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Isolation and Characteristics of Methanosaeta in Paddy Field Soils

Article  in  Bioscience Biotechnology and Biochemistry · May 2006

DOI: 10.1271/bbb.70.828 · Source: PubMed

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Biosci. Biotechnol. Biochem., 70 (4), 828–835, 2006
Isolation and Characteristics of Methanosaeta in Paddy Field Soils
Satomi M IZUKAMI,1; y Kiyoshi T AKEDA,2 Shinji A KADA,2 and Takashi F UJITA2
1
National Institute of Advanced Industrial Science and Technology,
Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
2
Hirosaki University, Faculty of Agriculture and Life Science,
Bunkyocho 3, Hirosaki City, Aomori 036-8560, Japan
Received August 25, 2005; Accepted December 5, 2005
The population of filamentous acetate-utilizing
methanogens in paddy field soils was 2:0  104 MPN/g
dry soil in the submerged condition. They were able to
form colonies in a deep agar medium, but not in a roll
tube. Filamentous acetate-utilizing methanogens isolat-
ed from Kanagi, Japan (strain K-5) and Tsukuba, Japan
(strain T-3) were divided into two types based on length
of filaments. One type, strain K-5, formed a short chain
which was dispersed easily by weak shaking. The other
type, strain T-3, formed a long chain, which formed
cotton-like flocs and was not dispersed by weak shaking.
They had sheaths composed of a pair of adjacent
membranes on the outside of the cell membranes.
The 16S rRNA gene similarities of strain T-3 and K-5
to Methanosaeta concilii strain Opfikon were 100% and
99.5% respectively. Filamentous acetate-utilizing meth-
anogens were also isolated from paddy field soils in
various other regions of Japan. Our results suggest that
Methanosaeta is universal in paddy soils and that it
plays an important role in methane production from
acetate.
Key words: Methanosaeta; acetate; paddy field soil;
colony formation
Methane is a major greenhouse gas, ranking second
only to carbon dioxide, and about 20% of methane is
released from paddy fields into the atmosphere.1) One of
the characteristics of paddy field soil is that the supply of
oxygen is limited after submersion, resulting in a
lowering of the redox potential. Therefore organic
matter in flooded paddy field soils is finally decomposed
anaerobically into methane and carbon dioxide. In this
process, acetate is the most important substrate for
methanogenesis, along with carbon dioxide and hydro-
gen. It appears that acetate is the most common organic
acid in paddy field soils, and 50% to 70% of the methane
released into the atmosphere from this environment is
produced from acetate.2,3) Thus acetotrophic methano-
gens play important roles in methanogenesis in paddy
field soils. There are two genera of acetate-utilizing
y
To whom correspondence should be addressed. Fax: +81-29-861-6066; E-mail: samy-mizukami@aist.go.jp
methanogens according to Bergey’s Manual,4) Metha-
nosarcina and Methanosaeta. Methanosarcina has been
isolated from paddy field soils and characterized
(Rajagopal et al.,5) Fetzer et al.,6) Asakawa et al.,7)
and Joulian et al.8)). In Japan, Kudo et al.9) reported that
Methanosaeta was found in paddy fields by PCR
analysis of 16S rRNA genes extracted directly from
soil samples. Grosskopf et al.10) reported that Methano-
saeta was dominant with respect to its archaeal cell
number and was the most important acetoclastic
methanogen in paddy field soils. These studies focused
on not physiology but on molecular biology. Janssen11)
developed a selective enrichment method for Methano-
saeta spp., which competes with Methanosarcina for
acetate in paddy soils using acetone or isopropanol as
the growth substrate. It had a pure culture by dilution
culture. But, there have been few reports on isolation by
colony formation in a solid medium or characterization
of Methanosaeta from paddy field soils. In this paper,
