thermacetogenium phaeum gen. nov., sp. nov., a strictly anaerobic, thermophilic, syntrophic...

International Journal of Systematic and Evolutionary Microbiology (2000), 50, 16011609 Printed in Great Britain

Thermacetogenium phaeum gen. nov., sp. nov.,a strictly anaerobic, thermophilic, syntrophicacetate-oxidizing bacterium

Satoshi Hattori,1,2 Yoichi Kamagata,1 Satoshi Hanada1

and Hirofumi Shoun2

Author for correspondence: Yoichi Kamagata. Tel : 81 298 61 6591. Fax: 81 298 61 6587.e-mail : kamagata!nibh.go.jp

1 National Institute ofBioscience and HumanTechnology, Agency ofIndustrial Science andTechnology, 1-1 Higashi,Tsukuba, Ibaraki 305-8566,Japan

2 Institute of AppliedBiochemistry, University ofTsukuba, 1-1-1 Tennodai,Tsukuba, Ibaraki 305-8572,Japan

A novel anaerobic, thermophilic, syntrophic acetate-oxidizing bacterium, strainPBT, was isolated from a thermophilic (55 SC) anaerobic methanogenic reactorwhich had been treating kraft-pulp waste water. The bacterium oxidizedacetate in co-culture with a thermophilic hydrogenotrophic methanogen.Strain PBT, a Gram-positive, spore-forming, rod-shaped bacterium grewoptimally at 58 SC and pH 6

S. Hattori and others

genetically and chemotaxonomically well charac-terized, the phylogenetic position of the thermophilicstrain AOR is not known. Moreover, the isolate hasnot been deposited in any culture collection and is nolonger available. Considering the other findings thatsyntrophic oxidation may be involved in methano-genesis from acetate in thermophilic methanogenicreactors (Petersen & Ahring, 1991; Uemura & Harada,1993), the phylogenetic position and the physiologicaltraits of thermophilic acetate-oxidizing syntrophs areof great interest to elucidate their importance in theenvironment. Ten years ago, we found methanogenesisfrom acetate in a thermophilic methanogenic en-richment in which neither Methanosarcina- norMethanosaeta-like cells were observed (Kamagata &Mikami, 1989). In this report, we describe the isolationand characterization of a new thermophilic, syntrophicacetate-oxidizing bacterium, strain PBT, from theenrichment and propose a new name for the isolate.

METHODS

Source of organisms. The syntrophic acetate-oxidizingbacterium, strain PBT, and a thermophilic hydrogen-and formate-utilizing methanogen, Methanothermobacterthermoautotrophicus strain TM (previously Methano-bacterium thermoautotrophicum) were isolated from athermophilic (60 C) anaerobic methanogenic reactor thattreated waste water from a kraft-pulp production plant inJapan. M. thermoautotrophicus strain DH (flDSM 1053)was obtained from the DSMZ (Braunschweig, Germany).

Media and cultivation. The composition of the basal mediumused for all experiments was based on DSM 334 medium(DSMZ, 1983). The basal medium contained (per litre) :10 g NH

%Cl, 03 g KH

#PO

%, 06 g NaCl, 01 g MgCl

#\2H

#O,

008 g CaCl#\2H

#O, 35 g KHCO

$, 1 mg sodium resazurin,

10 ml vitamin solution and 5 ml trace element solution. Thevitamin solution was replaced with that of DSM 141 medium(DSMZ, 1983). The composition was as follows (per litre) :20 mg biotin, 20 mg folic acid, 100 mg pyridoxine hy-drochloride, 50 mg thiamin hydrochloride, 50 mg ribo-flavin, 50 mg nicotinic acid, 50 mg dl-calcium panto-thenate, 01 mg vitamin B

"#, 50 mg p-aminobenzoate and

50 mg lipoic acid. The trace element solution was based onDSM 318 medium (DSMZ, 1983) and contained (per litre) :128 g nitrilotriacetic acid, 135 g FeCl

