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Sterolibacterium denitrificans gen. nov., sp. nov., a
novel cholesterol-oxidizing, denitrifying member of
the b-Proteobacteria
Silvana Tarlera
1
and Ewald B. M. Denner
2
Correspondence
Silvana Tarlera
starlera@fq.edu.uy
1
Ca
´tedra de Microbiologı´a, Facultad de Quı´mica y Facultad de Ciencias, Universidad de la
Repu´blica, C. C. 1157, Montevideo, Uruguay
2
Institut fu
¨r Mikrobiologie und Genetik, Universita¨t Wien, A-1030 Wien, Austria
A bacterial strain (Chol-1S
T
) that is able to oxidize cholesterol to CO
2
and reduce nitrate to
dinitrogen was enriched and isolated from an upflow sludge bed (USB) anoxic reactor that treats
sanitary landfill leachate from the city of Montevideo, Uruguay. Cells of strain Chol-1S
T
were
Gram-negative, rod-shaped to slightly curved, measured 0?5–0?661?0-1?3mm and were motile
by a single polar flagellum. Strain Chol-1S
T
grew optimally at 30–32 ˚C and pH 7?0, with a
doubling time of 44–46 h when cholesterol was used as the sole carbon and energy source. The
metabolism of strain Chol-1S
T
was strictly respiratory, with oxygen or nitrate as the terminal electron
acceptor. The presence of ubiquinone Q-8 as the sole respiratory lipoquinone indicated that strain
Chol-1S
T
belonged to the b-subclass of the Proteobacteria. Phosphatidylethanolamine was the
predominant polar lipid and the G+C content of the DNA was 65?3 mol%. The fatty acid profile
of strain Chol-1S
T
, cultivated under denitrifying conditions by using a defined mineral medium
supplemented with cholesterol, was characterized by the following major components: summed
feature 4 (C
16 : 1
v7cand/or iso C
15 : 0
2-OH), C
16 : 0
,C
18 : 1
v7cand hydroxy acid C
10 : 0
3-OH.
Minor components included C
10 : 0
,C
11 : 0
,C
12 : 0
,C
14 : 0
,C
15 : 0
,C
19 : 0
,C
19 : 0
10-methyl and
hydroxylated acids C
8:0
3-OH and C
16 : 0
3-OH. Analysis of the 16S rDNA sequence showed that
strain Chol-1S
T
represents a separate lineage within the Thauera,Azoarcus,Zoogloea and
Rhodocyclus assemblage of the b-Proteobacteria. Strain Chol-1S
T
had highest sequence similarity
(96?5 %) with strain 72Chol, a denitrifying b-Proteobacterium. On the basis of polyphasic evidence,
strain Chol-1S
T
(=DSM 13999
T
=ATCC BAA-354
T
) is proposed as the type strain of
Sterolibacterium denitrificans gen. nov., sp. nov.
Cholesterol and related sterols are natural recalcitrants in
anaerobic treatment of the highly pollutant effluent pro-
duced by the wool-scouring process (Gutie
´rrez et al., 1999).
This can be attributed to the low number of functional
groups (a carbon–carbon double bond and a hydroxyl
group), its low solubility and the complexity of its spatial
conformation. Aerobically, various genera of bacteria can
degrade cholesterol completely to carbon dioxide by
employing mono- and dioxygenases (Kieslich, 1985). How-
ever, less is known about the mechanisms that operate in the
absence of oxygen. The best-studied anaerobic reaction so
far is the reduction of cholesterol to coprostanol by
intestinal fermentative bacteria (Freier et al., 1994).
Due to their facultative metabolism, denitrifying bacteria are
more versatile than other groups of bacteria and can serve as
potential sources for novel biotransformations of natural
substances. Previous work has demonstrated that denitrify-
ing conditions can support the mineralization of cholesterol
(Taylor et al., 1981). A denitrifying bacterial strain, 72Chol,
which oxidizes cholesterol to carbon dioxide in the absence
of molecular oxygen, was isolated from a leaf-covered ditch
(Harder & Probian, 1997).
A denitrifying post-treatment step of anaerobically pre-
treated wastewater can be envisaged to complement the
removal of organic matter, in order to comply with environ-
mental discharge standards. The purpose of this investi-
gation was to determine whether organisms capable of
anaerobic mineralization of cholesterol were present in
different anaerobic man-made ecosystems that treat wool-
scouring effluent and sanitary landfill leachate. The latter
ecosystem has high contents of readily degradable carbon
but also of recalcitrant organic compounds, such as
Abbreviations: FAME, fatty acid methyl ester; USB, upflow sludge bed.
