Download Caloranaerobacter ferrireducens sp. nov., an anaerobic

International Journal of Systematic and Evolutionary Microbiology (2015), 65, 1714–1718
DOI 10.1099/ijs.0.000165
Caloranaerobacter ferrireducens sp. nov., an
anaerobic, thermophilic, iron (III)-reducing
bacterium isolated from deep-sea hydrothermal
sulfide deposits
Xiang Zeng,1,2,33 Zhao Zhang,1,2,33 Xi Li,1,2,3 Mohamed Jebbar,4,5,6
Karine Alain4,5,6 and Zongze Shao1,2,3
Correspondence
Zongze Shao
[email protected]
1
Key Laboratory of Marine Biogenetic Resources, the Third Institute of Oceanography SOA,
Xiamen, Fujian 361005, PR China
2
Collaborative Innovation Center of Deep Sea Biology, Xiamen, Fujian 361005, PR China
3
Key Laboratory of Marine Genetic Resources of Fujian Province, Xiamen, Fujian 361005,
PR China
4
Université de Bretagne Occidentale (UBO, UEB), Institut Universitaire Européen de la Mer
(IUEM)–UMR 6197, Laboratoire de Microbiologie des Environnements Extrêmes (LM2E),
Place Nicolas Copernic, F-29280 Plouzané, France
5
CNRS, IUEM–UMR 6197, Laboratoire de Microbiologie des Environnements Extrêmes (LM2E),
Place Nicolas Copernic, F-29280 Plouzané, France
6
Ifremer, UMR 6197, Laboratoire de Microbiologie des Environnements Extrêmes (LM2E),
Technopôle Pointe du diable, F-29280 Plouzané, France
A thermophilic, anaerobic, iron-reducing bacterium (strain DY22619T) was isolated from a sulfide
sample collected from an East Pacific Ocean hydrothermal field at a depth of 2901 m. Cells were
Gram-stain-negative, motile rods (2–10 mm in length, 0.5 mm in width) with multiple peritrichous
flagella. The strain grew at 40–70 6C inclusive (optimum 60 6C), at pH 4.5–8.5 inclusive
(optimum pH 7.0) and with sea salts concentrations of 1–10 % (w/v) (optimum 3 % sea salts) and
NaCl concentrations of 1.5–5.0 % (w/v) (optimum 2.5 % NaCl). Under optimal growth conditions,
the generation time was around 55 min. The isolate was an obligate chemoorganoheterotroph,
utilizing complex organic compounds, amino acids, carbohydrates and organic acids including
peptone, tryptone, beef extract, yeast extract, alanine, glutamate, methionine, threonine, fructose,
mannose, galactose, glucose, palatinose, rhamnose, turanose, gentiobiose, xylose, sorbose,
pyruvate, tartaric acid, a-ketobutyric acid, a-ketovaleric acid, galacturonic acid and glucosaminic
acid. Strain DY22619T was strictly anaerobic and facultatively dependent on various forms of
Fe(III) as an electron acceptor: insoluble forms and soluble forms. It did not reduce sulfite, sulfate,
thiosulfate or nitrate. The genomic DNA G+C content was 29.0 mol%. Phylogenetic 16S rRNA
gene sequence analyses revealed that the closest relative of strain DY22619T was
Caloranaerobacter azorensis MV1087T, sharing 97.41 % 16S rRNA gene sequence similarity. On
the basis of physiological distinctness and phylogenetic distance, the isolate is considered to
represent a novel species of the genus Caloranaerobacter, for which the name Caloranaerobacter
http://dx.doi.org/10.1601/nm.4081ferrireducens sp. nov. is proposed. The type strain is
DY22619T (5JCM 19467T5DSM 27799T5MCCC1A06455T).
3These authors contributed equally to this work.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain DY22619T is KC794016.
A supplementary figure and a supplementary table are available with the
online Supplementary Material.
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Iron minerals are abundant at deep-sea hydrothermal vents.
