Carboxydothermus islandicus sp nov., a thermophilic, hydrogenogenic, carboxydotrophic bacterium isolated from a hot spring

Abstract

An anaerobic, thermophilic bacterium, strain SET IS-9(T), was isolated from an Icelandic hot spring. Cells of strain SET IS-9(T) are short, slightly curved, motile rods. The strain grows chemolithotrophically on CO, producing equimolar quantities of H(2) and CO(2). It also grows fermentatively on lactate or pyruvate in the presence of yeast extract (0.2 g l(-1)). Products of pyruvate fermentation are acetate, CO(2) and H(2). Growth occurs at 50-70 °C, with an optimum at 65 °C, and at pH 5.0-8.0, with an optimum at pH 5.5-6.0. The generation time during chemolithotrophic growth on CO under optimal conditions is 2.0 h. 16S rRNA gene sequence analysis suggested that the organism belongs to the genus Carboxydothermus. On the basis of phenotypic features and phylogenetic analysis, Carboxydothermus islandicus sp. nov. is proposed, with the type strain SET IS-9(T) ( = DSM 21830(T)  = VKM B-2561(T)). An emended description of the genus Carboxydothermus is also given.

Full text

Carboxydothermus islandicus sp. nov., a
thermophilic, hydrogenogenic, carboxydotrophic
bacterium isolated from a hot spring
Andrey A. Novikov,
1
Tatyana G. Sokolova,
2
Alexander V. Lebedinsky,
2
Tatyana V. Kolganova
3
and Elizaveta A. Bonch-Osmolovskaya
2
Correspondence
Andrey A. Novikov
setchemist@gmail.com
1
Gubkin Russian State University of Oil and Gas, Leninskiy Prospect 65, 117485 Moscow, Russia
2
Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60 Let Oktyabrya
7/2, 117312 Moscow, Russia
3
Bioengineering Center, Russian Academy of Sciences, Prospect 60 Let Oktyabrya 7/1,
117312 Moscow, Russia
An anaerobic, thermophilic bacterium, strain SET IS-9
T
, was isolated from an Icelandic hot spring.
Cells of strain SET IS-9
T
are short, slightly curved, motile rods. The strain grows
chemolithotrophically on CO, producing equimolar quantities of H
2
and CO
2
. It also grows
fermentatively on lactate or pyruvate in the presence of yeast extract (0.2 g l
”1
). Products of
pyruvate fermentation are acetate, CO
2
and H
2
. Growth occurs at 50–70 6C, with an optimum at
65 6C, and at pH 5.0–8.0, with an optimum at pH 5.5–6.0. The generation time during
chemolithotrophic growth on CO under optimal conditions is 2.0 h. 16S rRNA gene sequence
analysis suggested that the organism belongs to the genus Carboxydothermus. On the basis of
phenotypic features and phylogenetic analysis, Carboxydothermus islandicus sp. nov. is
proposed, with the type strain SET IS-9
T
(5DSM 21830
T
5VKM B-2561
T
). An emended
description of the genus Carboxydothermus is also given.
Microbial conversion of CO to H
2
by thermophiles can
be accomplished by phylogenetically diverse prokaryotes
(Sokolova et al., 2009) and has been known since the
isolation of Carboxydothermus hydrogenoformans (Svetlichny
et al., 1991). At the time of writing, the genus Carboxy-
dothermus contains three species with validly published
names: C. hydrogenoformans (Svetlichny et al., 1991), C.
ferrireducens (Slobodkin et al., 2006) and C. siderophilus
(Slepova et al., 2009). Strain R1
T
5DSM 7242
T
,initially
described as ‘Carboxydothermus restrictus’ (Svetlichny et al.,
1994), was later shown to belong to a different phylogenetic
lineage and was reclassified as Thermolithobacter carboxydi-
vorans (Sokolova et al., 2007). Here, we describe a novel
species of the genus Carboxydothermus.