Published online ahead of print on 6 December 2002 as DOI 10.1099/
ijs.0.02039-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain Chol-1S
T
is AJ306683.
02039 G2003 IUMS Printed in Great Britain 1085
International Journal of Systematic and Evolutionary Microbiology (2003), 53, 1085–1091 DOI 10.1099/ijs.0.02039-0
aromatics (Wang & Barlaz, 1998). In the study presented
here, we report the isolation and polyphasic characteriza-
tion of a novel nitrate-reducing bacterium that oxidizes
cholesterol to CO
2
.
Source, enrichment and isolation
Enrichment and cultivation were performed in a bicarbo-
nate/CO
2
-buffered, basal anaerobic mineral medium,
modified from that of Tarlera et al. (1997) as follows
(l
21
): 1?2gK
2
HPO
4
,0?4gKH
2
PO
4
,1?0 g KNO
3
and 1 ml
(25 g Na
2
S.9H
2
Ol
21
) solution. Prior to inoculation, 1 ml
vitamin solution (Touzel & Albagnac, 1983), 20 ml (11?6g
MgCl
2
.6H
2
Ol
21
,3?7 g CaCl
2
.2H
2
Ol
21
) solution and
substrates were added. Oxic liquid media did not contain
nitrate or bicarbonate and were incubated with agitation.
Soluble substrates were added from anaerobic filter-
sterilized stock solutions. Insoluble substrates were added
either as a solid before autoclaving or as 1 % (w/v) solution
in 2,2,4,4,6,8,8-heptamethylnonane as the inert carrier
phase. All incubations were carried out at 30–32 uC and
pH 7?0, except for tests to determine temperature and pH
ranges for growth. For quantification studies, acetylene
(10 %) was added to the culture headspace to block the last
step of denitrification (N
2
ORN
2
). Total N
2
O content was
calculated from the headspace concentration as described by
Christensen & Tiedje (1988). Growth tests were considered
to be positive for substrate utilization when an increase in
optical density above that of control tubes with no added
substrate was detected and confirmed by nitrate and nitrite
quantification.
Denitrification in the presence of oxygen was tested by
measuring the reduction of nitrate in anoxic and oxic
conditions (incubated with agitation at 80 r.p.m.). Nitrate,
nitrite and N
2
O were measured as described by Etchebehere
et al. (2001). Volatile fatty acids, alcohols and sugars were
analysed by HPLC as described by Menes & Muxı
´(2002).
For quantification of cholesterol, samples were extracted
with ethyl acetate, evaporated under vacuum conditions,
redissolved in methanol and analysed by UV (SPD-10AV;
Shimadzu)-HPLC (Waters) at 210 nm, by using a C
18
column with a mobile phase of 60/40 % (v/v) acetonitrile/2-
propanol at a flow rate of 1?5 ml min
21
. Biomass was
calculated from the total protein content of the cultures by
using a protein per cell dry weight ratio of 50 % and
assuming the simplified cell formula C
4
H
7
O
3
(M
r
=103).
Protein determination was performed according to the
Lowry method, by using BSA as the standard (Daniels et al.,
1994).
Strain Chol-1S
T
was isolated from an upflow sludge bed
(USB) denitrifying reactor that treats sanitary landfill
leachate, after enrichment with cholesterol as the carbon
source and nitrate as the electron acceptor (Barrandeguy &
Tarlera, 2001). Other enrichments, set up with inocula from
anaerobic lagoons and reactors that treat wool-scouring
effluent, failed to denitrify cholesterol.
Isolation of a pure culture was attempted from the
cholesterol-degrading enrichment by: (a) streaking onto
nutrient, brain–heart infusion (BHI) and R2A agar plates
under air; (b) streaking onto anoxic plates of mineral
medium with 10 mM nitrate and an agar overlay of chole-
sterol; and (c) anoxic agar dilution and liquid dilution series
of mineral medium with 1 mM cholesterol and 10 mM
nitrate. Colonies that grew on solid media were transferred
to liquid mineral medium with cholesterol as the sole carbon
and energy source. None of the isolates was able to grow on
cholesterol.