$\6H

#O, 01 g

MnCl#\4H

#O, 0024 g CoCl

#\6H

#O, 01 g CaCl

#\2H

#O,

01 g ZnCl#, 0025 g CuCl

#\2H

#O, 001 g H

$BO

$, 0024 g

Na#MoO

%\4H

#O, 10 g NaCl, 012 g NiCl

#\6H

#O, 40 mg

Na#SeO

$\5H

#O, 40 mg Na

#WO

%\2H

#O. The trace element

solution was adjusted to pH 65 with 1 M KOH. For growthof the syntrophic acetate-oxidizing co-culture, the mediumcontained 80 mM sodium acetate. For the isolation andmaintenance of strain PBT, 80 mM sodium pyruvate wasused as the substrate. The medium was anaerobicallydispensed into serum vials or bottles under N

#}CO

#(4 :1,

v}v) atmosphere. The vials or bottles were sealed with butylrubber stoppers fitted with caps. The medium was auto-claved for 20 min at 121 C. The pH of the autoclavedmedium was approximately 6971. Prior to inoculation, themedium was reduced with sterile stock solutions of Na

#S and

cysteine}HCl (final concentration 03 g l" each). Unlessotherwise noted, cultures were incubated at 55 C in the darkwithout shaking.

Determination of growth. To determine the growth, a 25 ml

Hungate tube containing 10 ml medium was used. Growthwas determined by measuring OD

!with a Hitachi spectro-

photometer model 100-10. Unless otherwise noted, thegrowth experiments were performed at least in duplicate.

Temperature, pH and NaCl ranges for growth. Mediumcontaining 20 mM sodium pyruvate was used, and a late-logculture of strain PBT was inoculated into the fresh basalmedium. For determination of the temperature ranges forgrowth, cultures were incubated at 3070 C. For the pHexperiment, the pH of the medium was adjusted with sterilestock solutions of 10% K

#CO

$or 1 M HCl. The pH value

was calibrated at 25 C. The range of pH tested was5491.To determine the effect of salt concentrations ongrowth, sodium chloride was added to the medium beforeautoclaving to give final concentrations of 005 to 5%.Determination of acetate-oxidation rate. The acetate-oxi-dation rate [mmol acetate consumed h" (g dry cell wt)"] wasdetermined by using washed-cell suspension. Strain PBT wasco-cultured with Methanothermobacter thermoautotrophicusstrain TM on medium containing 80 mM acetate (500 ml)for a month. The cells were harvested anaerobically, sus-pended in the acetate medium (20 ml) and incubated at55 C. Methane and acetate were determined at appropriateintervals.

Utilization of substrates and electron acceptors. Severalcompounds including organic acids, alcohols, methoxylatedaromatics, sugars, amino acids and hydrogen were used forthe substrate-utilization tests. Substrates were added fromfilter-sterilized or autoclaved stock solutions (pH 70) to givethe desired concentrations. When hydrogen was used as thesubstrate, the head space (15 ml) of the tube was replacedwith hydrogen}CO

#(4 :1, v}v, 151 kPa). For determining

electron acceptors, 40 mM sodium acetate was used as theelectron donor, and the following organic or inorganiccompounds were tested as electron acceptors (mM): sulfate(20), sulfite (25), nitrate (20), nitrite (25), thiosulfate (20),amorphous Fe(III) (5) and fumarate (10). Two negativecontrols were incubated simultaneously: (1) medium con-taining strain PBT and acetate but without an electronacceptor and (2) the medium containing acetate and electronacceptor but without cells.

Microscopy. Cultures were routinely examined with anOlympus model AX-80 microscope equipped with phase-contrast and epifluorescence apparatus. For transmissionelectron microscopy, cells were centrifuged and fixed with5% (v}v) glutaraldehyde and 1% (v}v) osmium tetroxide.The samples were embedded in Spurr resin and the ultrathinsections of the samples were prepared with a Reinchert-Nissei ultramicrotome. The samples were stained with 1%uranyl acetate and 03% lead citrate. After treatment ofvapor deposition with carbon, the samples were observedusing a Hitachi model H-7000 electron microscope operatingat 75 kV.