Strain SET IS-9
T
was isolated from a sample of water, mud
and grey filaments from an Icelandic hot spring with a
temperature of 68.6 uC and a pH of 6.5 in the Hveragerdi
area (64u09530N, 21u119180W). For the enrichment and
isolation of this strain, medium 1 was used (l
21
): 0.66 g
NH
4
Cl, 0.16 g MgCl
2
.6H
2
O, 0.1 g CaCl
2
.6H
2
O, 0.33 g
KCl, 0.5 g KH
2
PO
4
, 1 ml trace element solution (Kevbrin
& Zavarzin, 1992) and 1 ml vitamin solution (Wolin et al.,
1963). After boiling, the medium was flushed with N
2
and
cooled, NaHCO
3
(0.5 g l
21
) and Na
2
S.9H
2
O (1.0 g l
21
)
were added, and the pH was adjusted to 6.8–7.0 with 6 M
HCl. Medium 1 was dispensed into 55 ml bottles in 10 ml
portions. Bottles were inoculated with approximately 1 ml
sample per bottle under an N
2
flow, then the gas phase was
changed to 100 % CO at ambient pressure. The bottles
were incubated at 65 uC. After 5 days incubation, the
pressure in the inoculated bottles increased to 130–
140 kPa, the CO content in the gas phase decreased and
H
2
appeared. The concentrations of CO, H
2
,CO
2
and
volatile fatty acids were determined by using GC,
performed as described previously (Sokolova et al., 2002).
A pure culture was obtained by using sequential serial
dilutions and isolation of single colonies. Single colonies
were obtained in medium 1 solidified with 5 % agar in roll
tubes with 100 % CO in the gas phase.
Light microscopy, performed as described previously
(Sokolova et al., 2002), revealed the growth of rod-shaped
cells. Colonies were brownish-yellow and 0.5 mm in
diameter on the ninth day after inoculation. Electron
Abbreviations: AQDS, 9,10-anthraquinone-2,6-disulfonate; CFA, cellular
fatty acid; DMA, dimethylacetal.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain SET IS-9
T
is GQ324698.
Three supplementary figures are available with the online version of this
paper.
International Journal of Systematic and Evolutionary Microbiology (2011), 61, 2532–2537 DOI 10.1099/ijs.0.030288-0
2532 030288 G2011 IUMS Printed in Great Britain

microscopy (negative staining of whole cells with 1 %
uranyl acetate; JEOL 100B electron microscope) revealed
that cells were 0.5–1.0 mm wide and 1.5–2.5 mm long and
had peritrichous flagella (Supplementary Fig. S1, available
in IJSEM Online). The KOH test (Gregersen, 1978) was
negative and cells stained Gram-positive, allowing us to
suggest a Gram-positive cell-wall structure of strain SET
IS-9
T
.
The effects of temperature and pH on growth were studied
in the same medium as was used for culture isolation.
Growth was estimated by direct cell count. Growth of
strain SET IS-9
T
occurred within a temperature range of
50–70 uC, with an optimum at 65 uC, and at pH 5.0–8.0,
with an optimum at pH 5.5–6.0. No growth was observed
at 45 or 75 uC, or at pH 4.5 or 8.5. Strain SET IS-9
T
grew
chemolithotrophically on 100 % CO, producing H
2
and
CO
2
according to the equation CO+H
2
OACO
2
+H
2
(Fig.
1). No other products were detected. The generation time
under optimal conditions on 100 % CO was 2.0 h. Growth
of the new isolate on various substrates was tested in
medium 1 at optimal pH and temperature values under
100 % N
2
in the gas phase in the case of non-gaseous
substrates. Growth on H
2
+CO
2
(80 % H
2
) or on the
following substrates (2 g l
21
) was tested: pyruvate, glucose,
cellobiose, sucrose, galactose, lactose, fructose, starch,
glycerol, formate, acetate, peptone, yeast extract, lactate,
ethanol and methanol, in both the presence and the
absence of yeast extract (0.2 g l
21
). After three sequential
transfers, growth was observed only in medium containing
yeast extract (0.2 g l
21
) with lactate or pyruvate. During
growth on pyruvate, the isolate produced acetate, CO
2
and
H
2
. Growth with various electron acceptors was tested in
medium 1 containing yeast extract (0.2 g l
21
). In the case
of Fe(III), 9,10-anthraquinone-2,6-disulfonate disodium
salt (AQDS), NO^
3or S2O22
3, medium 1 devoid of Na
2
S
was used. Possible electron acceptors were added to a
concentration of 2 g l
21
;S
0
was added to a concentration
of 10 g l
21
. The following electron donors were tested: CO
(100 %), H
2
(80 % H
2
+CO
2
), acetate, lactate and pyruvate
(2 g l
21
). Reduction of AQDS was determined from
coloration change. Reduction of S
0
,S
2O22
3and SO22
4was
determined by S
22
measurements (Tru
¨per & Schlegel,
1964). Reduction of NO^
3was determined with NO^
2/NO^
3
Merck test paper. Among the electron acceptors tested,
only AQDS was reduced in the presence of CO, acetate,
lactate or pyruvate; however, growth was observed only in
the presence of pyruvate, and it was not stimulated by
AQDS. Penicillin, ampicillin, oxacillin or streptomycin
(100 mg l
–1
) inhibited growth on CO and production of
H
2
completely.
