1 2 3 4 Methanogenic archaea isolated from the Taiwan Chelungpu fault 5 6 Sue-Yao Wu and Mei ...

35 Pages·2010·0.46 MB·English

AEM Accepts, published online ahead of print on 10 December 2010
Appl. Environ. Microbiol. doi:10.1128/AEM.01539-10
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1
2
3
4
5 Methanogenic archaea isolated from the Taiwan Chelungpu fault
6
D
o
w
7 Sue-Yao Wu and Mei-Chin Lai*
n
lo
a
8 Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, R.O.C. d
e
d
9 fr
o
m

h
10 t
t
p
:
/
/
11 *For correspondence. E-mail: [email protected]; Tel: [phone]; Fax a
e
m
12 (+ 886) 4-2287 4740. .
a
s
m
13 .
o
r
g
14 Running Title: Methanoarchaea from Chelungpu fault /
o
15 n

J
a
16 Key words: Subsurface microbiology, Archaea, Methanogen, Methanobacterium, n
u
a
r
17 Methanolobus, Chelungpu Fault. y

4
,

2
0
1
9

b
y

g
u
e
s
t
1
1 ABSTRACT
2 Terrestrial rocks, petroleum reservoirs, faults, coal seams, and subseafloor gas hydrates
3 contain an abundance of diverse methanoarchaea. However, reports on the isolation,
4 purification and characterization of methanoarchaea in the subsurface environment are rare.
5 Currently, no studies investigating methanoarchaea within fault environments exist. In this
6 report, we succeeded in obtaining two new methanogen isolates, Methanolobus
D
o
7 chelungpuianus St545MbT and Methanobacterium palustre FG694aF, from the Chelungpu w
n
lo
a
8 fault, which is the fault that caused a devastating earthquake in central Taiwan in 1999. Strain d
e
d
9 FG694aF was isolated from a fault gouge sample obtained at 694 meter below land surface f
r
o
m
10 (mbls) and is an autotrophic, mesophilic, nonmotile, thin, filamentous-rod shaped organism
h
t
t
p
11 capable of using H2/CO2 and formate as substrates for methanogenesis. The morphological, :/
/
a
e
12 biochemical and physiological characteristics and 16S rRNA gene sequence analysis revealed m
.
a
13 that this isolate belongs to Methanobacterium palustre. The mesophilic strain St545MbT, s
m
.
o
14 isolated from a sandstone sample at 545 mbls is a nonmotile, irregular coccoid organism that r
g
/

o
15 uses methanol and trimethylamine as substrates for methanogenesis. The 16S rRNA gene n

J
a
16 sequence of strain St545MbT was 99.0% similar to Methanolobus psychrophilus strain R15 n
u
a
r
17 and was 96-97.5% similar to the other Methanolobus species. However, the optimal growth y

4
,
18 temperature and total cell protein profile of strain St545MbT were different from M. 2
0
1
9
19 psychrophilus strain R15, and whole genome DNA-DNA hybridization revealed less than
b
y

20 20% relatedness between these two strains. On the basis of these observations, we propose g
u
e
s
21 that strain St545MbT (DSM 19953T; BBRC AR10030; JCM15190) be named Methanolobus t
22 chelungpuianus sp. nov. Moreover, the environmental DNA database survey indicates that
23 both Methanolobus chelungpuianus and Methanobacterium palustre are widespread in the
24 subsurface environment.
25
2
1 INTRODUCTION
2 The strict anaerobic, methane-producing archaea exhibit marked habitat diversity. They
3 have been isolated from virtually every habitat in which anaerobic biodegradation of organic
4 compounds occurs, including freshwater and marine sediments, digestive and intestinal tracts
5 of animals, and anaerobic waste digesters (7,14,22,68). Methanogens have also been isolated
6 from extreme environments such as geothermal springs, both shallow and deep-sea
D
o
w
7 hydrothermal vents, psychrophilic waters of Antarctica, and salty solar salterns
n
lo
a
8 (6,18,21,43,57). d
e
d
9 Subsurface ecosystems have been postulated to possess more than 70% of the Earth’s f
r
o
m
10 biomass (67). Evidence suggests that viable methanogenic archaea are present deep
h
t
t
p
11 underground in rocks and surrounding ground waters (49). In this habitat, methanogens may :
/
/
a
e
12 represent chemoautolithotrophic organisms that initiate food chains in the oligotrophic deep m
.
a
13 subsurface environment at the expense of geologically-produced molecular hydrogen. s
m
.
o
14 Although methanoarchaea comprise a major group of microorganisms that live deep r
g
/

o
15 underground, few have been characterized. The first methanogen isolated from the deep n