The surfaces of active chimneys are frequently covered with
deposits of iron oxides in different oxidative states. Thus,
deep-sea hydrothermal vents can provide an ecological
niche for Fe(III)-reducing micro-organisms (Slobodkin
et al., 2001). Dissimilatory Fe(III)-reducing bacteria (DIRB)
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Caloranaerobacter ferrireducens sp. nov.
conserve energy to support growth by coupling the oxidation
of organic compounds and/or H2 to the reduction of ferric
iron (Lovley et al., 1997). The process of dissimilatory Fe(III)reducing influences several biogeochemical element cycles,
causes the release of soluble Fe(II), phosphate and trace
metals, and affects sediment properties (Lovley, 1995).
At the time of writing, only a few Fe(III)-reducing bacteria
have been isolated and characterized from deep-sea hydrothermal areas, including Deferribacter abyssi (Miroshnichenko
et al., 2003) and Deferribacter autotrophicus (Slobodkina et al.,
2009) within the order Deferribacteres, and Geothermobacter
ehrlichii (Kashefi et al., 2003) and Deferrisoma camini
(Slobodkina et al., 2012) in the class Deltaproteobacteria. So
far, only one species, Tepidimicrobium ferriphilum (Slobodkin
et al., 2006), isolated from a hot spring, was described as an
iron-reducing thermophilic prokaryote within the order
Clostridiales. The genus Caloranaerobacter falls into the cluster
XII of the Clostridium subphylum. At the time of writing, this
genus comprises only one species of hydrothermal origin,
Caloranaerobacter azorensis, which was isolated from the MidAtlantic Ridge (Wery et al., 2001b). In this paper, we describe
an anaerobic, thermophilic, Fe(III)-reducing micro-organism, strain DY22619T, isolated from hydrothermal sulfide
deposits, and characterized as representing a novel species of
the genus Caloranaerobacter.
In July 2011, during the DY125-22 oceanographic cruise
onboard the R/V Da Yang Yi Hao, fragments of hydrothermal
sulfide deposits were collected at a depth of 2901 m on the
East Pacific Rise (102.6u W 3.1u S). Samples were collected
using a benthic seabed grab, stored hermetically in sealed
sterile vials, and transported at 4 uC to the laboratory. X-ray
diffraction analysis indicated that these samples were mainly
composed of pyrite (FeS2) and sphalerite (ZnS). One
subsample was used to inoculate (at 1/10th w/v) a sterile
liquid medium referenced as FRPFO, prepared anaerobically
and kept under an atmosphere of highly purified 100 %
nitrogen. The FRPFO medium contained (concentrations in
g l21, unless stated otherwise): peptone, 10; sea salts (Sigma),
30; PIPES, 6.05; cysteine-HCl, 0.5; resazurin, 1 mg; and
amorphous Fe(III) oxyhydroxide (pH 7.0), 50 mmol as an
electron acceptor. Enrichment cultures were incubated at
60 uC. Between 4 to 7 days of incubation, the colour of the
precipitates changed from brown to black, indicating Fe(III)
reduction. The population was composed of motile, long and
short rods. One strain, designated DY22619T, was unable to
form colonies in medium solidified by 1.5 % agar or 0.2 %
Gelrite, and was purified by three repeated dilution-toextinction series. The purity of this isolate was confirmed
routinely by microscopic examination and by cloning and
sequencing of ten 16S rRNA gene clones. Stock cultures were
stored at –80 uC in FRPFO medium supplemented with 5 %
(v/v) DMSO.
Morphological characteristics of cells of the novel isolate were
determined by using light microscopy (CX21; Olympus) and
transmission electron microscopy (JEM-1230 and JEM2100;
JEOL). Cells of strain DY22619T were motile, round-ended
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long rods of 2–10 mm in length and 0.5 mm in width, bearing
flagella. Most cells were 2–6 mm in length and a few were
10 mm in length (Fig. S1a, available in the online Supplementary Material), which are longer than cells of Caloranaerobacter azorensis MV1087T (0.5–2 mm in length). Strain
DY22619T was motile by means of multiple flagella, stained
Gram-negative (confirmed by conventional Gram staining
and KOH test) and divided with a pinching mechanism.
Electron microscopy of ultra-thin sections of cells of strain
DY22619T revealed the presence of two layers characteristic
of Gram-negative bacteria (Fig. S1b). Spores were never
observed, even after heat stimulation.