To determine the phylogenetic position of strain SET
IS-9
T
, DNA from its cells was isolated by a method based
on a modified alkaline extraction procedure (Birnboim &
Doly, 1979) and Promega Wizard technology. The G+C
content of DNA was 37.7±1.0 mol%, as determined by the
thermal-denaturation method (Owen et al., 1969). The 16S
rRNA gene was amplified by PCR using the primers Bact
27f (59-GTTTGATCMTGGCTCAG79-39) and Univ 1492r
(59-TACGGYTACCTTGTTACGACTT-39) (Lane, 1991).
The PCR products were purified from low-melting-
temperature agarose by using a Wizard PCR-Prep kit
(Promega) according to the manufacturer’s instructions.
Sequencing was performed by using primers recommended
by Lane (1991) and a BigDye Terminator v. 3.1 sequencing
reaction kit on an ABI 3730 DNA automatic sequencer
(Applied Biosystems). The 16S rRNA gene sequence of
strain SET IS-9
T
(1447 nt, corresponding to Escherichia coli
positions 27–1450) has been deposited in GenBank under
accession no. GQ324698. Preliminary phylogenetic analysis
of the newly determined sequences was done with the
NCBI BLAST server (Altschul et al., 1997; http://blast.ncbi.
nlm.nih.gov/Blast.cgi). The 16S rRNA gene sequence
similarity values with the closest relatives revealed by
BLAST were redetermined using the EzTaxon server 2.1
(Chun et al., 2007; http://www.eztaxon.org/) in cases where
BLAST failed to compare sequences along the entire lengths
of their overlaps. Both BLAST and EzTaxon values were then
recalculated so as to obtain similarity values with and
without taking into account insertions/deletions; distinc-
tions due to uncertainties (Ns etc.) were disregarded in
both cases.
The close relatives of strain SET IS-9
T
were determined
to be the three currently recognized Carboxydothermus
species. C. siderophilus exhibited 94.3 % 16S rRNA gene
sequence similarity to strain SET IS-9
T
if insertions/
deletions were taken into account and 97.3 % if they were
disregarded. The corresponding values for C. hydrogenofor-
mans ranged, for the four gene copies present in its
completely sequenced genome (Wu et al., 2005), from 96.6
to 97.0 % and from 97.5 to 97.9 %, respectively. The values
for the type strain of C. ferrireducens were 95.5 and 96.5 %,
respectively. In all cases, the sizes and positions of the
Fig. 1. Growth of strain SET IS-9
T
in medium 1 under an
atmosphere of 100 % CO at 65 6C. h, Cell number; m,H
2
production; &, CO consumption. CO and H
2
are shown as their
quantities in the gas phase [(ml liquid culture)
”1
].
Carboxydothermus islandicus sp. nov.
http://ijs.sgmjournals.org 2533

fragments compared coincided with those of the sequenced
fragment of the 16S rRNA gene of strain SET IS-9
T
. Thus, if
insertions/deletions were taken into account, the similarity
values were not above the value (97 %) set by Tindall et al.
(2010) as the threshold above which DNA–DNA hybrid-
ization is mandatory to claim species-level genotypic
distinction. If insertions/deletions were disregarded, the
similarity values were still not above the value of 98.7 %,
noted by Stackebrandt & Ebers (2006) to make DNA–DNA
hybridization unnecessary. The sequence similarity of the
16S rRNA gene of strain SET IS-9
T
to corresponding genes
of representatives of genera other than Carboxydothermus
was low (the closest were Moorella spp. strains, exhibiting
no more than 87 and 89 % 16S rRNA gene sequence
similarity as calculated with and without taking into
account insertions/deletions). Phylogenetic trees con-
structed by the neighbour-joining (Fig. 2) and max-
imum-parsimony (not shown) methods implemented in
MEGA version 4 (Tamura et al., 2007) after alignment
(Supplementary Fig. S2, available in IJSEM Online) with
built-in CLUSTAL W exhibited the same topology and
confirmed the distinct phylogenetic position of strain
SET IS-9
T
within the genus Carboxydothermus. Therefore,
we conclude that the phylogenetic position of strain SET
IS-9
T
corresponds to the status of a novel species in the
genus Carboxydothermus.