we describe colony formation, cell-morphology, and
phenotypic and phylogenetic characteristics of filamen-
tous acetate-utilizing methanogens isolated from five
paddy field soils. This is the first report of filamentous
acetate-utilizing methanogens isolated from paddy field
soils in Japan.
Materials and Methods
Soil samples. The soils were collected from five
Japanese paddy fields in the following regions: Hako-
date (Hokkaido), Kanagi (Aomori Prefecture), Mobara
(Chiba Prefecture), Tsukuba (Ibaraki Prefecture) and
Hita (Oita Prefecture). The soil samples’ lower 3–15 cm
(the reduced layer) were taken and put in plastic bags to
keep anaerobically. The soil samples’ upper 1 cm (the
oxidized layer) were also used to examine the number of
acetate-utilizing methanogens in paddy field soils.
Media and cultivation conditions. Filamentous ace-
tate-utilizing methanogens from the paddy soils were
cultivated in a mineral salt (MS) medium with sodium
 Isolation and Characteristics of Methanosaeta
acetate as a sole carbon and energy source. The basal
medium had the following composition (g/l): KH2 PO4 ,
.
0.2; NH4 Cl, 0.25; NaCl, 1.0; MgCl2 6H2 O, 0.4; KCl,
.
0.5; CaCl2 2H2 O, 0.15. This medium was sterilized for
15 min at 121
C. Sodium bicarbonate, sodium sulfide, a
trace-element solution, a selenite and tungstate solution,
and vitamin solutions were added to 1 liter of the
autoclaved, cooled basal medium from stock solutions.
NaHCO3 and Na2 S were added to give final concen-
trations of 30 mM and 0.5 mM respectively. The above-
mentioned solutions were: (a) 2 ml of trace-element
solution (mg/l) sterilized by autoclaving: ZnCl2 , 70;
. .
MnCl2 4H2 O, 100; Na2 MoO4 2H2 O, 36; CuCl2 2H2 O, .
. .
17; FeCl2 4H2 O, 1500; CoCl2 6H2 O, 190; H3 BO3 , 62;
.
NiCl2 6H2 O, 24; (b) 1 ml of selenite and tungstate
solution (mg/l) sterilized by autoclaving: Na2 SeO3
.
5H2 O, 3; Na2 WO4 2H2 O, 4; NaOH, 500; (c) 1 ml of B12
solution (50 mg/l) sterilized by autoclaving; and (d) 1 ml
of mixed-vitamin solution (mg/l) by filtration: D(+)-
biotin, 1; 4-aminobenzoic acid, 4; thiaminiumdichloride,
10. Sodium acetate was prepared as a 1 M stock solution,
sterilized by autoclaving, and added to the basal medium
before incubation. The MS medium containing 20 mM
sodium acetate was referred to as an AM medium.
Ampicillin and vancomycin (each at 30 mg) were added
to the MS medium. The gas phase was N2 , and the pH of
the medium was adjusted to between 7.1 and 7.3. Cells
were cultivated in the dark without shaking at 30
C.
Filamentous cells and coccoid or pseudosarcina cells
were observed with a phase-contrast photomicroscope
and a UV-epifluorescence microscope (Olympus model
BX50, Tokyo, Japan).
Enrichment of filamentous acetate-utilizing methano-
gens in paddy soils. Enrichment cultures of filamentous
acetate-utilizing methanogens from the paddy soils were
carried out with 50 mM acetate medium. A total of 1 g of
soil samples plus 100 ml of acetate medium were put
into a 140 ml flask, and the gas in the headspace of
the flask was replaced with N2 . The flasks were closed
with butyl rubber stoppers and sealed with aluminum
cups. They were incubated at 30
C. Cell numbers were
estimated with a microscope using a blood cell counting
chamber after hard shaking in order to disperse the
filamentous cells. The number of filamentous cells was
calculated as length of filament/one cell (2 mm). The
diameter of sarcina cells was 2–4 mm and the length of
filaments was 5–8 mm throughout the whole enrichment
cultivation after shaking. Filamentous methanogens
(width 0.8 mm) that showed weak autofluorescence at
F420 were counted as Methanosaeta-like cells. Coccoid
or pseudosarcina methanogens with strong autofluores-
cence were counted as Methanosarcina-like cells.
Enumeration of bacterial numbers. The numbers of
acetate-utilizing methanogens were counted by the
