- 1200 medium was dispensed (10 ml) into the tubes under an N, Ex I1 transmission electron microscope at 80 kV. atmosphere and autoclaved. The complex substrates, bovine albumin (Sigma), xylan (Sigma), cellulose (Sigma), gelatin DNA base composition. DNA was isolated (Sambrook et a/., (Sigma), peptone (Difco), yeast extract (Difco), casein 1989) and the G + C content was determined by HPLC at the (Merck) and starch (Fluka), were added to a final con- DSMZ (Mesbah et al., 1989). centration of 5 g lF1. For the other substrates, a final DNA-DNA hybridization. The genetic relatedness of strains concentration of 2 g was used. Growth was measured 1-' was determined by DNA-DNA hybridization on nylon spectrophotometrically (Genesys 5 ; Spectronic, Milton Roy) at 660 nm. In cultures containing insoluble substrates, membranes (Johnson, 1991). Serial dilutions of DNA in growth was monitored by microscopic examination and by denaturation solution (0.5 M NaOH, 1.5 M NaC1) were applied to a Pall Biodyne nylon membrane. The membrane analysis of end products (volatile fatty acids). The effect of sulfate, nitrate and thiosulfate on growth was tested using was neutralized with 1.5 M NaC1,0.5 M Tris/HCl pH 8 and baked for 2 h at 80 "C. Prehybridization (2 h) and hybrid- BCYT medium, supplemented with equimolar concen- ization (18 h) were performed at 65 "C in hybridization trations of glucose (20 mM) and electron acceptor (20 mM). buffer containing 0.15 M NaC1, 1 % (w/v) SDS, 0.3 'YOskim Analytical procedures. Fermentation products, volatile fatty milk, using as a probe chromosomal DNA from strain 3RT, acids and alcohols were measured by HPLC using a digested with the AIuI endonuclease and 32P-labelled using refractive index detector (Waters Millipore) with an OA1000 the Random Primers DNA Labelling System (Gibco BRL).

1298 In terna tionaI Journa I of Systematic Bacteriology 48 Coprothermobacter platensis sp. nov. ___

Fig. 1. (a) Phase-contrast micrograph of strain 3RT grown on PY medium, showing straight rods ocurring as single cells, in pairs or in chains. Bar, 10 pm. (b) Ultrathin section electron micrograph of strain 3RT showing the cell wall structure. An intensely stained inner layer (i) and a less densely stained outer layer (0)were observed. Bar, 0.2 pm.

