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1 23
Extremophiles
Microbial Life Under Extreme
Conditions
ISSN 1431-0651
Volume 19
Number 1
Extremophiles (2015) 19:39-47
DOI 10.1007/s00792-014-0701-6
Desulfosporosinus acididurans sp. nov.:
an acidophilic sulfate-reducing bacterium
isolated from acidic sediments
Irene Sánchez-Andrea, Alfons
J.M.Stams, Sabrina Hedrich, Ivan
Ňancucheo & D.Barrie Johnson
1 23
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ORIGINAL PAPER
Desulfosporosinus acididurans sp. nov.: an acidophilic
sulfate-reducing bacterium isolated from acidic sediments
Irene Sa
´nchez-Andrea •Alfons J. M. Stams •
Sabrina Hedrich •Ivan N
ˇancucheo •
D. Barrie Johnson
Received: 17 July 2014 / Accepted: 14 October 2014 / Published online: 5 November 2014
ÓSpringer Japan 2014
Abstract Three strains of sulfate-reducing bacteria (M1
T
,
D, and E) were isolated from acidic sediments (White river
and Tinto river) and characterized phylogenetically and
physiologically. All three strains were obligately anaero-
bic, mesophilic, spore-forming straight rods, stained Gram-
negative and displayed variable motility during active
growth. The pH range for growth was 3.8–7.0, with an
optimum at pH 5.5. The temperature range for growth was
15–40 °C, with an optimum at 30 °C. Strains M1
T
, D, and
E used a wide range of electron donors and acceptors, with
certain variability within the different strains. The nomi-
nated type strain (M1
T
) used ferric iron, nitrate, sulfate,
elemental sulfur, and thiosulfate (but not arsenate, sulfite,
or fumarate) as electron acceptors, and organic acids
(formate, lactate, butyrate, fumarate, malate, and pyruvate),
alcohols (glycerol, methanol, and ethanol), yeast extract,
and sugars (xylose, glucose, and fructose) as electron
donors. It also fermented some substrates such as pyruvate
and formate. Strain M1
T
tolerated up to 50 mM ferrous
iron and 10 mM aluminum, but was inhibited by 1 mM
copper. On the basis of phenotypic, phylogenetic, and
genetic characteristics, strains M1
T
, D, and E represent a
novel species within the genus Desulfosporosinus, for
which the name Desulfosporosinus acididurans sp. nov. is
proposed. The type strain is M1
T
(=DSM 27692
T
=JCM
19471
T
). Strain M1
T
was the first acidophilic SRB isolated,
and it is the third described species of acidophilic SRB
besides Desulfosporosinus acidiphilus and Thermodesulf-
obium narugense.
Keywords Acidophile Anaerobe Sulfate reduction
Isolation Characterization Desulfosporosinus
Introduction
Microorganisms that catalyse the dissimilatory reduction of
sulfate to sulfide include representatives of five phyloge-
netic lineages of bacteria (Deltaproteobacteria,Clostridia,
Nitrospiriae,Thermodesulfobiaceae, and Thermodesulfo-
bacteria) and the two major sub-groups (Crenarcheota and
Euryarchaeota) of the domain Archaea (Muyzer and Stams
2008). Sulfate-reducing bacteria (SRB) are highly diverse
in terms of the range of organic substrates they utilize,
which includes aromatic compounds and sugars, as well as
small molecular weight acids and alcohols, though poly-
meric organic materials generally are not utilized directly
by SRB (Muyzer and Stams 2008). In addition, some SRB
can grow autotrophically using hydrogen as electron donor
and fixing carbon dioxide, though others have a require-
ment for organic carbon, such as acetate, when growing on
Communicated by A. Oren.
Electronic supplementary material The online version of this
article (doi:10.1007/s00792-014-0701-6) contains supplementary
material, which is available to authorized users.
I. Sa
´nchez-Andrea (&)A. J. M. Stams
Laboratory of Microbiology, Wageningen University,
Dreijenplein 10, 6703 HB Wageningen, The Netherlands
e-mail: irene.sanchezandrea@wur.nl
S. Hedrich
Federal Institute for Geosciences and Natural Resources (BGR),
Stilleweg 2, 30655 Hannover, Germany
I. N
ˇancucheo
Universidad Arturo Prat, Iquique, Chile & Instituto Tecnolo
´gico
Vale, Bele
´m, Brazil
D. B. Johnson
College of Natural Sciences, Bangor University,
Bangor LL57 2UW, UK
123
Extremophiles (2015) 19:39–47
DOI 10.1007/s00792-014-0701-6
Author's personal copy
hydrogen (Widdel 1988). Many SRB can also use electron
acceptors other than sulfate for growth, such as sulfur,
sulfite, thiosulfate, nitrate, arsenate, iron or fumarate. Some
species of the Desulfovibrio genus can temporarily respire
oxygen, increasing resistance to oxic conditions (Mogen-
sen et al. 2005; Santana 2008). In environments that con-
tain relatively little sulfate, some SRB grow by
fermentation of organic substrates alone or in symbiosis
with methanogens (Plugge et al. 2011).
Most described SRB grow optimally at pH values
between 6 and 8 (Widdel 1988) and are mesophilic, but
several SRB have been isolated from extreme environ-
ments. Many thermophilic species have been described
(e.g., Thermodesulforhabdus norvegicus,Archaeoglobus
fulgidus,Desulfotomaculum spp.) (Beeder et al. 1995;
Zellner et al. 1989), while some species are psychrotolerant
(e.g., Desulfobacter psychrotolerans,Desulfovibrio cune-
atus and Desulfovibrio litoralis) (Tarpgaard et al. 2006;
Sass et al. 1998). Halophilic and haloalkalophilic species
have also been isolated e.g., Desulfonatronovibrio hy-
drogenovorans (Zhilina et al. 1997), Desulfonatronospira
thiodismutans (Sorokin et al. 2008).