The affiliation of strain SET IS-9
T
to a novel species of the
genus Carboxydothermus is supported by its phenotypic
features (Table 1). Strain SET IS-9
T
resembles other species
of the genus Carboxydothermus in its cell morphology, the
ability to grow lithotrophically on 100 % CO, and pH and
temperature growth ranges. However, significant differ-
ences in metabolic patterns were observed. As distinct from
other Carboxydothermus species, strain SET IS-9
T
was
unable to use any electron acceptor tested (AQDS, if
reduced, did not stimulate growth or even inhibited it). In
contrast to C. hydrogenoformans and C. ferrireducens, strain
SET IS-9
T
did not utilize H
2
and lactate as electron donors.
In contrast to C. siderophilus, strain SET IS-9
T
grew on
pyruvate without any acceptors.
To obtain additional chemotaxonomic information, cellular
fatty acid (CFA) profiles were determined for strain SET IS-
9
T
and Carboxydothermus species by GC-MS. For CFA
analysis, cells were centrifuged at 3300 gfor 15 min,
resuspended in 4 ml H
2
O and centrifuged again at
12 000 gfor 15 min, and the cell pellet (approx. 50 mg)
was frozen and then processed according to instructions for
the Microbial Identification system (Sasser, 1990). The
obtained extract of CFA methyl esters was then filtered, and
a1ml sample was injected into the ThermoScientific Trace
GC Ultra DSQ II GC-MS system (column TR-5MS
15 m60.25 mm; helium flow 1.2 ml min
21
; inlet 280 uC
with split flow 20 ml min
21
; oven temperature pro-
grammed as 5 min at 120 uC, 10 uCmin
21
to 250 uC,
3 min at 250 uC, 20 uC min
21
to 300 uC, 1.5 min at 300 uC;
transfer line 300 uC; mass-selective detector in EI 70 eV
mode). CFA content was determined as the percentage of
the total ion current peak area. The predominant fatty acid
species detected were C
14 : 0
(14.3 %), iso-C
15 : 0
(20.7 %) and
C
16 : 0
(41.9 %); significant amounts of dimethylacetals
(DMAs) and a low but detectable amount of nonadecane
were also detected (Table 2). Polar lipids were extracted and
analysed by two-dimensional TLC according to Tindall
(1990). The polar lipid pattern was fairly simple, with only
one predominant phospholipid in all species tested and
minor amounts of other polar lipids, barely detectable with
phosphomolybdic acid only (Supplementary Fig. S3,
available in IJSEM Online). Thus, strain SET IS-9
T
has a
CFA profile different from those of known Carboxy-
dothermus species: it exhibits a higher content of C
14 : 0
and
Fig. 2. 16S rRNA gene-based neighbour-joining dendrogram showing the relationships between strain SET IS-9
T
and
Carboxydothermus species. The dendrogram is a subtree of a tree constructed by using 16S rRNA gene sequences of the type
strains of Carboxydothermus species, Moorella thermoacetica ATCC 39073, taken as the closest outgroup relative revealed by
BLASTN, and type strains of Firmicutes species capable of hydrogenogenic carboxydotrophy and representing five families (the
latter strains are not shown in the figure, except for Caldanaerobacter subterraneus subsp. pacificus). C. hydrogenoformans
Z-2901
T
rrsA,B,Cand Dare the four gene copies present in the completely sequenced genome. The tree was constructed by
using the neighbour-joining method implemented in MEGA version 4 at default values of all parameters. There were 1333
positions in the final dataset. Bootstrap percentages (based on 500 replicates) are given at branching points. Bar, 0.02 base
substitutions per site.
