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Isolation Of A Novel Acidiphilic Methanogen
From An Acidic Peat Bog
By: Suzanna L. Brauer, Hinsby Cadillo-Quiroz, Erika Yashiro, Joseph B.
Yavitt & Stephen H. Zinder
Abstract
Acidic peatlands are among the largest natural sources of atmospheric methane and harbour a large
diversity of methanogenic Archaea1. Despite the ubiquity of methanogens in these peatlands, indigenous
methanogens capable of growth at acidic pH values have resisted culture and isolation2–4; these
recalcitrant methanogens include members of an uncultured family-level clade in the Methanomicrobiales
prevalent in many acidic peat bogs in the Northern Hemisphere1,5,6. However, we recently succeeded in
obtaining a mixed enrichment culture of a member of this clade7. Here we describe its isolation and initial
characterization. We demonstrate that the optimum pH for methanogenesis by this organism is lower
than that of any previously described methanogen.
Suzanna L. Brauer, Hinsby Cadillo-Quiroz, Erika Yashiro, Joseph B. Yavitt & Stephen H. Zinder. (2006).
"Isolation Of A Novel Acidiphilic Methanogen From An Acidic Peat Bog." Nature
, 442:192-194. [ISSN:
00280836] [DOI 10.1038/nature04810] Version Of Record Available At www.nature.com
It has been estimated that northern peatlands contain one-third of
the carbon found in soils worldwide
8
. They are important sources of
atmospheric methane, a greenhouse gas whose concentration has more
than doubled in the past 200 years (ref. 9), reaching levels unprece-
dented in the past 650,000 years (ref. 10). Sphagnum-dominated bogs,
the predominant peatlands
11
, are typically acidic (pH , 5) with low
concentrations of mineral nutrients. Acidiphilic aerobic methane-
oxidizing organisms have been isolated
12
, whereas attempts to isolate
acidiphilic methanogens from bogs were unsuccessful
2,13
.
16S rRN
A-based culture-independent studies indicate that the
methanogens present in acidic bogs comprise several novel genera
and species
1,5,6,11
. A family-level clade in the H
2
/CO
2
-using methano-
archaeal order Methanomicrobiales
—
originally called the R10
group
5
, and also called the fen cluster
7,14
or E1/E2 cluster
15
—
is usually
abundant in 16S rRNA clone libraries from acidic peat bogs in diverse
geographical locations
1,5,6,16
. For example, in McLean Bog, New York
State, a small acidic (pH 4.1) bog we study, 66 out of 84 methanogen
16S rRNA gene clones belonged to this clade
1
. More recent studies
using quantitative polymerase chain reaction (PCR) showed nearly
10
9
cells per gram dry weight of this group in active layers of McLean
Bog peat, about half of the total Archaea
15
. Recently, sequences in
the R10 group were detected as minority members of low-pH
methanogenic enrichment cultures from German or Siberian bogs
3,4
.
We recently demonstrated that the antibiotic rifampicin allowed
enrichment of H
2
/CO
2
-using methanogenic Archaea in McLean Bog
peat, and prevented growth of H
2
/CO
2
-using acetogenic Bacteria
7,17
.
Using results from experiments examining the physical and chemical
conditions that favoured methanogenesis
17
, we designed a low-ionic-
strength medium, designated PM1, that allowed repeated transfer of
H
2
/CO
2
-using methanogens at pH 5 (ref. 7). Dilutions in liquid
medium to 10
28
, the correct order of magnitude for a dilution to
extinction, produced a culture, designated 6A8, containing two mor-
photypes
—
thin straight rod cells (0.2–0.3
m
mdiameter,0.8–3.0
m
m
long) and irregular coccoid cells (0.3–0.8
m
m in diameter) (Fig. 1a, b).
The coccoid cells represented 5–8% of total cells and were present in
all growth phases.