Sulfate reduction in low pH natural and engineered
environments has been reported by a number of researchers
(Johnson et al. 1993; Moreau et al. 2010; Johnson et al.
2009;Sa
´nchez-Andrea et al. 2012b). Despite evidence of
their activity in situ, acidophilic SRB have been difficult to
isolate and cultivate in vitro. The first SRB isolated from
acidic sites were neutrophilic and inactive below pH 5
(Kusel et al. 2001; Tuttle et al. 1969; Lee et al. 2009; Gyure
et al. 1990). Various hypotheses were suggested to account
for this apparent incongruity, such as the possibility that
acid-tolerant or acidophilic SRB may not exist, and that
those present in acidic environments survive by creating
circum-neutral pH micro-environments in the sediment or
around wood or other suspended particles (Tuttle et al.
1969; Gyure et al. 1990). However, the use of inappropriate
enrichment and cultivation techniques (e.g., the use of
substrates such as lactate that occur predominantly as toxic
undissociated acids in low pH media) is one of the more
frequent reasons for problems encountered with isolating
acidophilic SRB (Kimura et al. 2006; Koschorreck 2008;
Alazard et al. 2010;Sa
´nchez-Andrea et al. 2014).
The first acidophilic SRB isolated and partially charac-
terized were Desulfosporosinus-like bacteria (strains P1
and M1
T
), which were isolated from the White River
(Monserrat, WI) and the abandoned Mynydd Parys copper
mine (Wales) (Sen and Johnson 1999). These isolates
shared [99 % 16S rRNA gene identity, and were affiliated
with the genus Desulfosporosinus, though strain P1 was
subsequently lost. Both isolates grew at pH as low as pH
3.8 using glycerol as electron donor. Kimura et al. (2006)
showed that strain M1
T
grew in co-culture with the non-
sulfidogenic acidophilic heterotroph Acidocella aromatica,
and provided evidence of syntrophic interaction, involving
hydrogen transfer, between the two bacteria (Kimura et al.
2006). Later, two other acidophilic SRB were described:
Thermodesulfobium narugense (Mori et al. 2003), a ther-
mophile isolated from a hot spring in Japan which grows
between pH 4.0 and 6.5 and Desulfosporosinus acidiphilus
(Alazard et al. 2010), a mesophile isolated from an acid
rock drainage environment. Other acidophilic Desulfosp-
orosinus strains (D and E), more closely related to strains
M1
T
and P1 than to D. acidiphilus, were isolated from the
Tinto river by Sa
´nchez-Andrea et al. (2013). Other SRB,
more acidophilic, have also been isolated, such as CL4
(Rowe et al. 2007), CEB3 (N
ˇancucheo and Johnson 2012)
and strain I (Sa
´nchez-Andrea et al. 2013) and await com-
plete characterization. Here, we report that the acidophilic
SRB isolates M1
T
, D, and E are strains of the same species,
which is described and for which the name Desulfosporo-
sinus acididurans is proposed.
Materials and methods
Source of the organisms
Strain M1
T
was isolated from the White river (pH 3.2,
T
a
=30 °C, Eh =?500 mV), which drained the Soufriere
hills in Montserrat, just before the cataclysmic volcano in
1995 (Atkinson et al. 2000). An enrichment culture of
sediment from this river was plated onto a glycerol-con-
taining overlay solid medium and incubated anaerobically
(Sen and Johnson 1999; Rowe et al. 2007). Strains D and E
were isolated by Sa
´nchez-Andrea et al. (2013) from an
enrichment containing acidic sediments from JL dam
(37.691207N, 6.560587W) in Tinto River basin (south-
western Spain). Detailed physico-chemical information has
been published elsewhere (Sa
´nchez-Andrea et al. 2012a).
For comparison purposes, Desulfosporosinus orientis DSM
765
T
and D. acidiphilus DSM 22704
T
were purchased from
the DSMZ (Braunschweig, Germany).
Cultivation techniques
The general cultivation media and conditions used were as
follows. Samples were inoculated into an anoxic basal
medium prepared as described previously (Stams et al.
1993) containing 5 mM glycerol and 10 mM sulfate, and
supplemented with 0.1 g/L yeast extract and 0.5 g/L L-
cysteine as reducing agent. Bicarbonate-buffer was elimi-
nated from the original composition to allow pH modifi-
cations. Media were adjusted with HCl and NaOH to the
different experimental pH values. The gas phase was
1.5 atm of N
2
/CO
2
(80:20, v/v). All compounds were heat-
40 Extremophiles (2015) 19:39–47
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sterilized except for the vitamins, which were filter-steril-
ized. The samples were incubated statically at 30 °C in the
dark. During the process, to check purity, isolates were
inoculated into the corresponding medium containing 5 g/
L yeast extract (Becton–Dickinson, Cockeysville, MD,
USA). After incubation, the cultures were examined
microscopically and the 16S rRNA genes of the isolates
were sequenced.
Genetic-based methods: phylogenetic and genomic
analysis
Cloning of the 16S rRNA gene was performed to determine
the phylogenetic affiliation of the isolates. Total genomic
DNA was extracted using the FastDNA
Ò
SPIN Kit for Soil
and the FastPrep
Ò
Instrument (MP Biomedicals, Santa Ana,
CA, USA). The 16S rRNA genes were amplified with the
primers set 27F-1492R (T
a
=57 °C) for Bacteria and
cloned in Escherichia coli DH5acompetent cells by using
the pGEM-T vector (Promega, Madison, WI, USA).
