SPOROMUSA ACIDOVORANS sp. nov. was isolated from a pilot fermenter inoculated with effluent sample from the alcohol distillation industry. The isolate was a Gram-negative, motile, curved, spore-forming rod. The DNA base composition was 42 % G+C. The temperature range for growth was 20 to 40°C, with an optimum at 35°C ; growth occurred within a pH range of 5.4 to 7.5, with an optimum at pH 6.5. Growth substrates included methanol, H2-CO2, formate, fructose, ribose, fumarate, succinate and glycerol. Yeast extract was required for growth. The organism performed the homoacetogenic reaction. (Résumé d'auteur

Cord-Ruwisch, Ralf

Garcia, Jean-Louis

Lombardo, Anna

Ollivier, Bernard

Horizon / Pleins textes

4 
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Arch Microbio1 (1985) 142:307-310 
Archives of 
II 
(0 Sminrer-Verlag 1985 
Isolation and characterization of Sporoinusa acidovorans sp. nov., 
a met h ylo tr ophic homoace togenic bac terium 
Bernard Ollivier ', Ralf Cordruwisch ', Anna Lombardoi, and Jean-Louis Garcia * 
Laboritoire de Microbiologic OIISTOM, UnivcrsilL: de Provence, 3 Place Viclor-l-lugo, F-13331 Marseille CCdex 3, Francc 
,2 Istituto di Biologia del Mare, Riva 7 Martiri, 1-30122 Venezia, Italia 
Abstract. Sporomuw ackkuvorans sp.nov. was isolated from 
a pilot fermenter inoculated with effluent sample from the 
alcohol distillation industry. The isolate was a Gram-nega- 
tive, motile, curved, spore-forming rod. The DNA base 
composition was 42% G 4- C. The temperature range for 
growth was 20 to 40"C, with an optimum at 35°C; growth 
occurred within a pH range of 5.4 to 7.5, with an optimum 
at pH 6.5. Growth substrates included methanol, H2-C02, 
formate, fructose, ribose, fumarate, succinate and glycerol. 
Yeast extract was required for growth. The organism per- 
formed the homoacetogenic reaction. 
Key words: Sporotnusu ciciduvorcrns - Homoacetogenesis - 
Methanol - Hydrogen - Fructose 
In absence of sulfate, the anaerobic oxidation of Hz leads to 
methane or acetate production. Bacteria capable of reducing 
COz to acetate were first observed in non-defined mixed 
cultures by Fischer et al. (1931). In 1936 Wieringa described 
the enrichment and the isolation of the homoacetogenic 
bacterium: Clostridiuni aceticum. Within the sanie genus, 
other homoacetogenic H2 oxidizing bacteria have been dis- 
covered. C. thertiioaurotrophicutn was isolated from mud or 
soil samples (Wiege1 et al. 1981). A non-identified methanol 
degrading Clostridium strain has been recently described by 
Adamse and Velzeboer (1982). 
Other spore-forming . H,-oxidizing bacteria stained 
Gram negative and belonged to the newly described genus 
SporotnuSa (Möller et hl. 1984). Three non spore-forming 
acetate producing bacteria have been isolated on Hz and 
CO2: Acetobacterium woodii (Balch et al. 1977), A .  wieringae 
(Braun and Gottschalk 1982) and Acetogenium kivui (Leigh 
et al. 1981), a thermophilic homoacetogen. 
We report on the isolation of a new Gram negative 
spore-forming homoacetogenic bacterium. This rod-shaped 
bacterium used methanol or Hz as energy source and is 
proposed as a new species of lhe genus Sporomusa: 
S. acidovoruns. 
Materials and methods 
Chetniculs. All chemicals were of reagent quality unless 
otherwise noted. Gases were purchased from Airgaz 
(Marseille, France). 
OjJjwint requests to: J .  L. Garcia, Laboratoire de Microbiologie 
ORSTOM, Université de Provence, 3 Place Victor-Hugo, F-13331 
Marseille CCdex 3, France 
I Itionrlirm. Anoxic samples of LI pilot fermenter feeded with 
waste water of the alcohol distillation industry (INRA, 
Narbonne, France) served as inoculum. The major sub- 