three-tube most-probable-number (MPN) method using
a 10 fold serial dilution of soil in the AM medium and
829
agar (0.9%, w/v) in an N2 atmosphere. The agar shake
tubes were incubated at 30
C for six months. Colonies
in the tubes in which CH4 was detected were chosen and
observed microscopically. Coccoid or pseudosarcina
cells that showed blue-green autofluorescence were
counted as Methanosarcina-like cells, and filamentous
cells as Methanosaeta-like cells.
Electron microscopy. For thin-section electron mi-
croscopy, cells were placed in a Kellenberger buffer
containing 1% OsO4 to prefix the cells. The cells were
harvested by centrifugation for 15 min at 13,000 rpm and
fixed in the same buffer for 2 h at room temperature, and
then left overnight at 4
C. The fixed samples were
rinsed three times in the buffer, and twice in deionized
. water, and then soaked in 0.5% uranyl acetate in
deionized water for 2 h. The cells were embedded in
small agar blocks and dehydrated in increasing concen-
trations of ethanol, transferred to acetone, and infiltrated
with Spurr’s low-viscosity resin. Thin sections were
obtained with glass knives on an LKB2188 Ultrotome
NOVA (LKB, Broma, Sweden), and poststained with
uranyl acetate and lead citrate. Sections were examined
with an electron microscope (JOEL model JEM-
2000EX, Tokyo, Japan) operated at 80 kV.
Phenotypic and phylogenetic characterization. Using
the MS medium, formate, methanol, acetate (each at
10 mM) and H2 þ CO2 (80:20, 2 kg/cm2 ) were tested as
carbon and energy sources for archaeal growth. The
optimum temperature and pH for growth were deter-
mined using an AM medium. The additional effects
of NaCl, CO2 , and vitamins on growth were also tested
using the AM medium. Nitrogenase activity was tested
by acetylene reduction assay.12) Catalase activity
was tested by the procedures described in Beers and
Sizer.13)
Gas chromatography and high performance liquid
chromatography. CH4 , CO2 , N2 , and H2 were measured
with a gas chromatograph (Shimadzu model GC-8A,
Tokyo, Japan) using a WG-100 column connected to a
thermal conductivity detector. The oven temperature
was 60
C, and Ar was used as the carrier gas.
Ethylene was measured with a gas chromatograph
using a Porapack R column connected to a flame
ionization detector. The oven temperature was 50
C,
and N2 was used as the carrier gas.
The concentration on acetate was determined with a
high performance liquid chromatograph (Shimadzu
model LC-10A) using a Shim-Pack SCR-101H column
connected to an electroconductivity detector. The HPLC
was operated at a flow rate of 0.8 ml/min with a mobile
phase of 4 mM p-toluenesulfonic acid aqueous solution
and a buffer of 16 mM Bis-Tris aqueous solution
containing 4 mM p-toluenesulfonic acid and 0.1 mM
EDTA aqueous solution. The oven temperature was
40
C.
830
Table 1. Populations of Acetate-Utilizing Methanogens in Paddy Field Soils in Kanagi
Soil condition
Reduced layer
Submerged soil
Oxidized layer
Drained soil
DNA extraction. Cells were pelleted by centrifugation
and lysed in 0.5 ml DNA extraction buffer (Tris 100 mM,
EDTA 40 mM, pH 9.0) containing sodium dodecyl
sulfate (SDS) (final concentration, 2.8%) and 2.5 mg
dithiothreitol. Genomic DNA was purified by a proce-
dure described elsewhere,14) and used for determination
of the G þ C content and 16S rRNA genes of the
bacterial DNA. The G þ C content of the bacterial DNA
was measured using a DNA-GC-kit (Yamasa Shyoyu,
Tokyo, Japan).15)
16S rRNA gene analysis. The primer system, A109f
(50 -ACKGCTCAGTAACACGT-30 ) and A934b (50 -
GTGCTCCCCCGCCAATTCCT-30 ), was used based
on the sequences of the methanogen 16S rRNA gene,
obtained from a 16S rRNA gene data base10) to amplify
archaeal 16S rRNA genes 109 through 934, and to
determine the 16S rRNA sequences of strains H-3, K-5,
M-4, T-3 and O-3. PCR experiments were carried out in