Analytical methods. Methane was analysed by a Shimadzumodel GC-8AIT gas chromatograph equipped with amolecular sieve 5A 60}80 mesh column and a thermalconductivity detector. Argon was used as the carrier gas at aflow rate of 27 ml min". The column and the detectortemperatures were kept at 60 C and 110 C, respectively.Acetate and fermentation products were determined byusing a Shimadzu model LC-6A HPLC equipped with aShimadzu SCR-101H column and a model SPD-6A UVdetector. The column was operated at 40 C, and 17 mMHClO

%was used as the eluent at a flow rate of 1 ml min".

Sulfate, sulfite, nitrate, nitrite and thiosulfate were de-termined by using a Shimadzu model LC-6A HPLC

1602 International Journal of Systematic and Evolutionary Microbiology 50

Thermacetogenium phaeum gen. nov., sp. nov.

equipped with a Shimadzu IC-A3 ion-exchange column anda model CDD-6A electronic conductivity detector.Operating conditions were as follows: column temperature,40 C; eluent, 80 mM p-hydroxybenzoic acid and 32 mMbis-tris ; flow rate, 12 ml min". Utilization of amorphousFe(III) was assessed by measuring changes in the visiblecolour of the medium (Slobodkin et al., 1997a).

DNA extraction and purification. Genomic DNA wasextracted and purified according to the method of Hiraishi(1992). For determination of DNA base composition, thecrude DNA was further purified with an equal volume ofphenol}chloroform}isoamylalcohol (25:24:1, by vol).

DNA base composition. The GC content of strain PBT wasdetermined by enzymic digestion of genomic DNA andHPLC separation using a Yamasa GC kit as described byKamagata & Mikami (1991). The purified DNA sample wasseparated by using a Shimadzu model LC-6A HPLCequipped with a Shimadzu CLC-ODS reverse-phase columnand a model SPD-6A UV detector at 270 nm. The columnwas operated at 40 C, and 10 mM potassium phosphatebuffer (pH 35) containing 5% methanol was used as theeluent at a flow rate of 1 ml min".

Amplification and purification of 16S rDNA. Crude DNA wasused as template for PCR amplification of 16S rDNA. PCRwas performed by using an AmpliTaq Gold reagent kit(Perkin Elmer) according to the manufacturers protocol.The PCR primers used for amplification were the for-ward primer 8F (5-AGAGTTTGATCCTGGCTCAG-3,Escherichia colipositions 827) and the reverse primer 1491R(5-GGTTACCTTGTTACGACTT-3, E. coli positions15091491) (Weisburg et al., 1991). PCR conditions wereas follows: pre-heating at 95 C for 9 min, 35 cycles ofdenaturing at 95 C for 1 min, annealing at 50 C for 1 minand extension at 72 C for 2 min.

16S rDNA sequence determination and phylogenetic analy-sis. Purified PCR products of 16S rDNA were sequenceddirectly with a PRISM dRhodamine Terminator CycleSequencing Ready Reaction Kit (Applied Biosystems) ac-cording to the manufacturers protocol. Six primers wereused for sequencing according to the method of Hiraishi(1992). Both obtained and reference 16S rDNA sequenceswere aligned with the clustal w program, version 1.5(Thompson et al., 1994). The phylogenetic tree was con-structed by the neighbour-joining method (Saito & Nei,1987) with the mega program. Bootstrap values wereobtained for 100 replicates to estimate the confidence of treetopologies.

Quinone analysis. Quinone was determined according to themethod of Tamaoka et al. (1983). Approximately 2 g (wetweight) of the late-exponential-phase culture of strain PBT

grown on pyruvate was used for the analysis.

Nucleotide sequence accession numbers. The 16S rDNAsequence of strain PBT has been deposited in the DDBJ}GenBank}EMBL database under the accession numberAB020336. The accession numbers of the referencesequences used in the phylogenetic analysis are as follows:Thermoterrabacterium ferrireducens DSM 11225T, U76364;Moorella glycerini DSM 11254T, U82327; Moorella thermo-acetica ATCC 39073T, M59121; Moorella thermoauto-trophica DSM 1974T, L09168; Thermoanaerobacterethanolicus DSM 2355T, L09164; Thermoanaerobacteriumthermosulfurigenes DSM 2229T, L09171; Syntrophomonaswolfei DSM 2245T, AF022248; Syntrophospora bryantiiDSM 3014T, M26491; Thermosyntropha lipolytica DSM

11003T, X99980; Desulfotomaculum australicum DSM11792,M96665;Desulfotomaculum thermoacetoxidansDSM5813T, Y11573; Desulfotomaculum thermobenzoicum DSM6193T, Y11574; Desulfotomaculum nigrificans DSM 574T,X62176; Desulfotomaculum ruminis DSM 2154T, Y11572;Bacillus subtilis NCDO 1769, X60646; Clostridium ultunenseDSM 10521T, Z69293; Clostridium butyricum DSM 552T,M59085; Arthrobacter globiformis DSM 20124T, X80736.