However, the isolation of a pure culture was successful after
several successive anoxic liquid dilution series, which were
subcultured as soon as nitrite production was detected in
the highest dilutions. This strategy allowed enrichment of
the cholesterol-degrading bacteria with respect to other
bacteria. Finally, after consecutive series of dilutions that
took place over 1 year, a pure culture that consisted only of
straight or slightly curved short rods with rounded ends was
obtained. The purity of the culture was tested by sub-
culturing in mineral salt medium amended with volatile
fatty acids, amino acids and ethanol (growth substrates
commonly used by denitrifiers). In addition, the culture
was transferred into complex media [oxic and anoxic TSB
(trypticase soy broth), BHI and nutrient broth]and streaked
onto different oxic and anoxic agar plates (R2A, TSA and
nutrient agar). No growth was observed under the different
conditions tested after more than 2 months of incubation.
In a previous report, amplified rDNA restriction analysis
(ARDRA), performed on the clones from the enrichment,
revealed the presence of only two different restriction
patterns (Barrandeguy & Tarlera, 2001). The partial 16S
rDNA sequence (700 bp) that was representative of the
dominant member of the enrichment, according to ARDRA,
was identical to that determined later for strain Chol-1S
T
.
These results allow the conclusion that a pure culture was
obtained.
Morphological and physiological characteristics
Cells of strain Chol-1S
T
were Gram-negative, straight to
slightly curved rods (0?5–0?661?0–1?3mm) that were motile
by means of a single polar flagellum (Fig. 1a) and had lateral
fimbria-like appendages (Fig. 1b).
Physiological characterization showed that strain Chol-1S
T
used only a limited number of substrates, including sterols
and saturated long-chain fatty acids. Detailed physiological
characteristics are listed in the formal species description
below. Briefly, anaerobically grown cultures showed a lag
phase before they started to grow with oxygen as the electron
acceptor. Simultaneous reduction of oxygen and nitrate was
not observed. The co-respiration of nitrate and oxygen
under oxygen-saturated conditions is noted only for bac-
teria isolated from aerobic habitats, such as soil and
activated sludge (Patureau et al., 1998; Rainey et al., 1999;
Scholten et al., 1999).
1086 International Journal of Systematic and Evolutionary Microbiology 53
S. Tarlera and E. B. M. Denner
Oxidation of cholesterol
Strain Chol-1S
T
was not able to grow with cholesterol as the
electron donor, with sulphate, thiosulphate, fumarate or
Fe(III) as electron acceptors or under fermentative condi-
tions. Growth tests in an ammonium-free medium were
likewise negative. After 10 weeks incubation, incomplete
nitrate reduction (but no visible growth) was observed on
acetate and propionate. Partial reduction (2 mM) of nitrate
to nitrite was observed on lithocholic acid. Noticeably,
strain Chol-1S
T
has a narrow substrate-utilization profile
that is specific for non-polar, hydrophobic compounds as
carbon and energy sources.
To determine whether complete degradation of cholesterol
was taking place, the oxidation of cholesterol and reduction
of nitrate were quantified. The disappearance of 0?42 mM
cholesterol was accompanied by the consumption of
9?7 mM nitrate and the synthesis of 120 mg cell dry mass
l
21
. Nitrite accumulated temporarily during growth, but
was further consumed after near-depletion of nitrate
(Fig. 2). More than 80 % of nitrate reduced was recovered
as dinitrogen oxide in experiments with an acetylene block,
whereas only traces of dinitrogen oxide were detected in
vials with no added acetylene. Also, as an increase in growth
yield accompanied nitrate and nitrite reduction, a respira-
tory denitrification process could be ascertained. Time-
course analysis of growing cultures by HPLC showed no
accumulation of short-chain fatty acids or alcohols in the
culture broth of strain Chol-1S
T
. Synthesis of biomass
(120 mg l
21
; Fig. 2) can account for the consumption of
0?17 mM cholesterol and 1?29 mM nitrate based on the
assimilation equation:
20C27H46 Oz149NO{
3z149Hz?