50 ml, final volume, with 1 ml of template DNA, 5 ml of
Ampli Taq reaction buffer, 25 pmol of each primer,
10 mmol dNTP, and 1.25 units of Ampli Taq Gold
polymerase (Ampli Taq Gold DNA polymerase; Perkin-
Elmer, Foster City, CA, USA). The samples were
amplified with over 30 cycles under the following
conditions: 95
C for 1 min, 47
C for 1 min, and 72
C
for 1 min. A final extension was carried out for 10 min at
72
C. Amplified DNA fragments generated by PCR
were visualized by electrophoresis on a 0.8% agarose
gel and staining with ethidium bromide. The PCR
products were cloned into the bacterial plasmid vector
pBluescript II KS (+) at the EcoRV site. Sequencing of
the cloned 16S rRNA genes was carried out on an
automated sequencer (Li-Cor model 4000, Lincoln, NE,
USA) following the reaction prepared with a Sequi-
Therm thermostable DNA polymerase kit (Epicentre
Technologies, Madison, WI, USA). The sequence
determination was carried out on three clones to exclude
the possibility of misreading caused by artificial muta-
tion during PCR amplification. Sequences were assem-
bled using the DNAsis program (Hitachi Software
Engineering, Tokyo, Japan). The reference sequence
used in the phylogenetic analysis was obtained directly
from DDBJ under accession nos. S42679 and S42680
(Methanosaeta concilii strain GP6), X16932 (Methano-
saeta concilii strain Opfikon), Y15402 (Methanosaeta
concilii strain VeAc9), and M59146 (Methanosaeta
concilii strain FE).
S. M IZUKAMI et al.
Cell numbers of acetate-utilizing methanogen
(MPN/g d.w.soil)
Filamentous type Coccoid or sarcina type
4
2:0  10 5:2  105
4:5  103 6:1  103
3
2:3  10 1:5  104
Nucleotide sequence accession numbers. The partial
16S rRNA gene sequences obtained in this study for
strains K-5, T-3, M-4, O-1, and H-3 have been deposited
in DDBJ under accession nos. AB212060 through
AB212063, and AB212065.
Results
Enumeration of acetate-utilizing methanogens in
paddy field soils
The numbers of Methanosaeta-like cells and Meth-
anosarcina-like cells in Kanagi paddy field soil were
enumerated by a combination of the agar shake
technique and the MPN method. Filamentous methano-
gens in paddy field soils formed colonies in a deep-agar
medium. The filamentous cells possessed weak auto-
fluorescence peculiar to F420 -containing methanogens,
and the coccoid cells possessed strong fluorescence.
Table 1 shows the numbers of acetate-utilizing meth-
anogens in Kanagi paddy soil under submerged con-
ditions and drained conditions. The number of
Methanosaeta-like cells in the reduced layer under
submerged conditions was 2:0  104 MPN/g dry soil. In
the oxidized layer under submerged conditions and
drained soil, the numbers of Methanosaeta-like cell
were about 1/4 and 1/9 respectively of those of the
reduced layer. On the other hand, the numbers of
Methanosaeta-like cells were about 1/26 and 1/7 of
those of Methanosarcina-like cells in the reduced layer
under submerged conditions and drained soil respec-
tively, though the number of Methanosaeta-like cells
was almost the same as that of Methanosarcina-like
cells in the oxidized layer.
Isolation of filamentous acetate-utilizing methanogens
in paddy field soils
Soil samples were collected from the Kanagi and
Tsukuba paddy field soils in order to isolate filamentous
acetate-utilizing methanogens. The numbers of Meth-
anosaeta-like cells and Methanosarcina-like cells
changed according to the number of times of repeating
enrichments using the acetate medium, as shown in
Fig. 1. The number of Methansarcina-like cells increas-
ed quickly after the first enrichment. When little acetate
was detected in the first enrichment culture, the second
enrichment was kept by the addition of 50 mM acetate.
After the third successive enrichment, however, the
number of Methanosaeta-like cells became much higher
 Isolation and Characteristics of Methanosaeta 831