strates in the effluent were glycerol and. lactic acid. 
Etiriclitnetit and isolotioti. Enrichment cultures were in- 
cubated at 37°C with 10% inoculum from the pilot 
fermenler in 60 nil serum bottles containing 20 ml medium 
with methanol as substrate. Cultures were transferred (2 ml) 
every 2 weeks into fresh media. For isolation of axenic 
cultures of the homoacetogenic bacterium, the enrichment 
was serially diluted and inoculated into rol1,tubes (Hungate 
1969). 
Mediu. Sporotnusa ucidovorans was grown on complex 
' medium containing the following: NH4C1, 1.0 g; K2HP04 
6Hz0,  0.2 g; cystein-HC1, 0.5 g; 
fructose, 6 g; yeast extract (Difco), 1 g; resazurin, 0.001 g; 
mineral solution no 2 (Balch et al. 1979), 50 ml; trace ele- 
ment solution (Balch et al. 1979), 10 ml; distilled water, 
1,000 ml. For roll tubes, agar (2%) was added in liquid 
inedium. The medium was adjusted to pH 7.0 wilh'KOH 
and was boiled under N2. After cooling to room tem- 
perature, 20 nil of medium were transferred into 60 ml 
serum bottles inside an anaerqbic glove box (La Calhène, 
Bezons, France). The bottles were stoppered with black 
butyl rubber clasures (Bellco glass Inc., Vineland, NJ, USA) 
and outgassed with N 2  -COz. After sterilization (11O"C, 
35 min), 0.25 nil Na2C03 (l0Y0 w/v) and 0.2 nil Na2S * 
9 H z 0  (2% w/v) were dispensed into each bottle. 
For roll tubes preparation, agar was supplemented. Agar 
medium was dispensed anaerobically in portions of 4.5 ml 
into Hungate tubes (Bellco Glass Inc.). 0.08 ml Nazco, 
(10%) and 0.1 ml NazS (1%) were added to the medium. 
Stock solutions of Na2C03 and NazS were prepared 
anaerobically under Nz in 120 nil serum bottles and were 
autoclaved (llO°C, 35 min). Sugar solutions were sterilized 
by filtration. 
3H20,  0.4 g; MgC12 
Analytical techniques. Volatile fatty acids, organic acids and 
H2 were analysed as previously described (Garcia et al. 
1982). Formic acid was measured using the technique of 
Lang and Lang (1972) as  modified by Sleat and Mah (1984). 
Bacterial growth was quantified by measuring the optical 
density at 580 nm with a Spectronic 21 spectrophotometer 
(Baush and Lomb Inc., Rochester, NY, USA). 
Microscopy. A Zeiss microscope equipped with epifluo- 
rescence was used to detect methanogenic bacteria which 
exhibited a blue-green fluorescence under UV illumination. 
. ORSTOM Fonds Documentaire 
308, 
Phake contrast microscopy was performed using a Nikon 
microscope with an automatic camera for photography with 
Illford HPS film (ASA 400). Cells examined by electron 
microscopy were fixed with glutaraldehyde (1 % v/v) and 
osmium tetroxide (1 %O w/v) and included in Epon-Araldite 
resin. Ultrathin sections were examined with electronic 
Jeol JEM 100 V microscope. 
DNA4 preparation. DNA was isolated and purified by the 
method of Price et al. (1978). The mol %I G + C was deter- 
mined according to the method of De Ley (1970) from 
thermhl denaturation in 0.015 M NaCl and 0.015 M 
trisqdium citsate. 
Results 
Morphology. White and round colonies appeared after 
3 weeks incubation in agar roll tubes at 37°C. Older colonies 
I 
, -  
. '  4- a 
Fig. 1. Phase contrast photomicrograph of Sporotmsu widovornns. 
Bar is 5.0 pm. Note the curved rod and the subterminal spore 
were dark brownish and as large as 3-4 mm. Three pure 
cultures were isolated from the last positive dilution (strains 
Mol, Mol, and Molz). All strains appeared morphologically 
similar in the microscope. They produced only acetate from 
. complex medium containing sugars. They used fructose, 
ribose, methanol and H2 -CO2. Strain Mol was subjected 
to further characterization. 