135C4H7O3z74.5N2z62H2O
Initial and final cholesterol concentrations were 1 and
0?58 mM, respectively. Considering the cholesterol and
nitrate consumed for cell synthesis, 0?25 mM cholesterol
was dissimilated while 8?41 mM nitrate was consumed and
not recovered as nitrite. An electron recovery of 90 % was
calculated (the ratio of number of electrons produced by
complete oxidation of the dissimilated amount of choles-
terol to CO
2
to number of electrons consumed by nitrate
reduction to dinitrogen). The complete oxidation of 1 mol
cholesterol yields 152 mol electrons; 5 mol electrons are
considered to be accepted for 1 mol nitrate to be reduced
(a)
(b)
Fig. 1. Transmission electron micrographs of strain Chol-1S
T
,
showing its morphology and flagellar position (a) and fimbria-
like appendages (b). Bars, 0?3mm (a); 50 nm (b).
Fig. 2. Growth of strain Chol-1S
T
with cholesterol: OD
600
($),
nitrate consumption (&) and nitrite production (m). Data shown
are representative of repeated experiments with similar results.
Data are corrected for the amount of nitrate consumed in con-
trols that lacked cholesterol.
http://ijs.sgmjournals.org 1087
Sterolibacterium denitrificans gen. nov., sp. nov.
to dinitrogen. These findings are in good agreement with
the theoretical stoichiometry for cholesterol mineralization
according to the equation:
C27H46 Oz30.4NO{
3z30.4Hz?
27CO2z15.2N2z38.2H2O
Chemotaxonomy
Analysis of the respiratory lipoquinones was carried out
by using HPLC at 30 uC as described by Tindall (1990). The
HPLC system was equipped with a model 510 pump, a
model UK6 injector, a model 484 UV detector (all from
Waters) and a reversed-phase column (Hypersil ODS RP 18,
25064?6mm,5mmparticles;AgilentTechnologies). Analysis
revealed a single peak that corresponded to ubiquinone-8
(Q-8). This quinone system is a characteristic feature of the
b-Proteobacteria (Collins & Jones, 1981; Yokota et al., 1992).
Two-dimensional TLC of cellular lipids (Denner et al., 2001)
indicated only the presence of phosphatidylethanolamine
(PE) in strain Chol-1S
T
. PE is one of the most widely
occurring bacterial phospholipids (Wilkinson, 1988); solely
PE was reported for Alcaligenes faecalis (Ghanekar & Nair,
1974) and Azotobacter chroococcum (Reczek & Burton,
1979). All other species of b-Proteobacteria that have been
investigated so far have been shown to contain a more
complex polar lipid pattern (Wilkinson, 1988; Cox &
Wilkinson, 1989; Yabuuchi et al., 1992, 1998; Blumel et al.,
2001). Thus, the polar lipid pattern of strain Chol-1S
T
is a
potentially distinctive trait of this species.
For fatty acid analysis, strain Chol-1S
T
was grown on
anaerobic mineral medium supplemented with 1 mM
cholesterol and 10 mM nitrate, harvested by centrifugation,
washed with 20 mM phosphate buffer and freeze-dried.
Fatty acid methyl esters (FAMEs) were extracted and pre-
pared according to the standard protocol of the Microbial
Identification system (MIDI; Microbial ID). FAME extract
of strain Chol-1S
T
was analysed by GLC as described by
Ka
¨mpfer & Kroppenstedt (1996). The fatty acid profile of
strain Chol-1S
T
was characterized by 33?8%C
16 : 1
v7cand/
or iso C
15 : 0
2-OH (summed feature 4), 21?5%C
16 : 0
,14?5%
C
18 : 1
v7cand 14?0% C
10 : 0
3-OH. Other fatty acids that
were present in significant amounts included C
12 : 0
(5?3 %),
C
19 : 0
10-methyl (4?0 %) and C
19 : 0
(2?3 %). Fatty acids
present in minor amounts included hydroxylated fatty acids
C
8:0
3-OH (0?2 %) and C
16 : 0
3-OH (1?0 %) and saturated
fatty acids C
10 : 0
(0?9 %), C
11 : 0
(0?2 %), C
14 : 0
(0?9 %) and
C
15 : 0
(0?8 %). Some unknown fatty acids were also present
in minor amounts (data not shown). Similar fatty acid
profiles have been described for the genera Thauera and
Azoarcus (Song et al., 2001) and Zoogloea ramigera (Hiraishi
et al., 1992); the presence of C
8:0
3-OH, C
16 : 0
3-OH and
C
19 : 0
10-methyl was not reported (Hiraishi et al., 1992; Song
et al., 2001), although a direct comparison of fatty acid
profiles may be difficult because of the different cultivation
conditions. However, fatty acid analysis clearly allows
differentiation of strain Chol-1S
T
from these taxa.