The results were scored by autoradiography. Similarity margins, transparent to whitish. A single colony was values were calculated as described by Johnson (1991). picked and purified. This purified strain, designated Sequence analysis of 165 rDNA and phylogenetic analysis. 3RT (T = type strain), was characterized. DNA from strain 3R" was purified (Sambrook rt a/., 1989) and the ribosomal 16s genes were amplified by PCR Morphology and cell structure (Johnson, 1994), using the following universal primers: 1492R(5'-C;GTTACCTTGTTACGACTT-3'),correspond- Cells of strain 3RT grown in PY medium were non- ing to positions 1510-1 492 in reverse Esclzerichia coli motile, straight rods, occurring singly or in pairs (Fig. numbering. and 27F (5'-AGAGTTTGATCMTGGCTC- la). They were generally 1.5-2 pm long and 0-5 pm AG-37, corresponding to positions 8-27 in forward E. coli numbering. The following temperature cycles were per- wide. Long chains were also observed in old cultures. Spores were never observed, and the cultures did not formed: 94 "C for 5 min, 30 cycles of 94 "C for 60 s, 60 "C for 60 s and 72 "C for 60 s, followed by a final 7 min survive a heat treatment of 2 h at 90 "C. Lysis was incubation 'it 72 "C. The PCR products were purified using observed in late stationary phase. Young cultures of WIZARD I'CR Preps columns (Promega). The PCR prod- strain 3RT stained Gram-negative but had a negative uct was manually sequenced using the fmol DNA Sequenc- KOH test (Gregersen, 1978). Electron micrography of ing kit (Inkitrogen). The 16s rDNA sequence was aligned, thin sections revealed a cell wall with an intensely using the LUJSTAL v program, with similar sequences stained inner layer and a less densely stained outer belonging 10 various members of the domain Bacteria layer (Fig. lb). retrieved from the EMBL database and the Ribosomal Database Project (Maidak eta/., 1994). Only unambiguously aligned positions were used for phylogenetic analysis (1235 Physiological characteristics positions, from 227 to 1416 by E. coli numbering, were selected). An unrooted tree was constructed using the (i) Growth requirements. Strain 3RT required strictly DNADIST (Jukes & Cantor option) and the NEIGHBOR-JOINING anaerobic conditions for growth. The addition of programs contained in the PHYLIP Phylogeny Inference 0.02% yeast extract was necessary for growth in Package, version 3.5 (Felsenstein, 1993). A bootstrap analy- medium with glucose as sole carbon source. The sis (1000 replicates) was also performed using a program temperature range for growth at pH 7 was 35-65 "C, included in the same package. with an optimum at 55 "C. The pH range for growth at 55 "C was 4.3-8.3, with an optimum at 7.0. During RESULTS growth at pH values close to neutrality, the pH decreased by not more than 0.4 units. The doubling Enrichment and isolation of strain 3RT time under optimal conditions in PY medium was 16.1 h. Growth was inhibited in PY medium supple- The bacterial strain used in this study was isolated mented with NaCl to a concentration of 0.4M or from a mesophilic anaerobic wastewater digester at a higher. baker's ye:ist factory located in Montevideo, Uruguay. Bacterial growth was evident after incubation in (ii) Substrate utilization and fermentation products. Cells of glucose-gelatin medium at 55 "C within 5-7 d after strain 3RT were proteolytic. Growth was observed inoculation. Microscopic examination of this primary using gelatin, casein, bovine albumin and other pro- enrichment culture revealed micro-organisms with teinaceous substrates, such as yeast extract and pep- diverse morphologies, but after eight subcultures tones, as energy sources. An extracellular protease small, rod-shaped cells were dominant. Purification activity was demonstrated for cells grown on gelatin. was performed on agar plates incubated at 55 "C under Protease activity increased during growth, showing a anaerobic conditions. After 10 d incubation, colonies maximum of 116220 U at the end of the exponential were about 1 mm in diameter, circular with smooth growth phase.

International Journal of Systematic Bacteriology 48 1299 C. Etchebehere and others

Table 1. Effect of thiosulfate on growth of Coprothermobacter proteolyticus BTT and strain 3RT

Optical density, sulfide and glucose were measured after 7 d incubation at 55 "C for C. proteolyticus BT'" and after 9 d for strain 3R'".

Growth conditions C. proteolyticus BTT Strain 3RT _.

OD,,, "2s Glucose OD,,, "2s Glucose (mM) consumed (YO) (mM) consumed (YO)

- Basal medium" 0,145 3-2 - 0.109 3.5 Basal medium + glucose? 0.387 3.2 58 0.2 10 3.8 19 Basal medium + glucose + thiosulfate? 0,756 20.6 100 1.132 23.5 100

* Basal medium BC supplemented with yeast extract (1 g 1-') and tryptone (1 g 1-l). t Glucose (20 mM) was used as substrate and thiosulfate (20 mM) as electron acceptor.