Sequences were assembled with the DNABaser software
3.5.3 and prior to phylogenetic analysis, vector sequences
flanking the 16S rRNA gene inserts were identified using
VecScreen tool (NCBI) (http://www.ncbi.nlm.nih.gov/
VecScreen/VecScreen.html) and removed. Clone sequences
were checked for chimeras (http://decipher.cee.wisc.edu/
FindChimeras.html), aligned with SINA (v1.2.11) (Pruesse
et al. 2012), and added to a database of over 230,000
homologous prokaryotic 16S rRNA gene primary structures
by using the merging tool of the ARB program package
(Ludwig et al. 2004). Sequences were then manually cor-
rected with the alignment tool of the same software and
added by parsimony to the tree generated in the Living Tree
Project (Yarza et al. 2008). Phylogenetic reconstruction was
performed using the three algorithms as implemented in the
ARB package. The maximum-likelihood method was cho-
sen for the generation of the consensus tree and bootstrap
analysis performance. Different copies of the 16S rRNA
genes sequences were detected, the more abundant copies of
each strain have been deposited in the EMBL database
under accession numbers HG316990-HG316992 (http://
www.ebi.ac.uk/ena/data/view/HG316990-HG316992)being
HG316990 the sequence of the type strain M1
T
. The G ?C
content of the DNA was determined via genome sequencing
(Germany) by Dr. Martin Mu
¨hling and Dr. Patrick Petzsch.
Phenotypic characterization: morphology, physiology,
and chemotaxonomy
Cell morphology, motility, and spore formation were
examined by phase contrast microscopy using a Leica
DM2000 microscope. The lengths and widths of several
cells were measured and mean dimensions recorded. Gram
staining was performed according to standard procedures
(Doetsch 1981). Gram-structure was also check by mixing
cells with a drop of 3 % (w/v) solution of KOH. A positive
KOH reaction is characteristic of the Gram-negative bac-
teria. Catalase activity was determined by reaction with
3 % (w/v) solution of H
2
O
2
. Oxidase test was performed
with a filter impregnated in 1 % (w/v) solution of tetra-
methyl-p-phenylenediamine in dimethyl sulfoxide (Sigma-
Aldrich, St Louis, MO, USA). Indole and urease formation
as well as gelatin and esculin hydrolysis were determined
with API
Ò
20A (bioMe
´rieux, France) according manufac-
turer’s instructions.
Growth experiments were performed in duplicate for
each strain, using 120 mL-serum bottles. Unless otherwise
indicated, the general conditions were pH 5.5, T=30 °C,
glycerol (5 mM), and sulfate (10 mM). Growth was mon-
itored by measuring optical density at 600 nm (OD
600
)
with a spectrophotometer (U-1500 Hitachi, Tokyo, Japan).
Soluble substrates and intermediates (sugars and volatile
fatty acids) were measured using a Thermo Electron
spectrasystem HPLC equipped with an Agilent Metacarb
67H column. Gaseous compounds (H
2
) were analyzed
using a Shimadzu GC-2014 Gas Chromatograph equipped
with a Molsieve 13X column. Sulfide was measured
photometrically with the methylene blue method (Cline
1969). Different organic electron donors were tested with
10 mM sulfate as electron acceptor at final concentrations
between 2 and 8 mM, in order to provide similar amounts
of carbon-equivalents. Different potential electron accep-
tors [30 mM iron (III)-nitrilotriacetate and elemental sul-
fur, and 5 mM nitrate, arsenate, thiosulfate, sulfite] were
tested using glycerol (2 mM) as the electron donor. Iron,
copper, and zinc toxicity was tested at different final con-
centrations of their sulfate salts of up to 50 mM. Growth
was also studied at different incubation temperatures (from
10 to 45 °C), pH (from 3.0 to 7.5), salinity (up to 0.5 M),
oxygen concentrations, and with different reducing agents:
L-cysteine (4 mM), sulfide (2 mM) and FeCl
2
(2 mM).
Sulfate reduction rates were determined during the expo-
nential phase of the cultures grown between pH 4 and 7 at
30 °C.
For appropriate comparison of cellular fatty acid
content, strain M1
T
and D. orientis weregrowninthe
same medium containing 5 mM of glycerol and sulfate,
as previously published for D. acidiphilus. Fatty acid
analyses were carried out by the Identification Service of
the DSMZ––Deutsche Sammlung von Mikroorganismen
und Zellkulturen GmbH (Braunschweig, Germany) using
the Sherlock MIS (MIDI Inc, Newark, USA) system
(Miller 1982) and identified by two different databases,
TSBA and BHILB. Analysis of respiratory quinones of
biomass grown on glycerol was also carried out by the
DSMZ.
Extremophiles (2015) 19:39–47 41
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Results and discussion
Phylogeny and genomic characteristics
Analysis of the almost complete sequences of the 16S rRNA
genes of the strains M1
T
,D,andEconfirmedthattheywere
phylogenetically affiliated to the genus Desulfosporosinus
(family Peptococcaceae,orderClostridiales) of the phylum
Firmicutes. Different copies of the 16S rRNA gene were
identified in the clone libraries performed. Other represen-
tatives of the Desulfosporosinus group show variable num-
bers of the 16S rRNA gene, ranging from 1 copy in
Desulfosporosinus sp. OT to 11 copies in Desulfosporosinus
meridei. Sequence identity calculations with neighbor-join-
ing analysis using the ARB package showed differences of
up to 2.9 % between the different 16S rRNA gene copies
identified in the three strains in the clone libraries per-
formed. The largest differences were due to insertions
(around 100 bp) detected in the sequences of some copies of
strain E. Pairwise comparison analysis showed that the M1
T
reference sequence (HG316990) shared 98.3 % identity with
the reference sequence of strain D (HG316991) and 98.5 %
with the reference sequence of strain E (HG316992) forming
a consistent cluster within the Desulfosporosinus genus
(Fig. 1). The same comparison indicated that the most clo-
sely related species of the three strains was D. acidiphilus
with a 96.4 % identity, followed by other Desulfosporosinus
spp., ranging from 92.3 to 96.4 % identity to the type strains
(Supplementary Table 1). Members of the genus Desul-
fosporosinus form a coherent phylogenetic group within the
family Peptococcaceae,orderClostridiales,phylumFirmi-
cutes (Fig. 1). This genus currently comprises eight validly
described species: D. orientis (Stackebrandt et al. 1997), D.
auripigmenti (Newman et al. 1997), D. meridiei (Robertson
et al. 2001), D. lacus (Ramamoorthy et al. 2006), D. hippei
(Vatsurina et al. 2008), D. youngiae (Lee et al. 2009), D.
acidiphilus (Alazard et al. 2010), and the recently described
D. burensis (Mayeux et al. 2013). The type species of the
genus is D. orientis. The cluster consisting of Desulfosp-
orosinus species is strongly supported by maximum likeli-
hood, parsimony and neighbor-joining algorithms (Fig. 1).