108

60
Cell number (cells/g soil)
50

Acetate (mM)
107
40

30
106
20

10 Coccoid or sarcina cells

Filamentous cells
105 0 Acetate
0 20 40 60 80 100 120 140

Incubation time (d)

Fig. 1. The Effect of Enrichment Culture with Acetate on Increase in Number of Filamentous Acetate-Utilizing Methanogens.
The soil sample from the Kanagi paddy field was used. Cell numbers were enumerated with a blood-cell counting chamber.

(a) (d)

10 µm 500 nm

(b) (e)

10 µm
1 µm

(c)

10 µm

Fig. 2. Cells of Filamentous Acetate-Utilizing Methanogens Isolated from Paddy Field Soils.
a, Phase-contrast photomicrograph of strain K-5. b, Phase-contrast photomicrograph of strain T-3. c, Epifluorescence photomicrograph of
strain T-3. d, Thin sections of cells of strain K-5 within a filament. ‘‘S’’ shows the sheath and ‘‘M’’ shows the cell membrane. e, Scanning electron
micrograph of strain T-3.
832 S. M IZUKAMI et al.

than that of Methanosarcina-like cells. After five

successive enrichments, white flocs formed at the (a)
bottoms of the vials. The flocs were composed of
filamentous cells. No Methanosarcina-like cells, how-
ever, were observed in these cultures. Filamentous
methanogens formed colonies in a deep-agar medium.
The filamentous cells possessed weak autofluorescence
peculiar to F420 -containing methanogens. The length of
filamentous cells isolated from paddy field soils in 10 µm
Kanagi (strain K-5) was a short chain, from 2 to 45 mm
in length (Fig. 2a). The other strain, T-3, isolated from
Tsukuba, was a long chain over 150 mm long (Fig. 2b).
These strains were gram-negative on staining. A phase-
contrast micrograph (Fig. 2b) and an epifluorescence (b)
micrograph (Fig. 2c) of strain T-3 are shown in Fig. 2.

Cell morphology and colony formation of Methano-

saeta isolated from paddy field soils
Both strains were gram-negative, non-motile, and
filamentous cells which consisted of rods, 0.8 mm  2 to
3 mm with flat ends, as shown in Table 2. No spores 10 µm
were observed. Thin sections revealed that the filamen-
tous methanogens grew as chains of cells within a sheath
to form a filament (Fig. 2d). The sheath layers resided
on the outer side of the double layers of the cell
membrane, as seen in Methanothrix sohengenii16) and (c)
M. concilii.17) The cells were lysed by SDS and
dithiothreitol. Scanning electron microscopy revealed
that these strains had filaments composed of bamboo-
shaped cells (Fig. 2e), characteristic of Methanosaeta.
The short filaments were strains K-5 and a long filament
was strain T-3. The former formed flocs that could be
dispersed by weak shaking. The latter formed cotton-
like flocs that were not dispersed by weak shaking.
Methanosaeta-like cells from paddy field soils were
able to form colonies in a deep-agar medium, but not on 10 µm

Fig. 3. The Process of Colony Formation of Strain K-5.