Table 2. Main characteristics of strain 3RT, Coprothermobacter proteolyticus and Thermobacteroides leptospartum

Character Strain 3RT C. proteolyticus" T. Ieptospartumt

Morphology Short rods, pleomorph Short rods, pleomorph Long, thin rods Optimum pH 7.0 7.5 7.5 Optimum temperature ("C) 55 63 60 Maximum temperature ("C) 65 70 71 G + C content (mol YO) 43 45 43 Fermentation end products from glucose Acetate, H,, CO, Acetate, H,, CO, Ethanol, acetate Thiosulfate reduction to sulfide + + NR Proteolytic + + + Utilization of: Glucose + + Sucrose + - Fructose + Maltose + + Xylose + Starch + NR Growth in the presence of antibiotics Vancomycin (2.5 mg 1-l) + Neomycin (0.15 g 1-') Polymixin B (20 mg 1-l) + - Sodium azide (0.5 g 1-') + + + Kanamycin (600 ng 1-') + + NR Penicillin G (20 U ml-l) + + Isolated from Mesophilic anaerobic Thermophilic anaerobic Cattle manure reactor reactor

NR, Not reported. * Data from this study, Ollivier et ul. (1985) and Kersters et al. (1994). t Data from Toda et a/. (1987).

Cells of strain 3R' were also able to grow on glucose, was consumed and the major fermentation products fructose, sucrose, maltose and starch. However, xylose, were acetate (5.6 mM), hydrogen (1.1 mM) and pro- lactose, sorbitol, glycerol, inositol, xylans and cellulose pionate (1.9 mM). Butyrate (4.6 mM), isobutyrate were not fermented. After growth in BC medium (traces) and isovalerate (traces) were detected in similar supplemented with glucose (2 g 1-'), 10% of the amounts as in control cultures in BC medium. Growth glucose was consumed and the major fermentation on glucose was not affected by the addition of nitrate products were acetate (2.1 mM), hydrogen (2.3 mM) or sulfate. However, thiosulfate had a pronounced and CO, (not determined). After growth in BC medium effect on growth and glucose utilization by strain 3R" supplemented with gelatin (5 g 1-'), 80 YOof the gelatin and C. proteolyticus (BT') (Table 1).

1300 International Journal of Systematic Bacteriology 48 Coprothermobacter platensis sp. nov.

Deinococcus radiodurans

Therm us aquaticus

IThermomicrobium roseum 4 Chloroflexus a uran tiacus Thermodesulfobacterium commune 98.7 Coprothermobacter proteolyticus 100 I- I Strain 3RT

6 Fervidobacterium islandicum

Thermotoga maritima

Hydrogenobacter thermophilus 1007

Aquifex pyrophilus

...... Fig. 2. Phylogenetic tree derived from 165 rDNA sequence data analysis, showing the position of strain 3RT. The bar corresponds to evolutionary distance of 0.1. The numbers shown next to the nodes indicate percentage bootstrap values from 1000 data sets. The EMBL accession numbers for the sequences used in the phylogenetic analysis were as follows: Aquifex pyrophilus strain Kol 5aT, M83548; Choloroflexus aurantiacus strain J-1 O-flT, M34116; Coprothermobacter proteolyticus strain ATCC 35245T, X69335; Deinococcus radiodurans strain ATCC 35073, M21413; Fervidobacterium islandicum strain H21T, M59176; Thermotoga maritima strain MSB8T, M21774; Thermomicrobium roseum strain ATCC 27502T, M34115, Thermodesulfobacterium commune strain YSRA-lT, L10662; Thermus aquaticus strain X-1, X58340; Hydrogenobacter thermophilus strain TK-6T, 230214; Methanococcus jannaschii strain JAL-lT, M59126. ~-

(iii) Antibiotic susceptibility. Growth of strain 3RT was of Jukes & Cantor, obtained from the phylogenetic inhibited by vancomycin (2.5 mg 1-'), neomycin (0.15 g distances matrix, were 96-3% for Coprothermobacter 1-') and polymyxin B (20 mg 1-'). Inhibition was not proteolyticus, 76-1 % for Thermotoga maritima and detected in the presence of penicillin G (20 U ml-'), 75.2 YO for Fervidobacterium islandicum, followed by kanamycin (600 ng ml-l) or sodium azide (0.5 g 1-') Thermodesulfobacterium commune (73.4 YO)and Hy- (Table 2). drogenobacter thermophilus (73.3 %). According to the phylogenetic analysis (Fig. 2) strain 3RT and Copro- DNA base composition thervlzobacter proteolyticus are in the same clade. However DNA-DNA hybridization analysis demon- G C 3RT The + content of strain was 43 mol%. strated less than 12 % similarity between the chromosomal DNAs of these bacteria. Phylogenetic analysis