J
a
16 subterranean biosphere was Methanobacterium subterraneum, which was isolated from deep n
u
a
r
17 granitic groundwater at depths of 68, 409 and 420 m (30). PCR-amplified 16S rRNA gene y

4
,

18 sequence studies have indicated that Methanobacterium spp. dominates in the four to five 2
0
1
9
19 kilometer deep fault at Drietontein Consolidated Mine in Johannesburg, South Africa (42).
b
y

20 Investigations of microbial communities have used 16S rRNA gene sequence analysis to g
u
e
s
21 characterize archaea in the deep subsurface such as terrestrials rocks (50,59), petroleum t
22 reservoirs (9,10,20,44,45,47,65,56), faults (42,54,64), coal seams (13,53), gas field (41) and
23 subseafloor gas hydrates (19,36,40). These studies have demonstrated the abundance and
24 diversity of methanoarchaea. However, reports of the isolation, purification and
25 characterization of methanoarchaea in the subsurface environment are rare. Currently, no
3
1 studies investigating methanoarchaea within fault environments exist.
2 Taiwan is located at a boundary between the Philippine Sea Plate and the Eurasian Plate.
3 Continuous convergence between these two plates has resulted in an arc-continent collision
4 that further induced the uplift of the Eocene to Pleistocene strata above the sea level along a
5 series of east-dipping thrusts since 5 Ma (60). On September 21, 1999, a magnitude 7.7
6 Chi-Chi earthquake devastated central Taiwan. The severity of this earthquake triggered the
D
o
w
7 geo-structural survey in the faults of Taiwan, and the initiation of the Taiwan Chelungpu-fault
n
lo
a
8 Drilling Project (TCDP, details in www.idsp-online.de/sites/chelungpu/ news/news.html). d
e
d
9 Coring was used to retrieve samples from an active fault zone that triggered the Chi-Chi f
r
o
m
10 earthquake (63). The coring penetrated through Cholan, Chinshui, and Kueichulin formations
h
t
t
p
11 to a depth of 2000 mbls. These rock formations are mainly composed of sandstone and shale :
/
/
a
e
12 in different proportions. Two major fracture zones were encountered: one located within the m
.
a
13 Chinshui formation at around 1110 mbls and the other at the boundary between the s
m
.
o
14 Kueichulin formation and the underlying Cholan formation at around 1750 mbls. At depths r
g
/

o
15 greater than 1750 mbls, the Cholan formation was displaced underneath the Kueichulin n

J
a
16 formation by overthrusting along the deeper fracture zone (64). n
u
a
r
17 This drilling project provided a great opportunity to investigate subsurface microbiology. y

4
,

18 In this report, we succeeded in obtaining two new methanogen isolates, Methanolobus 2
0
1
19 chelungpuianus St545MbT (DSM 19953; JCM 15190; BCRC AR10030) and 9
b
y

20 Methanobacterium palustre FG694aF (JCM 15160; BCRC AR10031), from the Chelungpu g
u
e
s
21 fault. t
4
1 MATERIALS AND METHODS
2 Sample Core Handling. Coring was performed with a diamond-coated hollow bit with an
3 inner diameter of 8.3 cm (64). Drilling fluids consisted of water pumped from an adjacent
4 river, bentonite and barite (BaSO4) was used to reduce heat, remove rock fragments generated
5 during pulverization, and prevent groundwater intrusion. Rhodamine dye was added to the
6 drilling fluid at a concentration of 50 ppm (w/v) to monitor contamination introduced during
D
o
w
7 the coring (64). Each core section, with a maximum length of around three meters, was
n
lo
a
8 brought to the surface using a wireless core catcher. Intact cores without lithological d
e
d
9 interclasts or structural features were chosen and sectioned into a length of 20 to 30 cm for f
r
o
m
10 geomicrobiological study and prepared by Dr. Pei-Ling Wang at the Institute of Oceanography,
h
t
t
p
11 National Taiwan University. The sectioned core was anaerobically handled and immediately :
/
/
a
e
12 transported into an anaerobic glove chamber within three hours (64). Anaerobic samples from m
.
a
13 the central part of the cores from depths ranging from 545 mbls to 1675 mbls were trimmed s
m
.
o
14 and prepared inside a Coy anaerobic chamber by Dr. Pei-Ling Wang; these samples were r
g
/

o
15 given to us for further enrichment, isolation and characterization. n

J
a
16 Media and culture techniques. The modified anaerobic technique of Hungate was utilized n
u
a
r
17 (1,56). Sterilized media were prepared in an oxygen-free N :CO (4:1) atmosphere. The MB y
2 2
4
,