RESULTS

Enrichment and isolation

Strain PBT was originally derived from an enrichmentculture which converted acetate to methane. For theprimary enrichment of a syntrophic acetate-oxidizingco-culture, a sample taken from a thermophilic an-aerobic reactor was serially diluted and inoculated intothe basal medium containing 80 mM sodium acetate.After 2 months incubation at 55 C, the culturesreceiving 10" to 10 dilutions completely degradedacetate and produced nearly equimolar amounts ofmethane. Under microscopic observation, the culturesreceiving 10" to 10% dilutions contained severalkinds of microbes such as Methanobacterium- andMethanosaeta-like methanogens. However, neitherMethanosarcina nor Methanosaeta-like cells wereobserved in the cultures that received the 10& to 10dilutions. The highest-dilution culture was againserially diluted into fresh acetate medium.After severaltransfers, the culture was found to be composed of twomorphologically distinct rod-shaped bacteria. Oneorganism had a terminal endospore and showed non-autofluorescence. The other, showing autofluorescenceindicative of F

%#!, was morphologically similar to

Methanobacterium cells. Neither Methanosarcina norMethanosaeta types of methanogen were found. Theseobservations suggested that syntrophic acetate oxi-dation, as has been previously described by Zinder &Koch (1984) and Schnu$ rer et al. (1994) occurred in theculture. However, the culture was not still a defined co-culture, since other types of heterotroph appearedvigorously when the culture was incubated with yeastextract or other carbon sources such as glucose andtryptone. To eliminate those contaminants, we tried torepeat serial dilutions, roll-tube culture or antibiotic-containing culture. However, all attempts were un-successful. Therefore, we tried to isolate each memberseparately from the culture. For isolation of thesyntrophic acetate-oxidizing bacterium, several com-pounds such as ethylene glycol were tested accordingto the method previously described (Lee & Zinder,1988; Schnu$ rer et al., 1996). Of the substrates tested,80 mM sodium pyruvate allowed a spore-formingbacterium to grow after 7 d incubation at 55 C. Themicrobe resembled the bacterium which had pre-dominated as well as Methanobacterium-like cells inthe enrichment. We therefore focused on this bac-terium and inoculated the cells into a 2% agar (AgarNoble, Difco) medium containing 80 mM pyruvate asthe sole carbon and energy source. After 2 monthsincubation, the colonies which grew up to 2 mm in

International Journal of Systematic and Evolutionary Microbiology 50 1603


50

40

30

20

10

6 12 18 24 30

Ace

tate

or

met

han

e (m

M)

Time (d)

.................................................................................................................................................

Fig. 1. Syntrophic degradation of acetate to methane by areconstituted co-culture of strain PBT and M. thermoauto-trophicus strain TM. D, acetate; +, methane.

diameter were picked with a sterile Pasteur pipette andsubcultured in liquid medium containing 80 mMpyruvate and 20 mM BES. Purity of the strain,designated PBT, was checked by inoculating it into themedium containing 01% yeast extract and 1% BactoTryptone (Difco), and by microscopy. The thermo-philic hydrogen- and formate-utilizing methanogen,M. thermoautotrophicus strain TM was also isolatedfrom the primary enrichment culture by using the sameprocedure, except that pyruvate was replaced withhydrogen}CO

#(4 :1, v}v, 151 kPa) and BES was

omitted.