The G ?C content of the genomic DNA of the type
strain (M1
T
) was 41.8 mol % (Mu
¨hling and Petzsch, TU
Bergakademie Freiberg, personal communication), com-
parable to values quoted for others members of the genus
Desulfosporosinus (41.6–46.9 %).
Morphology
Cells of strains M1
T
, D, and E were observed as straight
rods, 3–7 lm in length and 0.7 lm in width (Fig. 2), and
occurred as single, motile cells during the exponential
growth phase. Endospores were readily observed under the
growth conditions tested. The spores were refractive and
appear mainly in sub-terminal position (Fig. 2b). Cells of
M1
T
, D, and E stained Gram-negative, although the test
with KOH was negative, indicating a Gram-positive com-
position cell wall. Desulfosporosinus spp. phylogenetically
cluster within the low G ?C Gram-positive Peptococca-
ceae group in the phylum Firmicutes, but the different
species forming the group mainly stained Gram-negative.
Other examples of phylogenetically Gram-positive spore-
formers which stain Gram-negative are found in literature
such as Propionispora vibroides (Biebl et al. 2000).
Physiology
Strains M1
T
, D, and E were strict anaerobes, growing with
L-cysteine, ferrous iron, and sulfide as reducing agents.
Short-term exposure to oxygen (e.g., manipulating cultures
Fig. 1 Phylogenetic affiliations of 16S rRNA gene sequences of
strains M1
T
, D, and E (bold type) and the related species in the
Peptococcaceae family of the Firmicutes phylum in the Living Tree
Project (Yarza et al. 2008). Maximum-likelihood tree was chosen
after applying the three algorithms as implemented in the ARB
package. Based on 1000 replications, significant values of each
branch (above 70 %) are indicated at the nodes by odd circles. The
bar indicates a 1 % estimated sequence divergence. The sequence of
Desulfotomaculum nigrificans was used as the out-group
42 Extremophiles (2015) 19:39–47
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under atmospheric air) did not result in loss of viability.
Optimal growth was observed at 30 °C, but the isolates
grew well between 15 and 40 °C. Growth occurred in the
presence of up to 0.3 M NaCl. All three strains grew in a
pH range between 3.8 and 7.0 accumulating sulfide (up to
5 mM in our tests). Experiments performed with strain D
showed that growth occurred at pH 4 and a starting con-
centration of 4 mM of sulfide (Sa
´nchez-Andrea et al.
2013). When grown in citrate/phosphate buffered media,
growth of strain D was inhibited at pH 4 in comparison
with the non-buffered control, possibly because of the citric
acid concentration (Sa
´nchez-Andrea et al. 2013). Besides
buffering of media, another way of pH-control can be the
use of metals as a source of protons (when hydrolysing)
and sink of sulfide (when precipitating). Growth of strain
M1
T
with 2 mM glycerol in the presence of 5 mM zinc
sulfate occurred at pH as low as 3.5 increasing slightly the
value to 3.8. Experiments with strain M1
T
in the presence
of an acidophilic acetate-consumer, A. aromatica strain
PFBC (Jones et al. 2013) showed that strain M1
T
could
grow on glycerol at pH as low as 3.0 when acetate inhi-
bition is avoided by consumption by a syntroph (Kimura
et al. 2006).
Sulfate reduction rates of strain M1
T
, D, and E showed
that the optimum pH for growth was 5.5, and that growth
was much slower at higher pH values (see Fig. 3). Rates
were calculated during exponential growth when OD
600
of
strain M1
T
was 0.140, which corresponded with approxi-
mately 5.510
7
cells/mL. Doubling time of strain M1
T
at
optimum conditions (pH 5.5, T=30 °C) was about 5 h.
The conventional definition of an extreme acidophile is that
it is a microorganism with a pH optimum for growth at (or
below) pH 3.0 (Norris and Johnson 1998), whereas mod-
erate acidophiles grow optimally between pH 3 and 6.
Therefore, the pH dependence of growth of strains M1
T
,D,
and E indicates that they are moderately acidophilic bac-
teria, rather than acid-tolerant. So far, with the exception of
D. acidiphilus, the rest of the characterized representatives
of the genus Desulfosporosinus such as D. orientis or D.