18 medium (pH 7.0) consisted of deionized with MgCl .6H O (1.0 g/L), KCl (0.5 g/L), NaCl (5 2
2 2 0
1
9
19 g/L), CaCl2.2H2O (0.1 g/L), K2HPO4 (0.4 g/L), NH4Cl (1.0 g/L), cysteine.HCl (0.25 g/L), b
y

20 NaHCO (4.0 g/L), yeast extract (2 g/L), tryptone (2 g/L) and resazurin (0.001 g/L). Vitamin g
3 u
e
s
21 and trace element solutions without tungstate were each added to a final concentration of 1% t
22 (vol/vol) (35). An MB/W medium was prepared by prepared by adding a 1% (vol/vol) trace
23 element solution containing tungstate (Na WO , 0.3 mg/l) to MB medium (35). MM medium
2 4
24 was prepared using MB medium without the addition of yeast extract and tryptone. All of the
25 constituents except sulfide were dissolved in water, boiled and cooled under an oxygen-free
5
1 atmosphere of N :CO (4:1). The medium was distributed to serum bottles (Wheaton
2 2
2 Scientific, Millville, NJ) or Hungate tubes (Belleco Glass, Inc., Vineland, NJ) under the same
3 atmosphere. The anaerobic tubes were then sealed and autoclaved at 121 ºC for 20 min.
4 Sodium sulfide from a sterilized anoxic stock solution was added to a final concentration of 1
5 mM before inoculation. For solid roll tube medium, 20 g/l of agar was added. To measure the
6 effect of pH on growth, the ratio of N to CO in the gas phase and the concentration of
2 2 D
o
w
7 NaHCO in the medium were modified to obtain pH values between 5.6 and 8.3.
3 n
lo
a
8 Enrichment and Isolation. Enrichment was started immediately after the sample was d
e
d
9 brought to our laboratory. In an anaerobic chamber, the powdered core samples were added to f
r
o
m
10 160-ml serum bottles that contained 45 ml of MB/W or MM/W medium with sodium formate
h
t
t
p
11 (50 mM) plus acetate (20 mM), acetate (50 mM) or methanol (50 mM), respectively, as the :
/
/
a
e
12 catabolic substrates. These enrichment cultures were incubated at 37 and 45 ºC for one month m
.
a
13 (Supplementary Table S1). Methane production was determined by Gas chromatography with s
m
.
o
14 a flame-ionization detector (32). Trace amounts of methane were detected in most of r
g
/

o
15 enrichment after subtransfers with a 1:1 ratio and incubated at 37 ºC for an additional month. n

J
a
16 Among them, enrichments of sample St545, from sandstone from a depth of 545 m, with n
u
a
r
17 methanol as a substrate, and sample FG694, from a fault gouge from a depth of 694 m, with y

4
,

18 formate plus acetate as a substrate, accumulated the highest amounts of methane. These two 2
0
1
9
19 cultures were further subtransferred (1:20) into MB/W medium in Hungate tubes with the
b
y