Reconstruction of syntrophic acetate-oxidizingco-culture

To prove strain PBT to be a syntrophic acetate-oxidizing bacterium, strains PBT and TM were co-inoculated into basal medium containing 80 mMsodium acetate as the sole carbon and energy source,and incubated at 55 C. As the control experiments,strains PBT and TM were separately inoculated underthe same conditions. Growth was observed only in themixed culture after 40 d incubation. Acetate wasconverted to methane. The co-culture was able toretain methanogenic activity from acetate after furthertransfer into the fresh medium. Fig. 1 shows that theco-culture completely degraded 40 mM acetate andstoichiometrically produced methane within 24 d. Theacetate-oxidation rate using washed-cell suspensionwas calculated to be 129 mmol acetate consumed h"(g dry cell wt)". Strain PBT could also be co-culturedwith a hydrogen-utilizing methanogen, M. thermo-autotrophicus strain DH.

Colony and cell morphology

Colonies of strain PBT were disc shaped, 1030 mm indiameter, smooth and brownish. Figs 2 and 3 show themorphology and ultrathin sections of the syntrophic

(a)

(b)

(c)

.................................................................................................................................................

Fig. 2. (a) Phase-contrast micrograph of strain PBT in pureculture grown on methanol; (b) co-culture of strain PBT with M.thermoautotrophicus strain TM grown on acetate; (c)epifluorescence micrograph of the same field as (b). Bars,10 lm.

co-culture and the pure culture of strain PBT. Gram-staining was negative. However, transmission electronmicroscopy indicated a Gram-positive type of cell wall(Fig. 3). Cells were straight or slightly curved rods withrounded ends, 0407 lm wide and 20126 lm long.The cells occurred singly, in pairs or in chainsdepending on the growth phase and growth conditions,i.e. under syntrophic conditions, strain PBT elongatedthe cell length at late-exponential phase and occurredsingly. In pure culture, the cells often occurred in pairsor in chains at the exponential phase. Terminalendospores were much more frequently observed inthe syntrophic co-culture than in the pure culture(Fig. 3).



(a)

(b)

.....................................................................................................

Fig. 3. Transmission electron micrographs ofpure culture (a) and syntrophic co-cultureshowing a terminal endospore (b). Thearrow in (b) indicates the strain PBT cell.Bars, 1 lm.

Growth properties

The temperature range for growth of strain PBT was4065 C, with an optimum temperature around 58 C(Fig. 4a). No growth was observed within 2 months at35 C and 70 C. The pH range for growth was 5984and optimal growth was at pH 68 (Fig. 4b). Growthwas not observed at pH 54 and pH 88. The doublingtime under the optimum conditions was calculated tobe 228 h. Strain PBT was able to grow at NaClconcentrations ranging from 005 to 45% (w}v).

Substrate utilization

The following substrates were utilized for growthin pure culture (unless otherwise noted, substrateswere added at a final concentration of 20 mM): meth-anol, ethanol, n-propanol, n-butanol, 2,3-butanediol,ethanolamine, pyruvate, 3,4,5-trimethoxybenzoate(10 mM), syringate (10 mM), vanillate (10 mM), gly-cine, cysteine, formate (40 mM) and hydrogen}CO

#(4 :1, v}v, 151 kPa). Acetate was the sole or majorfermentation product from these substrates. In co-culture with hydrogenotrophic methanogens, the bac-terium syntrophically oxidized acetate (40 mM) toform methane. No growth was observed on 1,2-propanediol, ethylene glycol, glycerol, citrate,

fumarate, malate, lactate, sarcosine, betaine, meth-ylamine, dimethylamine, trimethylamine, glucose,fructose, galactose, maltose, sucrose, lactose and yeastextract (01%).

Electron acceptors

In the presence of sulfate, strain PBT consumed 12 mMacetate (initial concentration, 40 mM) with concomi-tant reduction of sulfate. Acetate was not degradedfurther after prolonged incubation. Thiosulfate wasalso utilized as an electron acceptor. Fe(III), sulfite,nitrate, nitrite and fumarate were not utilized.

Chemotaxonomic characteristics

The genomic DNA GC content of strain PBT was535 mol% as determined by HPLC. Quinone analysisrevealed that strain PBT contained MK-7 as the majorquinone component.