auripigmenti, are neutrophilic, growing optimally at pH
ranges of 6.2–7.0 and 6.4–7.0, respectively. The substrates
tested as possible energy and carbon sources in the pre-
sence of sulfate as electron acceptor are listed in Table 1,
together with the electron acceptors tested in the presence
of glycerol and the fermentation substrates. The three
strains M1
T
, D, and E grew autotrophically with 1.5 atm of
H
2
/CO
2
(80:20, v/v) as gas phase with hydrogen serving as
electron donor for sulfate reduction and CO
2
as the carbon
source for growth. Growth yields were comparable to other
substrates (OD
600
=0.145 in 6 days) and acetate was
produced. Most species of Desulfosporosinus genus also
have been reported to grow as autotrophs. The three iso-
lated strains showed different physiological pattern, strains
M1
T
used ferric iron as electron acceptor, and glucose and
fructose as electron donor in the presence of sulfate, while
strains D and E did not. In the presence of sulfate, strain
M1
T
grew on complex substrates such as yeast extract; on
carbohydrates such as glucose, fructose and xylose; on
organic acids such as lactate, fumarate, butyrate, malate,
pyruvate, and formate and on alcohols such as glycerol and
ethanol. In addition to the data shown in Table 1, strain
M1
T
grew on rhamnose, galactose, mannitol, lysine, glu-
tamate, ribose, and mannose when reducing sulfate. The
three strains are all incomplete substrate oxidizers, with
larger molecular weight carbon sources being incompletely
oxidized to acetate/acetic acid. No growth was observed on
the following substrates with sulfate as the electron
acceptor for any of the three strains: acetate, citrate, suc-
cinate, propionate, benzoate, and 1-propanol. They used
Fig. 2 Phase-contrast micrograph of astrain M1
T
grown on glycerol
attached to a ZnS particle and bcells of strain D in the spore-forming
phase, bar 5lm
Fig. 3 Effect of pH on the sulfate reduction rate (SRR) of strain M1
T
at different pH values (grown on 5 mM glycerol at 30 °C)
Extremophiles (2015) 19:39–47 43
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Table 1 Main characteristics differentiating strains M1
T
, D, and E from their closest phylogenetic relatives
Characteristics Strains M1
T
, D and E D. acidiphilus DSM 22704
T
D. orientis DSM 765
T
Type strain M1
T
SJ4
T
Singapore
T
I
Isolation source River sediment draining
a volcanic mountain range
Acid mine drainage
sediments
Soil
Cell size (lm) 3–5 90.7 4–7 90.8–1 5 91.5
Endospore position Subterminal Subterminal Subterminal, paracentral
or central
Motility Variable -?
Temperature range (°C) 15–40 25–40 \/42
Temperature optimum (°C) 30 30 30–37
pH range 3.8–7 3.6–5.6 5.6–7.4
pH optimum 5.5 5.2 6.4–7
NaCl range (%) 0–1.5 0–0.6 \4.5
e
NaCl optimum (%) 0.6 1.5 NT
Electron donors with sulfate
H
2
/CO
2
???
Organic acids
Formate ?,(?), (?)
g
-?
Fumarate ?-?
e, f
Citrate ---
a
Pyruvate ??
a
?
d, e, f
Malate ?--
c, e
Acetate ---
d, e, f
Propionate --
a
-
e, f
Lactate ?(?)?
Butyrate ?-?
f
Succinate ---
a, f
Benzoate ---
a, e, f
Alcohols
Ethanol ?-?
e,f
Glycerol ??-
f
,?
a
Methanol (?)-?
e, f
Propanol --?
Yeast extract ?-,?
a
?
f
Sugars
Fructose ?,-,-? -
d, e
Glucose ?,-,-(?)-
f
Xylose ?,(?), (?)
g
--
a
Electron acceptors with glycerol
Sulfate ???
Sulfur ?-,?
a
?
e, f
Sulfite --?
e, f
Thiosulfate ??
1
?
a, d, e, f
Arsenate ---
a, e, f
,?
c
Fumarate ---,?
f
Fe(III) ?,-,-
g
-?
a, c, e, f
Nitrate ?(?)
a
-
c, f
Fermentation in the absence of sulfate
Lactate --?
Glucose ---,?
a
44 Extremophiles (2015) 19:39–47
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sulfate, elemental sulfur, and nitrate as electron acceptors
(nitrite and ammonia accumulate in the medium), but not
arsenate, sulfite or fumarate. Strain M1
T
used iron(III) as
electron acceptor but strains D and E did not. Strains M1
T
,
D, and E differed from their closest relative, D. acidiphilus,
in using formate, fumarate, malate, butyrate, ethanol, and
xylose as electron donors, and additionally strain M1
T
in
using fructose and glucose as electron donor and iron(III)
as an electron acceptor. In the description of D. acidiphilus,
it was reported that this SRB cannot use elemental sulfur
but the available genome on JGI showed a sulfur-reductase
gene, and our experiments have confirmed that it (as well
as the three strains of D. acididurans tested) can indeed use
elemental sulfur as an electron acceptor. Heavy metal
toxicity experiments showed that strain M1
T
grows in
media containing up to 50 mM ferrous iron, and 10 mM
aluminum, but growth was completely inhibited by 1 mM
copper (added as sulfate salts).
Chemotaxonomy
Cellular fatty acid composition profiles of strain M1
T
and its
phylogenetic closest relatives are listed in Table 2. The
overall composition of its CFAs was in agreement with those
of the other members of the Desulfosporosinus genus.
However, there were quantitative differences; while D. ori-
entis shows significant abundance of the unsaturated straight
chain acids C
18:1
cis11 DMA and C
18:1
w7c, their presence in
strain M1
T
is almost negligible. The abundance of the satu-
rated branched-chain acid iso-C
15:0
is common between
strain M1
T
and D. acidiphilus, being absent in D. orientis.
Major menaquinone of strain M1
T
was MK-7 (98 %) with a
minor presence of menaquinone MK-8 (2 %).
Desulfosporosinus spp. represent a clade of strictly
anaerobic, rod-shaped, mesophilic spore-forming bacteria
which oxidize organic substrates to acetate and CO
2
. They
show a great metabolic diversity: all the strains of this
genus can use thiosulfate as an alternative electron
acceptor, most of them are able to utilize sulfite (D.orien-
tis,D.burensis,D.lacus,D.auripigmenti,D.meridiei and
D. youngiae), some can also reduce arsenate (D. acidiph-
ilus,D.auripigmenti), iron (III) (strain M1
T
,D. orientis
Table 1 continued
Characteristics Strains M1
T
, D and E D. acidiphilus DSM 22704
T
D. orientis DSM 765
T
Glycerol --
a
?
a
Ethanol ---
a
,?
d
Methanol --?
d
Pyruvate ?--,(?)
a
Fumarate ?(?)
a
?
a
Formate --?