Physiological characterization of strain DY22619T was
carried out in YTG medium dispensed anaerobically in
50 ml vials sealed with butyl-rubber stoppers, reduced with
0.1 ml of a 10 % (w/v) Na2S . 9H2O sterile solution, just
before inoculation, as described previously (Wery et al.,
2001a). Unless stated otherwise, experiments were carried
out anaerobically, under an atmosphere of N2 (100 %,
1 bar), and incubation was performed in the dark at 60 uC
and pH 7.0. Growth was routinely monitored by direct
cell counting using a modified Thoma chamber (depth
0.02 mm), or by counting after fixation with 1 % (v/v)
glutaraldehyde and storage at –20 uC. All conditions were
tested in triplicate. Growth rates were calculated using
linear regression analysis of eight to ten points along the
linear portions of the log-transformed growth curves.
Determination of the temperature range for growth was
investigated over the range 30–75 uC (at 30, 37, 40, 45, 50,
55, 60, 65, 70 and 75 uC). Growth was observed from 40 to
70 uC and the optimum temperature for growth was 60 uC.
The pH range for growth was tested from initial pH 4.0 to
initial pH 10.0, at 60 uC, in medium buffered and adjusted
to the required pH (initial pH at 20 uC) with MES buffer
(pH 4.0–6.0), PIPES buffer (pH 7.0–8.0), HEPES buffer
(pH 8.0–9.0) or AMPSO buffer (pH 9.0–10.0). Strain
DY22619T grew from pH 4.5 to pH 8.5 and the optimum
pH for growth was 7.0. Salt tolerance was tested at 60 uC in
YTG medium prepared with various concentrations of
NaCl (0–10 % w/v, 0.5 % intervals) or various concentrations of sea salts (0–15 % w/v, 0.5 % intervals). Growth was
observed with 1–10 % (w/v) sea salts (Sigma) and with 1.5–
5.0 % (w/v) NaCl. The optimum salt concentration was
3.0 % for sea salts and 2.5 % for NaCl. Strain DY22619T
was unable to grow without salt, but is not strictly
halophilic. Under optimal growth conditions, the shortest
generation time was 55 min.
Among the dissimilatory Fe(III)-reducing bacteria, strain
DY22619T can be classified in the ‘fermentative’ group, which
use Fe(III) reduction as a minor pathway for electron flow
while fermenting sugars or amino acids to a mixture of
volatile fatty acids (acetate, butyrate) and hydrogen. Strain
DY22619T was obligately chemoorganoheterotrophic, utilizing complex organic compounds including peptone,
tryptone, beef extract and yeast extract. The ability of the
isolate to use single carbon sources for growth was tested in
triplicate, under optimal growth conditions, by using Biolog
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X. Zeng and others
AN plates in the anaerobic jar according to the manufacturer’s instructions. Strain DY22619T was able to utilize
amino acids (including alanine, glutamate, methionine and
threonine), carbohydrates (including fructose, mannose,
galactose, glucose, palatinose, rhamnose, turanose, gentiobiose, xylose and sorbose) and organic acids (including
pyruvate, tartaric acid, a-ketobutyric acid, a-ketovaleric acid,
galacturonic acid and glucosaminic acid). Methanol, ethanol,
mannitol, formic acid, acetic acid, maltose, cellobiose and
sucrose were not used. Strain DY22619T was not capable of
chemoautotrophic growth in a H2/CO2 gas atmosphere.
The ability of the novel isolate to use electron acceptors was
tested by adding sulfite (1 mM), thiosulfate (20 mM),
nitrate (10 mM), MnO2(20 mM), Fe(III) oxyhydroxide
(pH 7.0; 50 mM), amorphous iron(III) oxide (pH 9.0;
50 mM), goethite (a-FeOOH, pH 12.0; 50 mM); Fe(III)
citrate (20 mM), Fe(III) chlorite (20 mM), EDTAFe(III)(20 mM) or oxygen (0.05–0.5 % v/v) to the
medium, without inoculation as a control. Various forms
of Fe(III) were synthesized by using modifications of
previously described methods (Lovley & Phillips, 1986a).