Phylogenetic analysis

Nearly the full length (1462 bp) of the 16S rDNAsequence of strain PBT was determined. The sequencewas aligned and compared with 18 representative



004

003

002

001

00030 40 50 60 70

(a)

(b)

Temp. ( C)

002

001

000

Gro

wth

rat

e (h

1)

5 6 7 8 9pH

.................................................................................................................................................

Fig. 4. Effect of temperature (a) and pH (b) on the growth ofstrain PBT.

sequences of other bacteria. On the basis of thephylogenetic analysis (Fig. 5), strain PBT was found tobe a member of the BacillusClostridium subphylum ofthe Gram-positive bacteria, and the closest relativewas Thermoterrabacterium ferrireducens (874% simi-larity).

.....................................................................................................

Fig. 5. Phylogenetic position of strain PBT

within the BacillusClostridium subphylumof the Gram-positive bacteria. The tree wasbased on 16S rDNA sequences and wasconstructed using the neighbour-joiningmethod. Bar, 002 base substitutions pernucleotide position.

DISCUSSION

In this report, we have described a novel strictlyanaerobic, thermophilic acetate-oxidizing syntroph. Inpure culture, this strain grew acetogenically on severalalcohols, organic acids, methoxylated aromatics andsome amino acids. The strain could also grow auto-trophically on hydrogen}CO

#to produce acetate as

the major product.

With regard to the other syntrophic acetate-oxidizingbacteria, two syntrophs have been previously isolatedand characterized: a thermophilic syntroph, strainAOR (Lee & Zinder, 1988), and a mesophilic syntroph,Clostridium ultunense (Schnu$ rer et al., 1996). Table 1shows the properties of these two isolates and ourstrain. All the isolates show acetogenic growth in pureculture.

One of the very interesting features that distinguishstrain PBT from the other two strains was that thestrain can oxidize acetate in pure culture by usingsulfate as the electron acceptor. This trait is verysimilar to several sulfate-reducing bacteria belongingto the genus Desulfotomaculum. Desulfotomaculumacetoxidans and D. thermoacetoxidans are able tooxidize acetate completely with sulfate reduction(Widdel & Pfennig, 1977; Min & Zinder, 1990). StrainPBT is phylogenetically affiliated with the Gram-positive low GC group, which also includes Desulfo-tomaculum species (Fig. 5). Moreover, strain PBT, aswell as Desulfotomaculum species, contains MK-7 asthe major quinone component. These indicate that ourisolate harbours a sulfate-respiration system. Theother feature of such micro-organisms including ourisolate is that they are also capable of growingacetogenically on pyruvate or methoxylated aromaticsin the absence of sulfate (Min & Zinder, 1990; Tasakiet al., 1992, 1993; Nilsen et al., 1996). Considering thefact that those Desulfotomaculum strains harbour theenzymes of the carbon monoxide dehydrogenase path-way that allows the microbes to convert methoxylatedcompounds to acetate, it is very likely that our isolatehas a similar pathway.



Table 1. Characteristics of strain PBT and other syntrophic acetate-oxidizing bacteria.....................................................................................................................................................................................................................................

nd, Not determined.

Characteristic Strain PBT* Strain AOR C. ultunense

Cell size (lm) 04072126 040623 0507057Spore formation fi Gram stain Temperature range (C) 4065 5065 1550Optimum temperature (C) 58 60 37pH range 5984 nd 510Optimum pH 68 nd 7DNA GC content (mol%) 535 47 32Major menaquinone MK-7 nd nd

Supplement required None Yeast extract Yeast extract

Utilization of substrate :

In pure culture

Hydrogen}CO#

fiFormate Ethylene glycol fi Methanol fi fiEthanol fi fin-Propanol fi ndn-Butanol fi fi1,2-Propanediol fi fi2,3-Butanediol fi ndEthanolamine fi fiGlycerol fi fi fiPyruvate 3,4,5-Trimethoxybenzoate fi fiSyringate nd fiVanillate nd fiGlycine fi fiBetaine fi Methylamine fi fi fiCysteine nd Glucose fi fi Acetate fi fi fiAcetate plus sulfate fi fi

In co-culture with methanogen

Acetate

*This study.

Data from Lee & Zinder (1988).

Data from Schnu$ rer et al. (1996).