The isolate was a slightly curved, spore-forming rod 
(5 pm length x 0.7 pm width) (Fig. 1) and stained Gram 
negative. I t  was motile by laterally inserted flagella. Its 
motility showed some resemblance with that of moving 
vibrios (Fig. 2) .  It occurred singly or as arrangements of two 
or more cells. Electron microscopy showed a multilayered 
cell wall (Fig. 3): Spore was terminal to subterminal. Spore 
suspensions survived pasteurization for 20 min at 80°C 
Substrates und oplinml growth conditions. Nutritional studies 
were performed at 37°C. Growth occurred only in presence 
of yeast extract. Besides methanol, the bacterium fermented 
a variety of substrates as shown in Table 1. The pH and 
temperature profiles of strain Mol were examined during 
growth in basal medium with 0.1% yeast extract and 0.6% 
fructose as energy source. The pH optimum for growth was 
between 6.5 and 7.0 (Fig. 4). No growth occurred at pH 8.0 
or pH 5.4. The 0ptimu.m growth temperature was 35°C 
(Fig. 5). Nitrate, sulfate and sulfite were not used as electron 
acceptors when the bacteria were cultivated on methanol. 
In carbonate free medium, fructose was used, but not 
methanol. Acetate was the only organic end product formed. 
Alcohols or H2 were never produced. 
' 
D N A  hase coniposifioti. The mol YO G f C content was 42. 
Discussion 
Anaerobic enrichment cultures on methanol from a 
B'ermentcr feeded with alcohol distillation wastes led to the 
development of hohoacetogenic bacteria. Methylotrophic 
methanogenic bacteria (Methanos~zrcina sp.) were not ob- 
served. The ecological importance of the isolated homo- 
acetogen in the pilot fermenter was presumably related to 
the high amount of glycerol used as growth substrate in the 
effluent. 
Fig. 2 
Transmission cleclron microgrnph of 
Sporo~m~sa acidovorans. Note the flagella 
distributcd a t  the coilcave side. Bar is 0.5 pm 
Table 1. Utilization of various substrates by Spurumixu crcirlovorr~ns, 
S. spliueroirles and S. U V U I U  
Substrate S.  ucidovoruns S.  sp/ineroides" S. ovu~uu  '1 ' 
H2 - CO2 
Methaiiol 
' Ethanol 
Glycerol 
Formate 
Lactate 
Pyruvate 
Fumarate 
Succinate 
Malate 
Oxaloacetate 
Glutamate 
Trimethylamine 
L-Serine 
Fructose 
t + 
+ + 
t 
t + 
3- + + 
+ 
t 
- 
- 
- 
+ 
;t 
t + + 
3- + - 
- 
nd 
nd 
nd ' + + - 
. +  
4- 
-1- 
+ + + 
- 
- 
- 
nd 
nd 
nd 
- 
- 
+ 
10 
... 
æ - 
E 
i= 20 
(II ._ B 
n O 
30 
- - Ribose + 
Data from Möller'et i l .  (1984); -, no growth; +, growth; nd, 
not determined 
The medium conlnined 0.5% of lhe organic substrate and 0.05% 
yeast extract. Additional compounds tested which did not support 
growth: malonate, citrate, lysine, threonine, glucose, maltose, 
lactose, xylose, melibiosc, arabinose, dulcitol, yalactose, saccharose, 
cellobiose, starch, cellulose, gelatin 
The isolated homoacetogenic spore-forming bacterium 
showed a typical Gram-negative cell wall with lateral 
flagella. These features exclude il from the genus 
Clostridium. Other anaerobic H,-oxidizing genera were 
ruled out for the following reasons: Acetohricterium sp. 
(Balch et al. 1977; Braun and Gottschalk 1982) stained 
Gram-positive, did not form spores and was motile by 
peritrichous flagella. The therniophilic Acefugenium k iw i  
(Leigh et al. 1981) wiis nonmotile and did not produce 
spores. Unlike DwuUutorncrculum species (Campbell and 
Postgate 1965; Pfennig et al. 1981), strain Mol could not use 
sulfate as electron acceptor. 
The banana-shaped cell forni, the truly Gram-negative 
cell wall with laterally inserted flagella a s  well ils the reduc- 
tion of COz to acelate indicated that strain Mol is a member 
Fig. 3 
Electronmicrograph of thin sections 