20 same substrate and vancomycin (an antibiotic to inhibit bacteria). This procedure was g
u
e
s
21 repeated for four successive transfers. The culture was then diluted and transferred into t
22 molten MB agar, and the roll tube technique was performed. Colonies grew on the inner wall
23 of the glass tube after two to three weeks. Colonies were picked with disposable, sterilized
24 inoculation needles in a Coy anaerobic chamber and transferred to anaerobic tubes containing
25 5 ml of MB/W medium. Cultures from a single colony were further incubated at 37 ºC for 1-2
6
1 weeks. Two methane-producing cultures, St545Mb and FG694aF, derived from two
2 morphologically distinct colonies, were further purified with repeated serial dilutions with
3 vancomycin until they were free of contamination with non-methanogenic bacteria. The
4 axenic nature of the culture was confirmed by microscopic examination, the presence of
5 single colony type in roll tubes, and the absence of growth in anaerobically prepared Bacto
6 Thioglycollate medium.
D
o
w
7 Determination of catabolic substrates. The catabolic substrates tested under N :CO (4:1)
2 2 n
lo
a
8 were sodium formate (100 mM), sodium acetate (50 mM), trimethylamine (40 mM), d
e
d
9 dimethylamine (111 mM), methanol (50 mM), ethanol (48 mM), 2-propanol (48 mM), f
r
o
m
10 iso-butanol (48 mM), 2-butanol (48 mM), methylamine (50 mM) and methyl sulfide (50 mM).
h
t
t
p
11 H2 was tested by pressurizing the culture tubes with H2 (100%, 200 kPa). Utilization of these :/
/
a
e
12 substrates was determined in MB/W media by monitoring methane production. Methane m
.
a
13 production was determined by gas chromatography with flame ionization detection (32). s
m
.
o
14 Antibiotic susceptibility. Sensitivity to ampicillin, penicillin, spectinomycin, kanamycin, r
g
/

o
15 tetracycline and chloramphenicol (100 µg/ml of each) was tested in MB/W medium with n

J
a
16 sodium formate (100 mM) at 37 ºC. Growth was determined by methane production. n
u
a
r
17 Determination of growth rates. Specific growth rates were calculated from methane y

4
,

18 production, which was analyzed by linear regression of the logarithm of the total amount of 2
0
1
9
19 the methane that accumulated with time (32,51). Inocula were grown under conditions similar
b
y

g
20 to the experimental conditions. u
e
s
21 Microscopy. An Olympus BH-2 microscope was used for phase-contrast microscopy. t
22 Preparations for negative staining were performed as described previously (34). Electron
23 micrographs were obtained using model JEM-1200EXII and 200cx (Joel, Ltd.).
24 Whole-cell proteins profile. The mid-loge phase cells of Methanolobus chelungpuianus
25 strain St545MbT, Methanolobus psychrophilus strain R15, Methanolobus vulcani and
7
1 Methanohalophilus portucalensis strain FDF1T, were harvested and resuspended in loading
2 buffer containing 4% sodium dodecyl sulfate at a ratio of 1 ml buffer per OD unit. An OD
3 unit was the amount of cells found in 1 ml of culture with an absorbance of 1.0. SDS-PAGE
4 was performed as described by Laemmli (31). Gels were stained with Coomassie blue R-250.
5 Phylogenetic analysis. Chromosomal DNA preparations, PCR amplification of 16S rRNA
6 genes and sequencing were performed as described by Wu et al (70). PCR amplification
D
o
w
7 primers specific for methanogen were used as forward primer coccus 1
n
lo
a
8 (5’-CGACTAAGCCATGCGAGTC-3’) and reverse primer reverse 3 (5’-GTGACGGGCGGT d
e
d
9 GTGTGCAAG-3’). The sequences were corresponded to positions 2-20 and 1309-1329 in the f
r
o
m
10 16S rRNA nucleotide sequence of Methanofollis formosanus strain ML15T (AY186542). A
h
t
t
p
11 single PCR product of the expected size was detected from both cultures. The estimated :
/
/
a
e
12 1300-bp amplified fragments were further purified, cloned into pGEM-T easy vector and m
.
a
13 sequenced. The 1309-bp rRNA gene sequence from strain St545Mb (positions 44-1348 in the s
m
.
o
14 16S rRNA nucleotide sequence of Methanolobus psychrophilus strain R15) and the 1264-bp r
g
/

o
15 rRNA gene sequence from strain FG694aF (positions 87-1349 in the 16S rRNA nucleotide n

J
a
16 sequence of Methanobacterium palustre strain F) were obtained and compared with related n
u
a
r
17 methanogens. Gene sequences of the archaea used were obtained from the Ribosomal y

4
,

18 Database Project (RDP) Seqmatch (http://rdp.cme.msu.edu/seqmatch/ seqmatch_intro.jsp) (11) 2
0
1
9
19 and NCBI GenBank (http://blast.ncbi.nlm.nih.gov/GenBank). Multiple sequence alignments
b
y