a
Yeast extract ?-
a
NT
DNA G ?C mol % 41.8 42.3 41.7, 42.8
f
, 45.9
e
Quinones MK-7 NT MK-7
d
Strains: 1, M1
T
, D, and E (unless indicated otherwise, common features for the 3 strains); 2, Desulfosporosinus acidiphilus DSM 22704
T
,
otherwise indicated data from Alazard et al. (2010); and 3, D. orientis DSM 765
T
, otherwise indicated data from Klemps et al. (1985)
?supported growth, -did not support growth, (?) weak growth. All strains are rods, Gram-negative, oval-spore formers, catalase positive, use
lactate, glycerol, yeast extract, and hydrogen with sulfate as electron acceptor. None of the strains uses acetate, succinate, citrate or benzoate
a
This study,
b
Alazard et al. (2010),
c
Campbell and Postgate (1965),
d
Stackebrandt et al. (1997),
e
Robertson et al. (2001),
f
Ramamoorthy
et al. (2006),
g
different utilization in the three strains
Table 2 Relative abundance (% of total) of cellular fatty acids of
strain M1
T
and its phylogenetic closest relatives grown on glycerol
and sulfate
Fatty acids Strain
M1
T
D. acidiphilus
DSM 22704
T
D. orientis
DSM 765
T
C
14:0
3.6 15.7 2.6
Iso-C
15:0
31.1 28.1 –
Anteiso-C
15:0
0.4 2.4 –
C
15:2
– 2.8 1.7
C
16:0
ald 2.4 4.9 1.9
C
16:1
cis7 0.8 1.8 2.4
C
16:1
cis9 2.1 3.6 5.9
C
16:0
10.7 18.7 43.8
C
16:1
cis9 DMA 2.6 3 3.0
C
16:0
DMA 9.4 10.8 6.3
C
17:0
cyc 12.1 4.2 0.3
C
18:1
cis9 DMA 1.2 1.8 4.0
C
18:1
cis11 DMA 1.6 – 17.1
C
18:1
w7c 0.7 – 12.5
Strains: 1, strain M1
T
(this study); 2, D. acidiphilus, data from Ala-
zard et al. (2010); and 3, D. orientis (this study)
Significant values are highlighted in bold type
Extremophiles (2015) 19:39–47 45
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and D. lacus), nitrate (strain M1
T
,D. acidiphilus, and some
strains of D. meridiei), elemental sulfur (strain M1
T
,D.
acidiphilus,D. orientis,D. meridiei) and heavy metals as
electron acceptors (Spring and Rosenzweig 2006). So far,
the genus Desulfosporosinus is composed of seven neutr-
ophilic species and including strain M1
T
, two acidophilic
ones. Their prevalence in acidic environments suggests that
more acidophilic species might exist.
Description of Desulfosporosinus acididurans sp. nov.
Desulfosporosinus acididurans (a.ci.di. du’rans. N.L. neut.
n. acidum acid, L. part. adj. durans resisting, N.L. part.
adj. acididurans acid-resisting)
Gram-positive rods that stain Gram-negative. Cells are
0.7 lm in width and 3–7 lm in length. Motile in the
exponential phase, and occur as single rods. Sub-terminal,
oval endospores that swell the cells are produced. The pH
range for growth is pH 3.8–7, with an optimum at pH 5.5.
The temperature range for growth is 15–40 °C, with an
optimum at 30 °C. The upper limit for salt tolerance is
0.3 M NaCl. Strains M1
T
, D, and E used a wide range of
electron donors and acceptors presenting interspecies var-
iability. The proposed type strain (M1
T
) used iron, nitrate,
sulfate, elemental sulfur, and thiosulfate as electron
acceptors; but not arsenate, sulfite, or fumarate. Strain M1
T
utilizes H
2
, organic acids (formate, lactate, butyrate,
fumarate, malate, and pyruvate), alcohols (glycerol,
methanol, and ethanol), yeast extract, and sugars (xylose,
glucose, and fructose) as electron donors for sulfate
reduction. Strains D and E did not use ferric iron as elec-
tron acceptor or glucose and fructose as electron donors.
Organic substrates are oxidized incompletely to acetate and
CO
2
. The following substrates are not used as electron
donors for any of the three strains: acetate, succinate, cit-
rate, propionate, benzoate or propanol. They also ferment
some substrates such as pyruvate and formate. Strain M1
T
tolerates up to 50 mM of ferrous iron and 10 mM of alu-
minum being sensitive to 1 mM of copper. The predomi-
nant whole cell fatty acids of the type strain M1
T
are iso-
C
15:0
(31.1 %), C
17:0
cyc (12.0 %), C
16:0
(10.7 %) and C
16:0
DMA (9.4 %) and its genomic G ?C content is
41.8 mol %. Phylogenetically, it is a member of the order
Clostridiales within the Firmicutes phylum. The type strain
of Desulfosporosinus acididurans, strain M1
T
(=DSM
27692
T
=JCM 19471
T
), was isolated from acidic sedi-
ment of White river, Montserrat.
Acknowledgments This work was financed by an ERC Grant
(project 323009) attributed to A.J.M. Stams and the Gravitation Grant
(SIAM 024.002.002) of the Netherlands Ministry of Education,
Culture and Science and the Netherlands Science Foundation. Thanks
to Dr. Martin Mu
¨hling, Dr. Patrick Petzsch and Prof. Michael
Schlo
¨mann for sharing the data of the genome sequencing funded by
the European Social Fund (ESF) and the Federal State of Saxony
(Germany) and to Prof. Ricardo Amils and Prof. Jose Luis Sanz to
promote the isolation of strains D and E.