showing a multilaycred cell wall. 
Bar is 0.5 pm 
309 
Fig. 4. Effect of pH on generation time of Sporoniusu ~ciduvorunx. 
Cultures were incubated at 37°C. Medium contained 0.6'"~ fructose 
and 0.1 YU yeast extract 
Temperature ["Cl 
20 30 LO 
i 
Fig. 5. Effect of temperature o,n generation time of Sporoniusa 
uciduwrms. Medium contained 0.6% fructose and 0.1% yeast 
extriict . .  \ 
I 
of the newly described genus Sporonztisa (Möller et al. 1984). 
Within this genus, two species have been described. Strain 
Mol differed from S.  spliaeroides by using sugars. Further- 
more, strain Mol did not grow on trimethylamine. Unlike 
S.  ovutu, the current isolate used ribose, glycerol, L-serine 
and did not form oval spores. It could be distinguished from 
both species by the unability to use lactate, ethanol, and 
by requiring yeast extract for growth. S. .sp/taeroides and 
S. ovata grew on betaine in chemically defined medium 
containing minerals and vitamins. In contrast to S. sphue- 
roides and S.  ovum, strain Mol grew on fumarate or succi- 
nate. 
Thus we 'propose to place strain Mol in the genus 
Sporo usa and name it S.  acidovoruns in recognition to the 
utiliza ion of acidic compounds (succinate, fumarate.. .). 7 
acid from methanol. Antonie van Leeuwenhoek J Microbiol 
Serol 48: 305 -313 
Balch WE, Fox GE, Magrum LJ, Wolfe RS (1979) Methanogens: 
reevaluation of a unique biological group. Microbiol Rev 
Balch WE, Schoberth S, Tanner RS, Wolfe RS (1977) Acetobac- 
terium, a new genus of hydrogen oxidizing, carbon dioxide- 
reducing, anaerobic bacteria. Int J System Bacteriol 27: 355- 
361 
Braun M, Gottschalk G (1982) Acetobacterium wieriiigue sp.nov., a 
new species producing acetic acid from molecular hydrogen and 
carbon dioxide. Zbl Bakt Hyg, I Abt Orig C3:368-376 
Campbell LL, Postgate JR (1965) Classification of the spore forming 
' De fey J (1970) Reexamination of the association between melting 
point, buoyant density and the chemical base composition of 
deoxyribonucleic acid. J Bacteriol 101 :738-754 
43:260-2296 
ulfate reducing bacteria. Bacteriol Rev 29: 359-363 
Sp romzisn! acidovorutis sp.nov. (a.ci.do'vo.rans; L. neut. 
n. act um acid; L.v. vor0 to devour; M.L.part. adj. 
ncidov 1 runs acid-devouring). 
Morphology. Sporulating, curved rods 2 - 8 pm x 0.7 - 1 pm 
with #Gram-negative cell wall. Motile by laterally inserted 
flagella. Occurs singly or in short chains of cells. Colonies 
are white to darkly brown, entire and convex in shape. 
Metabolism. Obligate anaerobe. Degrades H2 - COz, meth- 
anol, formate, pyruvate, succinate, fumarate, malate, 
oxaloacetate, glutamate, glycerol, serin, fructose, ribose. 
Yeast extract is required for growth. The only fermentation 
product is acetate; COz is the only electron acceptor. 
DNA %i G + C. The ni01 % G + C of DNA is 42. 
Sourc . Pi1,ot fermenter feeded with effluent fr'om thte alcohol 
distill, tion industry. 
Type *traia. The type strain is Mol (DSM no. 3132). Its 
descri  tion is the saine as the species givfen above. 
Acknowledgenienfs. We thank H. E. Jones for puoviding us with 
the enrichment culture, C. Frehel and coworkers ([Institut Pasteur, 
Paris)l, D. Rainbaud and A. Bouleau (ORSTClM, B'ondy) for 
electron microscope facilities. We are grateful to F. Pichinoty and 
S. Raymond for assistance in determination of % G f C. 
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~ Möller B, OWnier R, Howard BH, Gottschalk G, Hippe H (1984) 
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, 


Isolation and characterization of SPOROMUSA ACIDOVORANS sp. nov., a methylogrophic homoacetogenic bacterium

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