Table 2. Characteristics of Strains K-5, and T-3 and Methanosaeta a, Cells elongated to about 50 mm. b, A colony developed from the
concilii Strain GP6 center of the filamentous cells. c, Irregularly shaped colonies formed
after 6 months of incubation.
M. concilii4,19,20)
Characteristics Strain K-5 Strain T-3
strain GP6
roll-tube agar, even after one year of cultivation. Colony
Single cell dimensions (mm) 0:8  2{3 0:8  2{3 0:8  2{6
Length of filament (mm) 2–45 2–167 150
formation was investigated using strain K-5. After 6
Motile    months of cultivation, it formed white or cream
Gram stain    irregularly-shaped colonies in a deep-agar medium
Energy and carbon source (Fig. 3c). First, filamentous cells were dispersed by
for growth acetate acetate acetate strong agitation in 2 (one cell)-6 mm of filament (2–3
Optimum pH 7.0–8.0 7.0–8.0 7.1–7.4
Optimum temperature (
C) 30–40 30–40 35–40
cells) in a 109 dilution. The cells elongated to about
Optimum NaCl (M) <0:02 <0:02 ND1 50 mm after 2 months (Fig. 3a), and colonies developed
Requirement of vitamines   +2 from the center of the filaments after 3 months (Fig. 3b).
Requirement of yeast extrac    A few colonies became interconnected, and small
Requirement of CO2   + colonies developed by elongation of the filaments of
Catalase + + ND
Nitrogenase + + ND
big colonies after 6 months.
G þ C (mol %) 51.8 51.9 49:0  1:25
Phenotypic and phylogenetic characteristics of Meth-
*1 Not detected
*2 Vitamins: pyridoxine–HCl, thiamine–HCl, riboflavin, nicotinic acid, anosaeta isolated from paddy field soils
p-aminobenzoic acid, lipoic acid, biotin, folic acid, and vitamin B12 Strains K-5 and T-3 utilized acetate as their sole
 Isolation and Characteristics of Methanosaeta 833
Table 3. G þ C Contents and 16S rRNA Sequence Similarities of investigated by analysis of the partial 16S rRNA
Strains K-5 and T-3 and Four Strains of Methanosaeta concilii sequences (approximately 750 bp) derived from them.
G þ C content Homology of 16S rRNA Strains H-3 and M-4 exhibited 100% similarity with the
Strain Habitat sequence of strain T-3, and strain O-1 exhibited 100%
(mol %) T-3 K-5
similarity with that of strain K-5.
T-3 51.9 100 99.5 Paddy soil
K-5 51.8 99.5 100 Paddy soil
Opfikon1 52.3 100 99.5 Digester
Discussion
VeAc92 ND5 100 99.5 Paddy soil
FE3 52.6 98.1 98.5 Digester The population of Methanosaeta in paddy field soils
GP64 49:0  1:25 97.7 97.7 Digester was determined by forming colonies in a deep agar
*1, DDBJ accession no.: X16932 medium. Methanosaeta colony formation is difficult. At
*2, DDBJ accession no.: Y15402 present, colonies have been formed for few strains of
*3, DDBJ accession no.: M59146
Methanosaeta,16,19–21) except for Methanothrix soehnge-
*4, DDBJ accession no.: S42679 and S42680
*5, ND, not determined nii strain VNBF.22) Kamagata and Mikami23) reported
that colonies developed in deep agar tubes containing
acetate medium at dilutions lower than 104 , but no
carbon and energy source and did not produce H2 during growth was obtained when colonies were transferred
incubation. Acetate was consumed to a concentration of into a liquid medium. We succeeded in forming colonies
less than 50 mM after 2 months of incubation. They of Methanosaeta in deep agar tubes containing an
metabolized acetate to methane even in the presence of acetate medium, but the attempt to form colonies by the
hydrogen. No growth occurred on the methanol, for- roll-tube method failed. In deep agar medium, a few
mate, or H2 þ CO2 . Both strains grew on a 10 mM colonies became interconnected, and one colony devel-
concentration of acetate to 300 mM. Yeast extract had oped into several when the culture was continued (data
no effect on cell growth. The optimum pH for growth not shown). These results indicate that Methanosaeta in
was 7.0–8.0. The optimum temperature for growth was flooded paddy soils can develop many additional
30–40
C. Neither salt, CO2 , nor vitamins was required colonies by the elongation of filamentous cells in
for growth (Table 2). Both strains were catalase- and existing colonies.
nitrogenase-positive. The G þ C contents of the DNAs The numbers of Methanosaeta-like cells in Kanagi
of T-3 and K-5 were 51.9 mol % and 51.8 mol % paddy field soil were 103 –104 MPN/g dry soil, lower