20 were analyzed using the ClustalW of MEGA4 (http://www.megasoftware.net/) (61), and g
u
e
s
21 phylogenetic trees were created using the neighbor-joining of MEGA4 with Bootstrap Test of t
22 Phylogeny with 500 replicates.
23 DNA-DNA hybridization and DNA G+C content. Cells of strain St545Mb,
24 Methanolobus psychrophilus R15, Methanolobus vulcani DSM3029T and Methanohalophilus
25 portucalensis FDF1T were harvested during the late exponential phase and used for DNA
8
1 isolation as described above. DNA-DNA hybridization experiments were performed by using
2 the dot-blot technique as described previously (35) with a DIG DNA Labeling and Detection
3 Kit (Roche Applied Science, Indianapolis, IN). Four target DNA samples were quantified by
4 A , diluted to 25, 50, 75, and 100 ng/µl and denatured by boiling at 100 ºC for 10 min. Two
260
5 microliters of each denatured DNA sample (50, 100, 150 and 200 ng/µl) was blotted on a
6 Biodyne® Nylon Membrane (Pall Corporation, Port Washington, NY). Two membranes
D
o
w
7 containing the same DNA samples were re-associated with the labeled DNA probes from
n
lo
a
8 strain St545Mb and strain R15 respectively at 45 ºC for overnight. Hybridization signals were d
e
d
9 detected and analyzed by TINA software (Version 2.09e; Raytest Isotopenmeßgeräte). f
r
o
m
10 Triplicate tests were performed for each assay, and self-hybridization of the probe with
h
t
t
p
11 homologous target DNA was set to 100%. :
/
/
a
e
12 The DNA G+C content was determined by high-performance liquid chromatography m
.
a
13 after enzymatic hydrolysis (39) by the Bioresource Collection and Research Center (BCRC) s
m
.
o
14 in the Food Industry Research and Development Institute (FIRDI) at Taiwan. r
g
/

o
15 Nucleotide sequence accession numbers. The 16S rDNA sequences of strain FG694aF n

J
a
16 and strain St545MbT determined in this study have been deposited in the GenBank database n
u
a
r
17 under accession numbers EU293795 and EU293796, respectively. y

4
,

2
0
1
9

b
y

g
u
e
s
t
9
1 RESULTS AND DISCUSSIONS
2 Methanogen enrichment from core samples of the Chelungpu-fault. The Taiwan
3 Chelungpu Drilling Project (TCDP) near Dai-Keng penetrated through the
4 Pliocene-Pleistocene sedimentary rocks to a depth of 2000 mbls and encountered the
5 Chelungpu and San-Yi fault zones (64). Anaerobic samples from the central part of the cores
6 at the depths ranging from 545 mbls to 1675 mbls were trimmed and prepared in a Coy
D
o
w
7 anaerobic chamber. Portions of powdered cores were added into serum bottles with
n
lo
a
8 methanogen basal medium (MB/W) or MM/W medium with sodium formate plus acetate, d
e
d
9 acetate and methanol as the catabolic substrate, respectively. f
r
o
m
10 After one month of incubation at 37 or 45 ºC respectively, trace amounts of methane
h
t
t
p
11 (below 20 µmoles per bottle) were detected in most of the enrichment (Supplementary Table :
/
/
a
e
12 S1). Among these cultures, enrichments of sample St545M, from sandstone from a depth of m
.
a
s
13 545 m using methanol as a substrate, sample FG694F, from a fault gouge from a depth of 694 m
.
o
14 m using formate as a substrate, and sample SS1033F, from siltstone from a depth of 1033 m r
g
/

o
15 using formate plus acetate as a substrate, accumulated the highest amounts of methane (2152, n

J
a
16 1246, and 113 µmole, respectively). These three cultures were further transferred (1:20) into n
u
a
r
y
17 MB/W medium in Hungate tubes with the respective substrate and vancomycin (100 µg/ml)
4
,

2
18 to inhibit bacterial growth. Methane was not detected upon transfer of culture SS1033F. 0
1
9
19 After successive transfers, serial dilution and the roll tube technique (33), a small b
y

g
20 transparent hemispherical colony (< 2 mm) was selected from the enrichment sample u
e
s
t
21 collected from sandstone from a depth of 545 m with methanol as a substrate. Upon
22 inoculation into thioglycollate (TGC) medium, no growth was observed, indicating that the
23 culture was free of anaerobic heterotrophic bacteria (33). This methanoarchaeal isolate (strain
24 St545Mb) was deposited into DSMZ Germany as DSM 19953, JCM Japan as JCM 15190 and
25 BCRC Taiwan as AR10030. Another methane-producing isolate (strain FG694aF) was
10