Strain DY22619T was found to be strictly anaerobic and
was able to grow only by fermentation. It was facultatively
dependent on various forms of Fe(III), including insoluble
forms such as amorphous Fe(III) oxyhydroxide (pH 7.0),
amorphous iron(III) oxide (pH 9.0), goethite (a-FeOOH,
pH 12.0), and soluble forms such as Fe(III) citrate, Fe(III)
chlorite, EDTA-Fe(III). 9,10-anthraquinone-2,6-disulfonate (AQDS; 5 mM) could be used as an electron shuttle
in Fe(III) respiration. Reduced Fe(II) was measured by
recording the accumulation of HCl-soluble Fe(II) over
time with ferrozine (Lovley & Phillips, 1986b). The
maximum concentration of reduced Fe(II) could reach
12.32 mM in the medium when strain DY22619T reached
the stationary phase, in medium supplemented with
amorphous iron(III) oxide (pH 9.0) as a terminal electron
acceptor. The Fe(III) reduction capability of strain
DY22619T seems higher than that of Caloranaerobacter
azorensis (4.12 mM). Various forms of Fe(III) did not
stimulate the growth of strain DY22619T, which may be
reduced as a minor pathway for electron flow. Strain
DY22619T also could reduce MnO2 to Mn(II). Strain
DY22619T did not reduce sulfite, sulfate, thiosulfate or
nitrate.
Determination of the whole-cell fatty acid composition
was performed on cultures of strain DY22619T and
Caloranaerobacter azorensis MV1087T grown at 60 uC on
YTG medium. YTG medium contained (g l21 unless
otherwise stated): yeast extract, 1; peptone, 1; glucose, 2.5;
artificial sea salts, 30; PIPES, 6.05; Wolf’s vitamin solution,
0.5 ml; Wolf’s trace elements solution, 5 ml; cysteine-HCl,
0.5; and resazurin, 1 mg. Cells were harvested at the end of
the exponential phase of growth (36 h of incubation). Fatty
acids were extracted and analysed following the instructions of the Microbial Identification System operating
manual (MIDI). Fatty acids in strain DY22619T comprised
three main species: iso-C15 : 0 (40.05 %), C14 : 0 (12.44 %)
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and iso-C14 : 0 3-OH (11.66 %). This differed from the fatty
acid profile of the closest relative Caloranaerobacter
azorensis MV1087T, which included principally iso-C15 : 0
(44.67 %) and iso-C14 : 0 3-OH (21.38 %). The detailed fatty
acid profiles of strain DY22619T and Caloranaerobacter
azorensis MV1087T are given in Table S1.
The G+C content of the chromosomal DNA was
determined according to the methods described using
reverse-phase HPLC (Mesbah & Whitman, 1989). The
DNA G+C content of the novel isolate DY22619T was
29.0 mol%, which was similar to the DNA G+C content
of Caloranaerobacter azorensis MV1087T (27.0 %). An
almost-complete 16S rRNA gene sequence (1471 nt) of
strain DY22619T was determined by double-strand sequencing and was deposited in the public database. The
identification of phylogenetic neighbours was initially
carried out using BLAST (Altschul et al., 1997) and
megaBLAST (Zhang et al., 2000) against the database of
type strains with validly published prokaryotic names
(Chun et al., 2007). A search of most similar 16S rRNA
gene sequences was also done against the web-based
EzTaxon-e Server (Kim et al., 2012). The 16S rRNA gene
sequence of strain DY22619T was most similar to that of
Caloranaerobacter azorensis MV1087T, with 97.41 %
sequence similarity. Comparisons of 16S rRNA gene
sequences with those of other members of the
‘Clostridia’, showed that strain DY22619T shared only
92.70 % similarity with Brassicibacter mesophilus BMT,
92.39 % with Clostridium purinilyticum DSM 1384T,
92.16 % with Sporosalibacterium faouarense SOL3f37T and
91.97 % with Thermohalobacter berrensis CTT3T. A phylogenetic tree of the representative members of cluster XII of
the Clostridium subphylum was reconstructed from 16S
rRNA gene sequences using 1374 conserved gene sequence
positions (Fig. 1). Alignment of all sequences was
performed using the software CLUSTAL X (version 2.3) and
the phylogenetic trees were reconstructed using the
neighbour-joining method with the software MEGA (version
5.1). Bootstrap analysis was performed with 1000 replications to provide confidence estimates for tree topologies.
These results indicate that strain DY22619T robustly
branches with Caloranaerobacter azorensis MV1087T.