respectively. The contents of these strains approximated than those of Methanosarcina-like cells. Methanosaeta-
the content of M. soehngenii strain Opfikon16,18) like cells isolated from agar shake tubes were closely
(Table 3). The strains were investigated by analysis of related to Methanosaeta concilii strains Opfikon and
the partial 16S rRNA gene sequences (747 bp) derived VeAc9. Asakawa et al. investigated methanogenic
from them. The 16S rRNA sequence of strain T-3 was populations in paddy field soil in situ over three
compared with strain K-5 and showed 99.5% similarity, successive years.24) The populations of acetate and
corresponding to 3 base differences of 144, 145, and hydrogen-utilizing methanogens were 104 –105 MPN/g
149 bp (E. coli 16S rRNA numbering). The 3 bases of dry soil, and 103 –104 MPN/g dry soil respectively,
strain T-3 were T, T, and T, and exhibited 100% although the acetate-utilizing methanogens were not
similarity with the sequence of M. concilii strains distinguished Methanosarcina from Methanosaeta.
Opfikon and VeAc9, as shown in Table 3. On the other They suggested that methanogenic populations in paddy
hand, strain K-5 was A, C, and G, and showed 99.5% field soil hardly changed even when the field was
similarity. flooded and drained and fertilizer was applied. In Italian
paddy soils, the number of hydrogen- and acetate-
Filamentous acetate-utilizing methanogens isolated utilizing metanogens was 2:3  107 MPN/g dry soil and
from paddy field soils in various regions 1:3  106 MPN/g dry soil respectively.10) The dominant
Soil samples were collected from the paddy fields in aceticlastic methanogens were Methanosaeta-like cells
various regions of Japan in order to isolate filamentous not affiliated morphologically or phylogenetically with
acetate-utilizing methanogens. After successive enrich- Methanocaeta concilii. By a molecular retrieval ap-
ments using 50 mM acetate medium, white flocs formed proach with archaeal 16S rRNA genes, the dominant
at the bottoms of the vials. Colonies formed in deep-agar methanogenic archaea on rice root were Methanobacte-
acetate medium. The length of filamentous cells isolated rium spp., Methanosarcina spp., and Rice cluster I. On
from paddy field soils in Hakodate (strain H-3), Mobara the other hand, Methanosaeta-like sequences were not
(strain M-4), and Hita (strain O-1) was a short chain detected from rice root, in contrast to the results
from 2 to 75 mm in length. A long chain over 150 mm obtained with surrounding anoxic rice paddy soils.25)
long was not isolated, except for strain T-3, isolated The methanogenic community was dominated by Meth-
from Tsukuba. Three strains were gram-negative non- anosaeteceae when rice paddy soil was incubated at
motile rods (0.8 mm  2 to 3 mm) that utilized acetate as 15
C, whereas at 30
C incubation, it was dominated by
their sole carbon and energy source. These strains were Methanosarcinaceae.26) These results were obtained
834 S. M IZUKAMI et al.
from Italian air-dried paddy soil. Kudo et al. inves-
tigated methanogen flora of soils taken from paddy fields
by a molecular retrieval approach with archaeal 16S
rRNA gene sequences in five areas of Japan.9) Meth-
anosarcina-like sequences were predominant in the
paddy field soils of four areas. Methanogenium-like
sequences and Methanosaeta-like sequences were pre-
dominant in one area. They suggested that acetate-
utilizing methanogens are dominant and the percentages
are in the range of 60–100% in many Japanese paddy
soils. Kruger et al. studied the population of methano-
gens in an Italian rice field over two consecutive
years.27) The populations were followed by terminal
restriction fragment polymorphism and real-time PCR
targeting archaeal SSU rRNA genes. The dominant
groups were methanogens affiliated with Rice cluster I
(unknown Methanosarcinales28)), Methanosaetaceae,
Methanosarcinaceae, and Methanobacteriaceae. Meth-
anosaetaceae were relatively abundant in the year in
which the acetate concentration was low. They suggest-
ed that the composition of the archaeal community
remained relatively constant through the seasons.
All strains were isolated by the formation of colonies