A. A. Novikov and others
2534 International Journal of Systematic and Evolutionary Microbiology 61

C
16 : 0
and a lower content of iso-C
15 : 0
and anteiso-C
15 : 0
(differences in DMA content between Carboxydothermus
species should be treated with caution, since, as seen from
Table 2, DMA content in strain SET IS-9
T
depended strongly
on growth conditions).
To conclude, based on phylogenetic evidence and pheno-
typic features, we propose that strain SET IS-9
T
should be
assigned to a novel species of the genus Carboxydothermus
with the name Carboxydothermus islandicus sp. nov.
Emended description of the genus
Carboxydothermus Slobodkin et al. 2006
Rod-shaped, Gram-positive bacteria. Anaerobic and thermo-
philic. Neutrophilic. CO is utilized either with or without
production of H
2
. Some species are able to couple the
reduction of an external electron acceptor such as Fe(III),
SO22
3,S
2O22
3,S
0
,NO
^
3, fumarate or AQDS with the
oxidation of organic acids, polyols and H
2
. In the presence
as well as the absence of electron acceptor, organic substrates
are oxidized incompletely to acetate as the main metabolic
product. Sulfate is not reduced.
Description of Carboxydothermus islandicus
sp. nov.
Carboxydothermus islandicus (is.lan9di.cus. N.L. masc. adj.
islandicus pertaining to Iceland, Icelandic).
Cells are Gram-positive, motile, straight to slightly curved
rods, 0.5–1.0 mm wide and 1.5–2.5 mm long with rounded
ends. Colonies are brownish-yellow and 0.5 mm in
diameter. Cells stain Gram-positive, occur singly or in
pairs and exhibit peritrichous flagella. Spores are not
observed. Strictly anaerobic. Growth occurs at 50–70 uC,
with an optimum at 65 uC, and at pH 5.0–8.0, with an
optimum at pH 5.5–6.0. Grows chemolithoautotrophically
and hydrogenogenically on CO; grows fermentatively on
lactate or pyruvate under N
2
in the presence of yeast
extract. Products of pyruvate fermentation are acetate, CO
2
and H
2
. Does not grow on H
2
+CO
2
(8 : 2) in the presence
Table 1. Characteristics useful for differentiating Carboxydothermus species and strain SET IS-9
T
Taxa: 1, C. hydrogenoformans Z-2901
T
;2,C. ferrireducens JW/AS-Y7
T
;3,C. siderophilus 1315
T
; 4, strain SET IS-9
T
. Data on CFA profiles and
substrate utilization were obtained in this work; other data for C. hydrogenoformans Z-2901
T
are from Svetlichny et al. (1991), for C. ferrireducens
JW/AS-Y7
T
from Slobodkin et al. (1997, 2006) and for C. siderophilus 1315
T
from Slepova et al. (2009). All taxa are unable to utilize lactate+sulfate.
ND, Not determined.
Characteristic 1 2 3 4
Morphology Slightly curved rods Straight to slightly
curved rods
Straight rods Straight to slightly
curved rods
Flagellation Lateral flagella Peritrichous flagella Non-motile Peritrichous flagella
Growth temperature range (optimum) (uC) 40–78 (70–72) 50–74 (65) 52–70 (65) 50–70 (65)
Growth pH range (optimum) 6.6–8.0 (7.0) 5.5–7.6 (6.0–6.2) 5.5–8.5 (6.5–7.2) 5.5–8.0 (5.5–6.0)
Generation time under optimal conditions (h) 2.0 ND 9.3 2.0
DNA G+C content (mol%) 39.0±1.0 41 41.5±0.5 37.7±1.0
Major fatty acids (% of total)
C
14 : 0
11.3 7.4 ND 14.3 (25.6)*
C
15 : 0
iso 30.1 30.8 ND 20.7 (12.4)
C
15 : 0
anteiso 9.8 8.6 ND 1.1 (4.1)
C
16 : 0
20.2 24.4 ND 41.9 (26.2)
DMA total 5.5 15.4 ND 6.8 (25.7)
Substrate utilization
100 % CO without yeast extract +22+
100 % CO +22+
Pyruvate ++2+
Lactate 2+2+
CO+Fe(III) 2++2
H
2
+Fe(III) ++22
CO+AQDS +++2
H
2
+AQDS ++22
Acetate+AQDS 2222
Lactate+AQDS +++2
Lactate+S2O22
3++22
Lactate+S
0
++22
Lactate+NO^
3++22
*Fatty acid data for strain SET IS-9
T
are given for cells grown on CO and cells grown on pyruvate (in parentheses).