Phylogenetic position of strain Chol-1S
T
Genomic DNA for 16S rDNA sequencing was extracted and
purified by using the Wizard Genomic DNA purification kit
(Promega) as described by the manufacturer. PCR ampli-
fication and sequencing were performed as described by
Menes & Muxı
´(2002). The primers used for sequencing were:
27F (positions 8–27: 59-AGAGTTTGATCCTGGCTCAG-39);
ST1F (positions 606–632: 59-GGCTCAACCTGGGAACT-39);
ST3F (positions 1273–1290: 59-GAGCCAATCCCAGAAAG-
39);ST4R (positions 1512–14 96: 59-ACGGCTACCTTGTTACG-
39); ST5R (positions 999–983: 5-GCATGTCAAGGGTAGGT-3);
and ST6R (positions 462–445: 59-CCCAGTCCGTTTCTTCC-
39)(Escherichia coli numbering). Sequencing was carried out
by the DNA Sequencing Core Laboratory at the University
of Florida (USA). Phylogenetic analyses were performed
with the PHYLIP 3.5c software package (Felsenstein, 1993) as
described by Menes & Muxı
´(2002).
A total of 1531 nt of the 16S rRNA gene of strain Chol-1S
T
were determined. Phylogenetic analyses based on a dataset
that comprised 1390 unambiguous nucleotides between
positions 53 and 1459 (E. coli numbering) showed that the
sequence of strain Chol-1S
T
is most similar to those of
species of the b-subclass of the Proteobacteria (Fig. 3).
Comparative evolutionary distance analysis showed that
strain Chol-1S
T
represents a separate lineage of descent
within the b-Proteobacteria. The nearest, albeit relatively
distant, phylogenetic relatives were species of the genera
Azoarcus,Thauera and Zoogloea. 16S rDNA sequence
similarities ranged from 91 % (with Z.ramigera ATCC
19544
T
) to 93 % (with Thauera linaloolentis DSM 12138
T
).
These similarity values are clearly below the usual criterion
of 95 % that is applied for genus delineation. The neighbour-
joining tree inference clustered the genera Thauera,Azoarcus
and Zoogloea at a significant level (86 %) in bootstrap
analysis. Also, the branching of genera Propionivibrio,
Rhodocyclus,Ferribacterium and Dechloromonas was sup-
ported by a high bootstrap value (100 %). Highest 16S rRNA
gene sequence similarity (96?5 %) was found to the already
mentioned denitrifying bacterial strain 72Chol (Harder &
Probian, 1997).
Comparative physiological investigations showed that strain
72Chol also uses only a limited number of substrates and
degrades cholesterol and other sterols, but not short-chain
fatty acids or sugars, under denitrifying conditions. In
contrast to strain Chol-1S
T
, strain 72Chol has been reported
to be non-motile, has a maximum growth temperature of
32 uC, has a doubling time of 37 h on cholesterol and is
unable to grow on palmitate (Harder & Probian, 1997).
To summarize, by considering the polyphasic taxonomic
data presented in this study, we could identify the novel
cholesterol-oxidizing denitrifying bacterial isolate Chol-1S
T
as a hitherto unknown taxon of the b-Proteobacteria, for
1088 International Journal of Systematic and Evolutionary Microbiology 53
S. Tarlera and E. B. M. Denner
which we propose the name Sterolibacterium denitrificans
gen. nov., sp. nov. Key taxonomic characteristics that
differentiate the genus Sterolibacterium from other related
genera are listed in Table 1.
Description of Sterolibacterium gen. nov.
Sterolibacterium (Ste.ro.li.bac.te9ri.um. N.L. neut. n. sterolum
sterol; Gr. dim. neut. n. bakterion small rod; N.L. neut. n.
Sterolibacterium sterol-utilizing small rod).