A total of 1446 nucleotides of the 16s rDNA gene were DISCUSSION sequenced from positions 30 to 1481 according to E. cofi numbering. Comparison with rDNA sequences The new isolate, strain 3RT, is an anaerobic, mod- available in databases revealed that strain 3RT is erately thermophilic, proteolytic bacterium. To our related to Coprothermobacter proteolyticus and peri- knowledge, six anaerobic, thermophilic, proteolytic pherically related to species of the genera Thermotoga micro-organisms of the domain Bacteria have been and Fervidobacterium. The percentage sequences simi- described in the past 10 years : Coprothermobacter larities, corrected for multiple changes by the method proteolyticus (Rainey & Stackebrandt, 1993), formerly

International Journal of Systematic Bacteriology 48 1301 C. Etchebehere and others

Tlzermobacteroides proteolyticus (Ollivier et al., 1985), reduction by bacteria of the Coprothermobacter genus Thermobacteroidgs leptospartum (Toda et al., 1988), as well as its ecological relevance. Clostridium P2 (Orlygsson, 1994), Anaerobranca lzori- According to the physiological and phylogenetic char- koshii (Engle et al., 1993, Thermobrnchium celere acteristics of strain 3RT, we propose the creation of a (Engle et al., 1996) and Calorarnutor proteoclasticus new species within the genus Coprothermobacter to be (Tarlera et al., 1997). The last four belong to the large named Coprothermobacter platensis sp. nov. low G+C content branch of the Gram-positive division of the domain Bacteria (Rainey et al., 1993; Description of Coprothermobacterplatensis sp. nov. Collins et al., 1994). The phylogenetic position of Thermobacteroides leptospurtum remains unknown, Coprothermobacter platensis (pla.ten'sis. L. masc. adj. while Coprotlzermobacter proteolyticus belongs to one pertaining to Rio de la Plata, a river between Uruguay of the deepest divisions of the domain Bacteria (Rainey and Argentina, the region from where the strain was & Stackebrandt, 1993). isolated). The phylogenetic analysis indicated that strain 3R' Cells are straight rods, 0-5 x 1.5-2.0 pm, that occur clusters with Coprothermobacter proteolyticus. This singly or in pairs in young cultures. Long chains are result was supported by the high bootstrap value present in old cultures. Lysis is observed in the (100% of 1000 replicates). The closest relatives are stationary phase. Colonies in PY agar plates are Thermotoga maritima and Fervidobacterium islan- circular, 1 mm in diameter, with an entire border, dicum, both members of the order Thermotogales. transparent to whitish. Stains Gram-negative but the However, the phylogenetic relationship between the cell wall under the electron microscope is atypical with genus Coprothermohacter and the Thermotogales is a dense inner layer and a less dense outer layer. Non- still uncertain, as suggested by the low bootstrap value motile, non-spore-forming. Obligately anaerobic. Pro- (69.5 Yo) and the short evolutionary distance of the teolytic. Ferments gelatin, casein, bovine albumin, common branch. peptone and yeast extract. Glucose, fructose, sucrose, maltose and starch are poorly fermented. Fermen- The level of 16s rDNA sequence similarity between tation products from glucose are acetate, H, and CO,. strain 3RT and C. proteolyticus, the only species in the The