Phylogenetic analysis based on 16S rDNA sequenceshowed that strain PBT was most closely related to aniron-reducing, non-spore forming bacterium, Thermo-terrabacterium ferrireducens (Slobodkin et al., 1997a)(Fig. 5). However, the sequence similarity was very low(874%). Moreover, strain PBT differed from Thermo-terrabacterium ferrireducens in several features : (1)strain PBT is not capable of utilizing Fe(III) as anelectron acceptor ; (2) it is a spore former; (3) it has amuch higher DNA GC content. Our strain is ratherrelated to the genus Moorella than to Thermo-

terrabacterium in several features such as cell mor-phology, substrate utilization, physiological proper-ties, quinone composition and the GC content ofDNA (Fontaine et al., 1942; Wiegel et al., 1981; Hippeet al., 1992; Collins et al., 1994; Slobodkin et al.,1997b). However, Moorella spp. do not have the abilityto reduce sulfate. These special features are clearlyenough to distinguish our isolate from the othergenera. On the basis of phenotypic and phylogeneticdistinctiveness of the isolate, we concluded that theorganism characterized here represents a novel and



distinct taxon for which we propose the name Therma-cetogenium phaeum gen. nov., sp. nov.

Description of Thermacetogenium gen. nov.

Thermacetogenium (therm.a.ce.to.geni.um. Gr. adj.thermos warm; L. n. acetum vinegar ; Gr. v. suff.genium producing; M.L. neut. n. Thermacetogeniumthermophilic vinegar producer).

Cells are rod shaped. Strictly anaerobic and thermo-philic. Chemoautotrophic and chemo-organotrophic.Gram reaction is negative, but shows a Gram-positivecell-wall structure. Round terminal endospores areformed. Colonies are disc shaped. Cells are able tooxidize acetate in co-culture with hydrogenotrophicmicro-organisms. Acetate can be utilized also bysulfate reduction in pure culture. MK-7 is the majorquinone. Grows acetogenically on several alcohols,methoxylated aromatics, organic acids, amino acidsandhydrogen}CO

#. Sugars are not utilized.Additional

supplements are not required.

Description of Thermacetogenium phaeum sp. nov.

Thermacetogenium phaeum (phaeum. Gr. adj. phaiosbrown, referring to the colour of the colonies ; M.L.neut. adj. phaeum brown).

Cells are straight or slightly curved and rod shaped,0407 lm wide and 20126 lm long. Occur singly, inpairs or in chains. Round terminal endospores areobserved. Colonies are disc shaped, 13 mm in di-ameter, smooth and brownish. Methanol, ethanol, n-propanol, n-butanol, 2,3-butanediol, ethanolamine,pyruvate, 3,4,5-trimethoxybenzoate, syringate, vanil-late, glycine, cysteine, formate, and hydrogen}CO

#are

utilized in pure culture. Acetate is the primary fer-mentation product. Sugars are not utilized. Organicnutrients such as yeast extract are not required forgrowth. Acetate is oxidized to CO

#in co-culture with

hydrogen-utilizing methanogens. Acetate is alsooxidized to CO

#in pure culture with reduction of

sulfate or thiosulfate as the electron acceptor. Sulfite,nitrate, nitrite, Fe(III) and fumarate are not utilizedwhen acetate is used as the electron donor. Tem-perature range for growth is 4065 C, with anoptimum at 58 C. Growth is observed at a pH rangefrom 59 to 84, and the optimum is pH 68. Growthoccurs in the presence of NaCl at 00545% (w}v).The GC content of DNA is 535 mol%. The majorquinone is MK-7. The type strain is Thermacetogeniumphaeum strain PBT (flDSM 12270T).

ACKNOWLEDGEMENTS

We thank Xian-Ying Meng at National Institute of Bio-science and Human Technology (NIBH) Agency ofIndustrial Science and Technology for taking the electronmicrographs. We also thank Yasuyo Ashizawa at NIBH forher help with the quinone analysis and Yoko Ueda at NIBHfor her assistance with the determination of DNA GC

content. We thank Yuji Sekiguchi at Nagaoka University ofTechnology for his critical reading of the manuscript.

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thermacetogenium phaeum gen. nov., sp. nov., a strictly anaerobic, thermophilic, syntrophic...

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