References
Alazard D, Joseph M, Battaglia-Brunet F, Cayol JL, Ollivier B (2010)
Desulfosporosinus acidiphilus sp. nov.: a moderately acidophilic
sulfate-reducing bacterium isolated from acid mining drainage
sediments. Extremophiles 14:305–312
Atkinson T, Cairns S, Cowan DA, Danson MJ, Hough DW, Johnson
DB, Norris PR, Raven N, Robinson C, Robson R (2000) A
microbiological survey of Montserrat Island hydrothermal
biotopes. Extremophiles 4:305–313
Beeder J, Torsvik T, Lien T (1995) Thermodesulforhabdus norvegi-
cus gen. nov., sp. nov., a novel thermophilic sulfate-reducing
bacterium from oil field water. Arch Microbiol 164:331–336
Biebl H, Schwab-Hanisch H, Spro
¨er C, Lu
¨nsdorf H (2000) Propio-
nispora vibrioides, nov. gen., nov. sp., a new gram-negative,
spore-forming anaerobe that ferments sugar alcohols. Arch
Microbiol 174:239–247
Campbell LL, Postgate JR (1965) Classification of the spore-forming
sulfate-reducing bacteria. Microbiol Mol Biol Rev 29:359–362
Cline JD (1969) Spectrophotometric determination of hydrogen
sulfide in natural waters. Limnol Oceanogr 14:454–458
Doetsch R (1981) Determinative methods of light microscopy. In:
Gerhardt P, Murray RDE, Costilow RN, Nester EW, Wood WA,
Krieg NR, Philips GB (eds) Manual of methods for general
bacteriology. American Society for Microbiology, Washington,
D.C., pp 21–33
Gyure RA, Konopka A, Brooks A, Doemel W (1990) Microbial
sulfate reduction in acidic (pH 3) strip-mine lakes. FEMS
Microbiol Lett 73:193–201
Johnson DB, Ghauri M, McGinness S (1993) Biogeochemical cycling
of iron and sulphur in leaching environments. FEMS Microbiol
Rev 11:63–70
Johnson DB, Jameson E, Rowe O, Wakeman K, Hallberg KB (2009)
Sulfidogenesis at low pH by acidophilic bacteria and its potential
for the selective recovery of transition metals from mine waters.
Adv Mater Res 71:693–696
Jones RM, Hedrich S, Johnson DB (2013) Acidocella aromatica sp.
nov.: an acidophilic heterotrophic alphaproteobacterium with
unusual phenotypic traits. Extremophiles 17:841–850
Kimura S, Hallberg KB, Johnson DB (2006) Sulfidogenesis in low pH
(3.8–4.2) media by a mixed population of acidophilic bacteria.
Biodegradation 17:57–65
Klemps R, Cypionka H, Widdel F, Pfennig N (1985) Growth with
hydrogen, and further physiological characteristics of Desulfo-
tomaculum species. Arch Microbiol 143:203–208
Koschorreck M (2008) Microbial sulphate reduction at a low pH.
FEMS Microbiol Ecol 64:329–342
Kusel K, Roth U, Trinkwalter T, Peiffer S (2001) Effect of pH on the
anaerobic microbial cycling of sulfur in mining-impacted
freshwater lake sediments. Environ Exp Bot 46:213–223
Lee YJ, Romanek CS, Wiegel J (2009) Desulfosporosinus youngiae
sp. nov., a spore-forming, sulfate-reducing bacterium isolated
from a constructed wetland treating acid mine drainage. Int J
Syst Evol Microbiol 59:2743–2746
Ludwig W, Strunk O, Westram R, Richter L, Meier H (2004) ARB: a
software environment for sequence data. Nucleic Acids Res
32:1363–1371
Mayeux B, Fardeau M, Bartoli-Joseph M, Casalot L, Vinsot A, Labat
M (2013) Desulfosporosinus burensis sp. nov., a spore-forming,
46 Extremophiles (2015) 19:39–47
123
Author's personal copy
mesophilic, sulfate-reducing bacterium isolated from a deep clay
environment. Int J Syst Evol Microbiol 63:593–598
Miller LT (1982) Single derivatization method for routine analysis of
bacterial whole-cell fatty acid methyl esters, including hydroxy
acids. J Clin Microbiol 16:584–586
Mogensen GL, Kjeldsen KU, Ingvorsen K (2005) Desulfovibrio
aerotolerans sp. nov., an oxygen tolerant sulphate-reducing
bacterium isolated from activated sludge. Anaerobe 11:339–349
Moreau JW, Zierenberg RA, Banfield JF (2010) Diversity of
dissimilatory sulfite reductase genes (dsrAB) in a salt marsh
impacted by long-term acid mine drainage. Appl Environ
Microbiol 76:4819–4828
Mori K, Kim H, Kakegawa T, Hanada S (2003) A novel lineage of
sulfate-reducing microorganisms: Thermodesulfobiaceae fam.
nov., Thermodesulfobium narugense, gen. nov., sp. nov., a new
thermophilic isolate from a hot spring. Extremophiles 7:283–290
Muyzer G, Stams AJM (2008) The ecology and biotechnology of
sulphate-reducing bacteria. Nat. Rev Microbiol 6:441–454
N
ˇancucheo I, Johnson DB (2012) Selective removal of transition
metals from acidic mine waters by novel consortia of acidophilic
sulfidogenic bacteria. Microb Biotechnol 5:34–44
Newman DK, Kennedy EK, Coates JD, Ahmann D, Ellis DJ, Lovley
DR, Morel FM (1997) Dissimilatory arsenate and sulfate
reduction in Desulfotomaculum auripigmentum sp. nov. Arch
Microbiol 168:380–388
Norris P, Johnson D (1998) Acidophilic microorganisms. In: Hori-
koshi K, Grant WD (eds) Extremophiles: microbial life in
extreme environments. Wiley-Liss, New York, pp 133–153
Plugge CM, Zhang W, Scholten JC, Stams AJ (2011) Metabolic
flexibility of sulfate-reducing bacteria. Front Microbiol 2:81
Pruesse E, Peplies J, Glo
¨ckner FO (2012) SINA: accurate high-
throughput multiple sequence alignment of ribosomal RNA
genes. Bioinformatics 28:1823–1829
Ramamoorthy S, Sass H, Langner H, Schumann P, Kroppenstedt R,
Spring S, Overmann J, Rosenzweig R (2006) Desulfosporosinus
lacus sp. nov., a sulfate-reducing bacterium isolated from
pristine freshwater lake sediments. Int J Syst Evol Microbiol
56:2729–2736
Robertson W, Bowman J, Franzmann P, Mee B (2001) Desulfosp-
orosinus meridiei sp. nov., a spore-forming sulfate-reducing
bacterium isolated from gasolene-contaminated groundwater. Int
J Syst Evol Microbiol 51:133–140
Rowe OF, Sa
´nchez-Espan
˜a J, Hallberg KB, Johnson DB (2007)
Microbial communities and geochemical dynamics in an
extremely acidic, metal-rich stream at an abandoned sulfide
mine (Huelva, Spain) underpinned by two functional primary
production systems. Environ Microbiol 9:1761–1771
Sa
´nchez-Andrea I, Knittel K, Amann R, Amils R, Sanz JL (2012a)
Quantification of Tinto River sediment microbial communities:
the importance of sulfate-reducing bacteria and their role in
attenuating acid mine drainage. Appl Environ Microbiol
78(13):4638–4645
Sa
´nchez-Andrea I, Triana D, Sanz JL (2012b) Bioremediation of acid
mine drainage coupled with domestic wastewater treatment.