major fermentation products from gelatin are genus so far, was 96.3 YO.It has been proposed that acetate, propionate, H, and CO,. Growth on glucose is strains sharing less than 97 YOsimilarity usually do not stimulated by thiosulfate, which is reduced to sulfide. belong to the same species (Stackebrandt & Goebel, Sulfate and nitrate are not reduced. Moderately 1994). Furthermore, the level of chromosomal DNA- thermophilic, optimum temperature 55 "C (range 35- DNA homology between the strains, as measured by 65 "C). Optimum pH 7-0 (range 4-3-8.3). Yeast extract hybridization techniques, was less than 12 YO,far below is required. Growth is inhibited by vancomycin (2.5 mg the threshold value of 70% recommended for species l-'), neomycin (0.15 g 1-') and polymyxin B (20 mg delineation (Wayne et al., 1987). According to these 1-'). Resistant to penicillin G (20 U ml-l), kanamycin results, strain 3RT can be considered as a new species (600 ng ml-') and sodium azide (0.5 g 1-l). NaCl, 0.4 M within the genus Coprothermohacter. or higher, is inhibitory. The G+C content of DNA is Table 2 shows some characteristics of the two species 43 mol Y as determined by HPLC. Phylogenetically of the genus Coprothermobacter and of Thermo- closely related to Coprothermobacter proteoljiticus bacteroides leptospartum, which originally belonged to according to the 16s rDNA sequence analysis. Both the same genus as Coprothermobacter proteolyticus. are included in one of the earlier branches of the There are no significant differences in optimum pH domain Bacteria. Isolated from a mesophilic upflow and G + C content of the chromosomal DNAs. Strain anaerobic sludge blanket reactor of a baker's yeast 3RTand Coprothermobacter proteolyticus show similar factory. The type strain is strain 3RT( = DSM 1 1748'). morphology and fermentation products, differing in both aspects from Thermobacteroides leptospartum. ACKNOWLEDGEMENTS Strain 3RT differs from C. proteolyticus and Thermo- in optimum temperature for We thank Dr Petruccelli from Universidad de la Plata, bacteroides leptospartum Argentina, for the electron microscopy studies and Dr J. growth, antibiotic susceptibility and sugar utilization. Castiglioni from LAFIDESU, Facultad de Quimica, We also tested the ability of strain 3RT and Copro- Uruguay, for hydrogen determinations. We also gratefully thermobacter proteolyticus strain BTT to reduce thio- acknowledge Dr F. Rainey for critically reading the manu- sulfate with glucose as substrate. Thiosulfate was script. This work was supported by a grant from PEDE- reduced to sulfide by both strains, and its addition CIBA-Quimica (a programme for the development of basic sciences in Uruguay). clearly stimulated glucose utilization and growth, as was reported for, among others, Thermotoga, Fervi- dobacterium (Fardeau et d., 1997; Ravot et al., 1995) REFERENCES and Thermosipho species (Ravot et al., 1996). This Bradford, M. M. (1976). A rapid and sensitive method for the property has not been previously reported for members quantitation of microgram quantities of protein utilizing the of the genus Coprothermobacter. Further studies are principle of protein-dye binding. Anid Biochem 72, 248-254. necessary to elucidate the mechanism of thiosulfate Brock, F. M., Forsberg, C. W. & Buchanan-Smith, J. G. (1982).