Based on the above analyses, strain DY22619T could be
assigned to the genus Caloranaerobacter, a member of
cluster XII of the Clostridium subphylum, within the
Gram-positive bacteria (Fig. 1). The 16S rRNA gene
sequence similarity value between strain DY22619T and
the type strain of Caloranaerobacter azorensis was wellbelow the threshold value (98.7–99 %) currently recommended as the need for DNA–DNA hybridization to test
for the genomic uniqueness of a novel species
(Stackebrandt & Ebers, 2006). Consequently, the novel
isolate displays sufficient molecular differences for delineation at the species level.
In summary, strain DY22619T shared many physiological
and chemotaxonomic characteristics with its closest phylogenetic relative, Caloranaerobacter azorensis. Nevertheless, it
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Caloranaerobacter ferrireducens sp. nov.
96
100
Caloranaerobacter azorensis MV1087T (AJ272422)
Caloranaerobacter ferrireducens DY22619T (KC794016)
56
0.01
70
88
Thermohalobacter berrensis CTT3T (AF113543)
74
Clostridiisalibacter paucivorans 37HS60T (EF026082)
Sporosalibacterium faouarense SOL3f37T (EU567322)
Brassicibacter mesophilus BMT (GU645013)
Proteiniborus ethanoligenes GWT (EF116488)
99
52
Alkaliphilus peptidifermentans Z-7036T (EF382660)
Alkaliphilus transvaalensis SAGM1T (AB037677)
100
Eubacterium angustum ATCC 43737T (L34612)
40
87
74
Clostridium acidurici ATCC 7906T (M59084)
81
Clostridium purinilyticum DSM 1384T (FR749894)
Sporanaerobacter acetigenes DSM 13106T (AF358114)
Tepidimicrobium ferriphilum SB91T (AY656718)
93
100
Tepidimicrobium xylanilyticum PML14T (EF522948)
Tissierella creatinini DSM 9508T (FR749955)
Peptostreptococcus hydrogenalis JCM 7635T (D14140)
Peptostreptococcus prevotii ATCC 9321T (D14153)
Fig. 1. Phylogenetic dendrogram obtained by neighbour-joining analysis based on 16S rRNA gene sequences (1374 bp,
omitting unaligned regions), showing the position of strain DY22619T and related strains within cluster XII of the Clostridium
subphylum of Gram-positive bacteria. The genus Peptostreptococcus was used as an outgroup. Bar, expected number of
changes per sequence position.
can be distinguished from Caloranaerobacter azorensis by its
clear phylogenetic distance, cell morphology, fatty acid
profile, doubling time under optimal growth conditions
and different use of soluble iron compounds as electron
acceptors (Table 1).
Therefore, based on the data presented and far phylogenetic distance with closest relatives (98.65 % 16S rRNA gene
sequence similarity can be used as the threshold for
differentiating two species; Kim et al., 2014), we suggest
that strain DY22619T represents a novel species of the
genus Caloranaerobacter, for which the name Caloranaerobacter ferrireducens sp. nov. is proposed.
Description of Caloranaerobacter ferrireducens
sp. nov.
Caloranaerobacter ferrireducens [fer.ri.re.du9cens. L. n.
ferrum iron; L. part. adj. reducens converting to a different
state; N.L. part. adj. ferrireducens reducing iron (III)].
Table 1. Characteristics that differentiate strain DY22619T from its closest relative Caloranaerobacter azorensis MV1087T
Strains: 1, DY22619T (data from this study); 2, Caloranaerobacter azorensis MV1087T (Wery et al., 2001b); +, Positive; 2, negative.
Characteristic
Origin
Cell diameter (mm)
Gram stain
NaCl range (optimum) (g l21)
Temperature range (optimum) (uC)
pH range (optimum)
DNA G+C content (mol%)
Doubling time (min)
Electron acceptors
Sulfur
Fe(III) chlorite
Fe(III) citrate
EDTA-Fe(III)
1
2
Hydrothermal vent from the East Pacific Rise
2.0–10.060.5
2
15.0–50.0 (25.0)
40.0–70.0 (60.0)
4.5–8.5 (7.0)
29.0
55
Hydrothermal vent from the Mid-Atlantic ridge
0.5–2.060.3–0.5
2
6.5–65.0 (20.0)
45.0–65.0 (65.0)
5.5–9.0 (7.0)
27.0
15
2
+
+
+
+
2*
2*
2*
*Data from this study.