Water Sci Technol 66(11):2425–2431
Sa
´nchez-Andrea I, Stams AJ, Amils R, Sanz JL (2013) Enrichment
and isolation of acidophilic sulfate-reducing bacteria from Tinto
River sediments. Environ Microbiol Rep 5:1758–2229
Sa
´nchez-Andrea I, Sanz JL, Bijmans MF, Stams AJ (2014) Sulfate
reduction at low pH to remediate acid mine drainage. J Hazard
Mater 269:98–109
Santana M (2008) Presence and expression of terminal oxygen
reductases in strictly anaerobic sulfate-reducing bacteria isolated
from salt-marsh sediments. Anaerobe 14:145–156
Sass H, Berchtold M, Branke J, Ko
¨nig H, Cypionka H, Babenzien H
(1998) Psychrotolerant sulfate-reducing bacteria from an oxic
freshwater sediment description of Desulfovibrio cuneatus sp.
nov. and Desulfovibrio litoralis sp. nov. Syst Appl Microbiol
21:212–219
Sen A, Johnson B (1999) Acidophilic sulphate-reducing bacteria:
candidates for bioremediation of acid mine drainage. Process
Metall 9:709–718
Sorokin DY, Tourova TP, Henstra AM, Stams AJM, Galinski EA,
Muyzer G (2008) Sulfidogenesis under extremely haloalkaline
conditions by Desulfonatronospira thiodismutans gen. nov., sp.
nov., and Desulfonatronospira delicata sp. nov.––a novel
lineage of Deltaproteobacteria from hypersaline soda lakes.
Microbiology 154:1444–1453
Spring S, Rosenzweig F (2006) The genera Desulfitobacterium and
Desulfosporosinus: taxonomy. In: Dworkin M, Falkow S,
Rosenberg E, Schleifer KH, Stackebrandt E (eds) The prokary-
otes: a handbook on the biology of bacteria, 3rd edn. Springer,
Singapore, pp 771–786
Stackebrandt E, Sproer C, Rainey FA, Burghardt J, Pauker O, Hippe
H (1997) Phylogenetic analysis of the genus Desulfotomaculum:
evidence for the misclassification of Desulfotomaculum guttoid-
eum and description of Desulfotomaculum orientis as Desul-
fosporosinus orientis gen. nov., comb. nov. Int J Syst Evol
Microbiol 47:1134–1139
Stams AJM, Van Dijk JB, Dijkema C, Plugge CM (1993) Growth of
syntrophic propionate-oxidizing bacteria with fumarate in the
absence of methanogenic bacteria. Appl Environ Microbiol
59:1114–1119
Tarpgaard IH, Boetius A, Finster K (2006) Desulfobacter psychro-
tolerans sp. nov., a new psychrotolerant sulfate-reducing bacte-
rium and descriptions of its physiological response to
temperature changes. Antonie Van Leeuwenhoek 89:109–124
Tuttle JH, Dugan PR, Macmillan CB, Randles CI (1969) Microbial
dissimilatory sulfur cycle in acid mine water. J Bacteriol
97:594–602
Vatsurina A, Badrutdinova D, Schumann P, Spring S, Vainshtein M
(2008) Desulfosporosinus hippei sp. nov., a mesophilic sulfate-
reducing bacterium isolated from permafrost. Int J Syst Evol
Microbiol 58:1228–1232
Widdel F (1988) Microbiology and ecology of sulfate- and sulfur-
reducing bacteria. In: Zehnder AJB (ed) Biology of anaerobic
microorganisms. Wiley, New York, pp 469–585
Yarza P, Richter M, Peplies J, Euzeby J, Amann R, Schleifer K,
Ludwig W, Glo
¨ckner FO, Rossello
´-Mo
´ra R (2008) The All-
species Living Tree project: a 16S rRNA-based phylogenetic
tree of all sequenced type strains. Syst Appl Microbiol
31:241–250
Zellner G, Stackerbrandt E, Kneifel H, Messner P, Sleytr UB,
Conway de Macario E, Zabel H, Stetter KO, Winter J (1989)
Isolation and characterization of a thermophilic, sulfate reducing
archaebacterium, Archaeoglobus fulgidus strain Z. Syst Appl
Microbiol 11:151–160
Zhilina T, Zavarzin G, Rainey F, Pikuta E, Osipov G, Kostrikina N
(1997) Desulfonatronovibrio hydrogenovorans gen. nov., sp.
nov., an alkaliphilic, sulfate-reducing bacterium. Int J Syst
Bacteriol 47:144–149
Extremophiles (2015) 19:39–47 47
123
Author's personal copy