1302 International Journal of Systematic Bacteriology 48 Copvothermohacter platensis sp. nov.

Proteolytic activity of rumen microorganisms and effects of terization of a methanogenic sludge to be used as inoculum for proteinase Inhibitors. Appl Environ Microbiol44, 561-569. a high-rate reactor. World J Microbiol Biotechnol8, 632-634. Collins, M. D., Lawson, P. A., Willens, A., Cordoba, 1. J., Fer- Ollivier, B. M., Mah, R. A., Ferguson, T. J., Boone, D. R., Garcia, 1. nandez-Garayzabal, J., Garcia, P., Hippe, H. & Farrow, J. A. E. L. & Robinson, R. (1985). Emendation of the genus Thermo- (1994). The phylogeny of the genus Clostridiurn : proposal of five bacteroides : Therinobacteroides proteolyticus sp. nov., a pro- new genera and eleven new species combinations. Int J S-vst teolytic acetogen from a methanogenic enrichment. Int J Syst Bucteriol44, 8 12-826. Bacteriol35, 425428. Engle, M., Li, Y., Woese, C. & Wiegel, 1. (1995). Isolation and Orlygsson, J. (1994). The role of' interspecies hydrogen transfer on characterization of a novel alkalitolerant thermophile, Anaero- the tlzerinophilic protein and amino acid metabolism. PhD thesis, hranca horikoslzii gen. nov., sp. nov. Int J S-yst Bacteriol 45, Swedish University of Agricultural Sciences. 45446 1. Rainey, F.A. & Stackebrandt, E. (1993). Transfer of the type Engle, M., Li, Y., Rainey, F., DeBlois, S., Mai, V., Reichert, A., species of the genus Thermobacteroides to the genus Thermo- Mayer, F., Messner, P. & Wiegel, 1. (1996). Thermobrachium anaerobacter as Thermoanaerobacter acetoetylicus (Ben-Bassat celere gen. nov., sp. nov., a rapidly growing thermophilic, and Ziekus 198 1) comb. nov., description of Coprothermobacter alkalitolerant, and proteolytic obligate anaerobe. Int J Sj>st gen. nov., and reclassification of Thermobacteroides proteo- Bacteriol46, 1025-1033. lyticus as Coprothermobacter proteolytictw (Ollivier et al., 1985) comb. nov. Int J Syst Bacteriol43. 857-859. Fardeau, M.-L., Ollivier, B., Patel, B. K. C., Magot, M., Thomas, P., Rimbault, A., Rocchiccioli, F. & Garcia, I.-L. (1997). Thermotoga Rainey, F. A., Ward, N. L., Morgan, H. W., Toalster, R. & Stacke- hypogeu sp. nov., a xylanolytic, thermophilic bacterium from an brandt, E. (1 993). Phylogenetic analysis of anaerobic thermo- oil-producing well. Int J Syst Bacteriol47, 1013-1019. philic bacteria: aid for their reclassification. Int J Syst Bacteriol Felsenstein, 1. (1993). PHYLIP (Phylogeny Inference Package) 175,4772-4778. version 3.517. Distributed by the author. Department of Gen- Rand, M. C., Greenberg, A. E. & Taras, M. 1. (1975). Sulfide, etics, University of Washington, Seattle, USA. methylene blue method. In Standard Methods for the Ekam- Gregersen, L. (1978). Rapid method for distinction of gram ination qf' Water and Wastewater, 14th edn, pp. 503-505. negative from gram positive bacteria. J Appl Biochem 5, Washington, DC : American Public Health Association, 123-126. American Water Works Association and Water Pollution Control Federation. Johnson, 1. L. (1991). DNA reassociation experiments. In Nucleic Acid Techniques in Bacteriul Systematics, pp. 21-44. Edited by Ravot, G., Ollivier, B., Magot, M., Patel, B. K. C., Crolet, 1. L., E. Stackcblandt & M. Goodfellow. New York: Wiley. Fardeau, M.-L. & Garcia, 1. L. (1995). Thiosulfate reduction, an important physiological feature shared by members of the order Johnson, J. L. (1994). Similarity analysis of rRNAs. In Methods Tlzermotogales. Appl Environ Microbiol61, 2053-2055. for Generd and Molecular Bacteriology, pp. 683-700. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Ravot, G., Ollivier, B., Patel, B. K. C., Magot, M. & Garcia, 1. L. Washington. DC : American Society for Microbiology. (1996). Emended description of Thermosipho africanus as a carbohydrate fermenting species using thiosulfate as an electron Kersters, I., Maestrojuan, G. M., Torck, U., Vancanneyt, M., acceptor. hit J Svst Bacteriol46, 321-323. Kersters, K. & Verstraete, W. (1994). Isolation of Copro- thermobwtc~rproteolyticus from an anaerobic digest and futher Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular characteriz'ition of the species. Sj9st Appl Microbioll7,289-295. Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Lee, Y. E., Jain, M. K., Lee, C., Lowe, 5. E. & Zeikus, 1. G. (1993). Taxonomic distinction of saccharolytic thermophilic anaer- Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characteriza- obes : description of Thermoanaerobacter xylarzolyticunz gen. tion. In Methods for General and Molecular Bacteriology, pp. nov., sp. nov., and Thermounaerobacterium saccharolyticum 611-651. Edited by P. Gerhardt, R. G. E. Murray, W. A. gen. nov., sp. nov., reclassification of Thermoanaerobium brokii, Wood & N. R. Krieg. Washington, DC : American Society for Clostridiurii therinosulfurogenes, and Clostridium thermohydro- Microbiology. sulfuricutn E 100-69 as Tlzer~noaiiaerobacter brokii comb. nov., Speece, R. E. (1 996). Anaerobic Biotechnology for Industrial Thermoc~Mirerobacteriuul.2 tliernzosulfurigenes comb. nov., and Wastewaters, pp. 3-6. Nashville, TN : Vanderbilt University Theriizoc~nLierobacter ther~~ohvdrosulfuricuscomb. nov., res- Press. pectively ; md transfer of Clostridium thermoliydrosulfuri~u~i Stackebrandt, E. & Goebel, M. (1994). Taxonomic note: a place 39E to Tlii~r.r~ioanaerobacteretlzanolycus. Int J Sjxt Bacteriol43, for DNA-DNA reassociation and 16rRNA sequence analysis 41-51. in the present species definition in bacteriology. Int J Syst Lettinga, G. (1995). Anaerobic digestion and wastewater treat- Bacteriol44, 846-849. ment systems. Antonie Leeuwenhoek 67, 3-28. Tarlera, S., Muxi, L., Soubes, M. & Stams, A. J. M. (1997). Mclnerney, M. J. (1988). Anaerobic hydrolysis and fermentation Caloramator proteoclasticus sp. nov., a new moderately thermo- of fats and proteins. In Biology of Anaerobic Microorganisms, philic anaerobic proteolytic bacterium. Int J Syst Bacteriol47, pp. 373-415. Edited by A. J. B. Zehnder. New York: Wiley. 65 1-656. Maidak, B. L., Larse, M. J., McCaughey, R., Overbeek, G. J., Fogel, Toda, Y., Saiki, T., Uozumi, T. & Beppu, T. (1988). Isolation and K., Blandy, 1. & Woese, C. (1994). The Ribosomal Database characterization of a protease-producing, thermophilic anaer- Project. Niiclclic Acids Res 22, 3485-3487. obic bacterium, Thermobacteroides leptospartum sp. nov. Agric Mesbah, M., Premanchandran, U. & Whitman, W. B. (1989). Biol Chem 52, 1339-1344. Precise mexiurement of the G + C content of deoxyribonucleic Touzel, J. P. & Albagnac, G. (1983). Isolation and charac- acid by hi9h performance liquid chromatography. bzt J Syst terization of Metlianococcus mazeii strain MC3. FEMS Micro- Bacteriol39, 159-1 67. hiol Lett 16, 241-245. Muxi, L., Zunino, L., Tarlera, 5. & Soubes, M. (1992). Charac- Van Lier, J. B., Hulsbeek, J., Stams, A. J. M. & Lettinga, G. (1993).

~ ~~ ~ ~~ ~~ International Journal of Systematic Bacteriology 48 1303 C. Etchebehere and others

Temperature susceptibility of thermophilic methanogenic Approaches to Bacterial Systematics. Int J Syst Bacteriol 37, sludge : implications for reactor start-up and operation. Bio- 463464. resource Techno143, 227-235. Wiegel, J. (1 992). The obligately anaerobic thermophilic bac- Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 others (1987). teria. In Thermophilic Bacteria, pp. 105-184. Edited by J. K. Report of the Ad Hoc Committee on Reconciliation of Kristjansson. Boca Raton, FL: CRC Press.

1304 International Journal of Systematic Bacteriology 48