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X. Zeng and others
Cells are motile, round-ended rods (2–10 mm in length,
0.5 mm in width) with flagellum. Endospores are never
observed. Cells grow at 37–70 uC (optimum 60 uC), at
pH 4.5–8.5 (optimum pH 7.0) and with 10–100 g l21 sea
salts (optimum 30 g l21). Doubling time is 55 min under
optimal growth conditions. Strictly anaerobic and obligately
chemoorganoheterotrophic. Can utilize complex organic
compounds, amino acids, sugars, and organic acids including
peptone, tryptone, beef extract, yeast extract, alanine,
glutamate, methionine, threonine, fructose, mannose, galactose, glucose, palatinose, rhamnose, turanose, gentiobiose,
xylose, sorbose, pyruvate, tartaric acid, a-ketobutyric acid, aketovaleric acid, galacturonic acid and glucosaminic acid.
Insoluble and soluble formed Fe(III) chemicals, including
amorphous Fe(III) oxyhydroxide (pH 7.0), amorphous
iron(III) oxide (pH 9.0), goethite (a-FeOOH, pH 12.0),
Fe(III) citrate, Fe(III) chlorite and EDTA-Fe(III) can be
reduced. Does not reduce sulfite, sulfate, thiosulfate or nitrate.
T
T
T
The type strain, DY22619 (5JCM 19467 5DSM 27799 5
MCCC1A06455T), was isolated from a hydrothermal sulfide
sample collected from an East Pacific Ocean hydrothermal
field (102.6u W 3.1u S) at a depth of 2901 m. The DNA G+C
content of the type strain is 29.0 mol%.
other metals. In Advances in Agronomy vol. 54, pp. 175–231. Edited by
D. L. Sparks. San Diego, CA: Academic Press.
Lovley, D. R. & Phillips, E. J. P. (1986a). Availability of ferric iron for
microbial reduction in bottom sediments of the freshwater tidal
potomac river. Appl Environ Microbiol 52, 751–757.
Lovley, D. R. & Phillips, E. J. P. (1986b). Organic matter
mineralization with reduction of ferric iron in anaerobic sediments.
Appl Environ Microbiol 51, 683–689.
Lovley, D. R., Coates, J. D., Saffarini, D. & Lonergan, D. J. (1997).
Diversity of dissimilatory Fe(III)-reducing bacteria. In Iron and
Related Transition Metals in Microbial Metabolism, pp. 187–215.
Edited by G. Winkelman & C. J. Carrano. Chur, Switzerland:
Harwood Academic Publishers.
Mesbah, M. & Whitman, W. B. (1989). Measurement of deoxyguanosine/thymidine ratios in complex mixtures by high-performance
liquid chromatography for determination of the mole percentage
guanine+cytosine of DNA. J Chromatogr A 479, 297–306.
Miroshnichenko, M. L., Slobodkin, A. I., Kostrikina, N. A., L’Haridon,
S., Nercessian, O., Spring, S., Stackebrandt, E., BonchOsmolovskaya, E. A. & Jeanthon, C. (2003). Deferribacter abyssi sp.
nov., an anaerobic thermophile from deep-sea hydrothermal vents of
the Mid-Atlantic Ridge. Int J Syst Evol Microbiol 53, 1637–1641.
Slobodkin, A. I., Campbell, B., Cary, S. C., Bonch-Osmolovskaya,
E. A. & Jeanthon, C. (2001). Evidence for the presence of
thermophilic Fe(III)-reducing microorganisms in deep-sea hydrothermal vents at 13u N (East Pacific Rise). FEMS Microbiol Ecol 36,
235–243.
Acknowledgements
We are very grateful to the R/V ‘Da-Yang Yi-Hao’ operation teams for
helping us to collect the samples from the deep-sea hydrothermal vent
field. This work was supported by the National Program on Key Basic
Research Project (973 Program, no.2012CB417304), the National
High Technology Research and Development Program of China (863
Program, no. 2012AA092102), the fund of National Infrastructure
of Microbial Resources (no. NIMR2014-9), the EU FP7 program
MaCuMBA (grant agreement no. 311975.), the PICS-CNRS-INEE
Phypress and the PHC Cai YuanPei 30412WG.
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