in agar shake tubes, and they were divided into two
types: those with a long-chain filament and those with a
short-chain filament. The cell morphology of both
strains was similar to that of M. soehngenii strains
Opfikon and Methanothrix soehngenii strain FE, whose
filaments broke into smaller fragments and individual
cells upon vigorous shaking, although those of M. con-
cilii strain GP6 did not break.16,18–21) There were cell-
morphological differences between the strains isolated
from paddy field soils in Japan and the Methanosaeta
dominating in the Italian paddy fields.10) The cells of the
former were 0.8 mm in diameter, and those of the latter
were about 0.5 mm. Strains T-3 and K-5 utilized acetate
up to 300 mM as sole carbon and energy source without a
negative effect on growth. It is generally recognized that
Methanosaeta has a higher affinity for acetate than
Methanosarcina; therefore, low acetate concentrations
favor Methanosaeta over Methanosarcina,23,29,30) but
there have been no studies on acetate utilization of
Methanosaeta at concentrations greater than
100 mM.18,22) Methanosaeta in paddy soils is considered
to be able to utilize acetate over a wide range of
concentrations. On the other hand, Methanosarcina did
not grow well on acetate isolated from paddy field soils,
although it can grow on methanol, methylamine, and
H2 þ CO2 .5,7) When acetate-utilizing methanogens from
paddy soil were enriched with acetate mineral medium,
Methanosaeta was more abundant than Methanosarcina
after the third enrichment culture. We believe that
Methanosarcina in paddy field soils does not grow well
on low concentrations of acetate, or else it requires a
growth factor. Although a biosynthetic requirement of
CO2 has been demonstrated in M. concilii strain GP6,
strains T-3 and K-5 did not require CO2 , nor do
M. soehngenii strains Opfikon and VNBF.18–20,22) Some
vitamins are known to be essential for growth of
M. concilii strain GP6.19,20) But strains T-3 and K-5
did not require vitamins when grown on acetate.
Catalase activities were detected in strains T-3 and K-
5. The catalase activities of Methanosaeta have not been
investigated, although M. soehngenii strain Opfikon has
been reported to have the ability to tolerate oxygen.18) It
is expected that catalase protects Methanosaeta from
oxygen toxicity in paddy field soils. Strains T-3 and K-5
grew on N2 as sole nitrogen source. Both strains are
considered to fix nitrogen in paddy field soil, like
Methanobacterium and Methanosarcina isolated from
paddy field soils.5)
The results for the 16S rRNA gene of five strains
isolated from paddy field soils in various regions of
Japan show that all strains have Methanosaeta concilii
as the closest phylogenetic neighbor. They were divided
two genogroups. Comparison of the 16S rRNA base
sequences of strains T-3, M-4, and H-3 showed that
these strains exhibit 100% similarity to M. concilii
strains VeAc910) and Opfikon.31) Strains T-3, M-4, and
H-3 are designated the TT-T genogroup. On the other
hand, strains O-1 and K-5 are designated the AC-G
genogroup. The AC-G genogroup was the same as the
base sequences of 16S rRNA of M. concilii strain FE.32)
No comparison of the genogroup with strain GP6 was
possible, because its partial sequence data could not be
obtained. Strains Opficon and FE were filamentous
acetate-utilizing methanogens isolated from methane
fermentation sludge. Strain VeAc9 was isolated from
Italian paddy soils. These results indicate that filamen-
tous acetate-utilizing methanogens isolated from paddy
field soils belong to the genus Methanosaeta. We could
not, however, determine the relationships between this
genogroup and the morphology of isolated Methanosae-
ta strains. More experiments are needed to determine the
meaning of these differences in 16S rRNA sequences.
In conclusion, our results indicate that filamentous
acetate-utilizing methanogens isolated from paddy field
soils belong to the genus Methanosaeta on the basis of
the cell-morphology, and the phenotypic and phyloge-
netic characteristics described above. They suggest that
Methanosaeta is universal in paddy field soils and that it
plays an important role in methane production from
acetate.
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