Carboxydothermus islandicus sp. nov.
http://ijs.sgmjournals.org 2535

or absence of Fe(III), AQDS, SO22
4,S
0
,NO
^
3or S2O22
3.
Does not reduce Fe(III), S
0
,NO
^
3or S2O22
3with CO, H
2
,
acetate, lactate or pyruvate. Does not ferment glucose,
cellobiose, sucrose, galactose, lactose, fructose, starch,
glycerol, formate, acetate, peptone, lactate, ethanol or
methanol, in either the presence or the absence of yeast
extract. Growth is inhibited completely by penicillin,
ampicillin, oxacillin and streptomycin. Major CFAs are
C
14 : 0
, iso-C
15 : 0
and C
16 : 0
. The DNA G+C content of the
type strain is 37.7±1.0 mol%.
The type strain, SET IS-9
T
(5DSM 21830
T
5VKM B-
2561
T
), was isolated from a hot spring in Iceland.
Acknowledgements
We are grateful to Anna Perevalova and Christa Schlepper for
providing samples, to Lyubov Didenko for electron microscopy and
to Ekaterina Detkova for DNA G+C content analysis. C. ferrireducens
JW/AS-Y7
T
was kindly provided by Sergey Gavrilov, Winogradsky
Institute of Microbiology. This work was supported by the Federal
Target Program ‘Scientific and Scientific-Pedagogical Personnel of
Innovative Russia 2009–2013’ and the Russian Academy of Sciences
‘Molecular and Cell Biology’ and ‘Origin and Evolution of the
Biosphere’ programmes.
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Table 2. Cellular fatty acid contents (%) of strain SET IS-9
T
and known Carboxydothermus species
Taxa: 1, C. hydrogenoformans Z-2901
T
;2,C. ferrireducens JW/AS-Y7
T
;
3, strain SET IS-9
T
grown on CO; 4, strain SET IS-9
T
grown on
pyruvate. Because of the absence of a common nutrient combination
supporting good growth of all of the strains studied, for CFA analysis
C. hydrogenoformans Z-2901
T
was grown on medium 1 supplemented
with 0.2 g yeast extract l
21
under 100 % CO for 72 h, C. ferrireducens
JW/AS-Y7
T
was grown on medium 1 supplemented with 20 mM
sodium fumarate, 20 mM glycerol and 0.2 g yeast extract l
21
under
100 % N
2
for 48h, and strain SET IS-9
T
was grown either on CO as for
C. hydrogenoformans Z-2901
T
or on medium 1 supplemented with
2.0 g sodium pyruvate l
21
and 0.2 g yeast extract l
21
under 100 % N
2
for 72 h. ND, Not detected; tr, ,1.0 %.
Fatty acid 1 2 3 4
C
12 : 0
iso 6.7 ND 1.0 1.0
C
14 : 0
11.3 7.4 14.3 25.6
C
15 : 0
iso 30.1 30.8 20.7 12.4
C
15 : 0
anteiso 9.8 8.6 1.1 4.1
C
14 : 0
DMA tr 1.4 tr 1.8
C
15 : 0
2.3 1.9 4.5 1.0
C
15 : 0
iso DMA tr 1.0 tr 1.6
C
16 : 0
amine tr 1.2 tr tr
C
16 : 0
iso 3.9 2.4 3.6 tr
C
16 : 0
20.2 24.4 41.9 26.2
C
16 : 0
iso DMA 3.7 9.3 5.8 21.8
C
17 : 0
iso 2.8 1.5 1.5 tr
C
17 : 0
anteiso 3.2 tr tr ND
C
18 : 0
DMA 1* tr 1.8 tr tr
C
18 : 0
DMA 2* ND 1.9 tr tr
C
18 : 0
3.0 1.0 2.0 1.8
C
19 : 0
alkane tr tr tr 1.2
*C
18 : 0
DMA 1 and C
18 : 0
DMA 2 are C
18 : 0
DMAs differing in their
structure (straight-chain or branched), which could not be deter-
mined from the mass spectra obtained.
A. A. Novikov and others
2536 International Journal of Systematic and Evolutionary Microbiology 61

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