Straight or slightly curved, small, rod-shaped, Gram-
negative cells. Mesophilic. Strictly respiratory type of
metabolism with oxygen or nitrate as terminal electron
acceptor. Nitrate is reduced to dinitrogen. Oxidase- and
catalase-positive. Chemo-organoheterotrophic. Does not
grow on complex media. Cholesterol is completely oxidized
to CO
2
; this reaction is coupled to nitrate reduction. Q-8 is
the sole respiratory lipoquinone. Phosphatidylethanolamine
is the predominant polar lipid. Major fatty acids are C
16 : 0
,
summed feature 4 (C
16 : 1
v7cand/or iso C
15 : 0
2-OH),
C
18 : 1
v7cand C
10 : 0
3-OH; minor amounts of C
8:0
3-OH
and C
16 : 0
3-OH are present. DNA G+C content is
65?3 mol% (HPLC). The type species of the genus is
Sterolibacterium denitrificans.
Description of Sterolibacterium denitrificans sp.
nov.
Sterolibacterium denitrificans (de.ni.tri9fi.cans. N.L. part.
adj. denitrificans denitrifying).
Cells are 1?0–1?360?5–0?6mm in size and are motile by
a single polar flagellum. Optimal pH and temperature for
growth are 7?0 and 30–32 uC, respectively. Temperature and
pH ranges for growth are 15–35 uC and 5?8–8?0, respectively.
Carbon sources used include cholesterol (5-cholesten-3b-ol),
Table 1. Differential characteristics of Sterolibacterium gen. nov. and related taxa
Genera: 1, Sterolibacterium;2,Thauera;3,Azoarcus;4,Zoogloea. Data for Thauera,Azoarcus and Zoogloea
were compiled from Unz (1984), Hiraishi et al. (1997), Song et al. (1998, 2001) and Reinhold-Hurek &
Hurek (2000). +, Positive; 2, negative.
Characteristic 1 2 3 4
Growth on nutrient agar, R2A or BHI agar 2++ +
Utilization as sole carbon source:
Succinate 2++ +
Acetate 2++ +
Glutamate 2++ +
Major quinone system* Q-8 Q-8 Q-8 Q-8, RQ-8
Diagnostic cellular fatty acids:
C
8:0
3-OH +22 2
C
16 : 0
3-OH +22 2
C
19 : 0
10-methyl +22 2
DNA G+C content (mol%) 65?3 64–69 64–68 67–69
*Q-8, ubiquinone-8; RQ-8, rhodoquinone-8.
Fig. 3. Phylogenetic tree derived from the analysis of 16S
rRNA gene sequences of strain Chol-1S
T
and other related b-
Proteobacteria. Numbers at nodes indicate levels of bootstrap
support, based on data for 100 resamplings. E. coli was used
as outgroup. Bar, 10 nucleotide substitutions per 100 nucleo-
tide positions.
http://ijs.sgmjournals.org 1089
Sterolibacterium denitrificans gen. nov., sp. nov.
4-cholesten-3-one, 5a-androstane-3,17-dione, 4-androstene-3,
17-dione, 3b-hydroxy-5a-cholestane, palmitate (C
16 : 0
) and
stearate (C
18 : 0
). Neither growth nor nitrate reduction was
demonstrated after 6 months with glucose, fructose, xylose,
ethanol, lactate, isobutyrate, succinate, malate, crotonate,
citrate, laureate, oleate, caproate, heptanoate, cyclohexanol,
cyclohexanone,glutamate,leucine, benzoate,phenol,cysteine,
dimethylmalonate, desoxycholate or 1,4-androstadiene-
3,17-dione. Doubling time for growth on cholesterol and
nitrate is 44–46 h.
The type strain is Chol-1S
T
(=DSM 13999
T
=ATCC BAA-
354
T
). Isolated from a USB denitrifying reactor that treats
sanitary landfill leachate in Montevideo, Uruguay.
Acknowledgements
We are grateful to Professor Dr Peter Ka
¨mpfer, Institut fu
¨r Angewandte
Mikrobiologie, Justus-Liebig-Universita
¨t Gießen, Germany, for per-
forming the fatty acid analysis and helpful suggestions. Professor Hans
Tru
¨per (University of Bonn, Germany) is acknowledged for advice on
Latin usage. S. T. also wishes to thank Lucı
´a Muxı
´for her encourage-
ment and valuable advice. This study was partly supported by
PEDECIBA, a Program for the Development of Basic Sciences in
Uruguay and by Universidad de la Repu
´blica (CSIC). Transmission
electron microscopy and analysis of DNA G+C content was carried
out by the Identification Service of the Deutsche Sammlung von
Mikroorganismen und Zellkulturen – DSMZ, Braunschweig, Germany.
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Sterolibacterium denitrificans gen. nov., sp. nov.