We initially assumed that the coccoid cells were heterotrophic
bacterial
contaminants; however, no growth or methanogenesis was
detected in medium amended with various organic substrates, and
PCR amplifications with the universal bacterial 16S rRNA gene
primers 27f and 1492r were negative. All of 39 clones amplified
with primers 1Af(-1) and 1100Ar for methanogens
1,5
were a single
restriction type, and a 92-clone library generated using broad-range
archaeal primers (1Af and ArchLSU47) that amplified a region
encoding 16S rRNA, the 16S–23S internal transcribed spacer (ITS)
region, and part of the 23S rRNA (1,730 base pairs in total), yielded
sequences with $99.7% identity, within the error rate expected for
PCR amplification
18,19
. The 6A8 16S rRNA gene sequence belonged to
the R10-Fen cluster-E1/E2 family, most closely related to sequences
from mixed cultures (Supplementary Fig. 1).
Figure 1 | Photomicrographs of culture 6A8. a, b, Phase contrast (a) and
fluorescence (b) images of cells in the same field of view, stained with the
DNA stain acridine orange (AO), showing thin rod and coccoid
morphologies. c, d, Fluorescence micrographs of rod-shaped (R) and
coccoid (C) cells in 6A8 stained with the DNA stain 4,6-diamidino-2-
phenylindole (DAPI) (c) or with a Cy-3-labelled 16S rRNA probe targeting
6A8, probe 6A8 644 (d). e, AO-stained cell apparently undergoing
asymmetric div ision to a coccoid cell (arrow). f, AO–stained cells passed
through a 0.45-
m
m membrane filter and then concentrated, showing short
rods and small cocci, including a cell apparently undergoing asymmetric
division (arrow).
Introduction
Fluorescence in situ hybridization (FISH) staining of rRNA allows
more positive identification of organisms. Initial attempts to stain
these cells failed, but a FISH protocol developed for SAR11 (ref. 20),
also difficult to stain, allowed visualization of both morphotypes
with the universal ARCH 915 probe, but not with the EUB 338 probe
(data not shown).ProbeMG12006A8, inwhich a single-base mismatch
with the ‘universal’ Methanomicrobiales probe MG1200 (ref. 21) was
corrected, hybridized with both morphotypes (Supplementary Fig. 2),
as did the strain-specific probe, 6A8 644 (Fig. 1c, d), which did not stain
cells from the most closely related isolate, Methanospirillum hungatei
JF1 (Supplementary Fig. 2).
During microscopic examination of growing cultures, cell division
in rod-shaped cells was observed, but not in coccoid cells. Division in
the rod-shaped cells was sometimes asymmetrical
22
, giving rise to
spherical cells (Fig. 1e). Filtration of cultures through a 0.45-
m
m filter
allowed passage of short rods and small coccoid cells (Fig. 1f). In
cultures grown out from this filtrate, long rods and large coccoid cells
returned, and indeed Fig. 1a, b shows such a culture. From these
observations, we conclude that both morphotypes are manifestations
of the same organism, and suggest that the large coccoid cells are
non-viable cultural artefacts.
6A8 used H
2
/CO
2
as a methanogenic substrate with a doubling
time near two days at pH 5.1 and 28 8C (standard growth conditions),
and did not use formate, ethanol, methanol or acetate. Besides
mineral nutrients, it required vitamins, yeast extract, coenzyme M
and low (,200
m
M) concentrations of acetate in the medium. The
optimum pH for methanogenesis was near 5 (Fig. 2), with little
methanogenesis in cultures incubated at pH 4.0 or 5.8. The tem-
perature optimum was near 37 8C (Supplementary Fig. 3), with slow
methanogenesis at 10 8C but not at 4 8C.
The most acidiphilic hydrogenotrophic methanogen described to
date is Methanobacterium espanolae
23
, with a pH optimum between 5.5
and 6.0 and a minimum near 4.7. Some strains of Methanosarcina spp.
can grow near pH 4.5, although their optimum is near neutrality
24
.
Microbial populations converting H
2
/CO
2
to methane in McLean Bog
showed an optimum pH near 5.0 (ref. 17), close to that of 6A8, but
produced considerably more methane relative to the optimum at pH
values near 4.3 and 5.8 than did culture 6A8, indicating that this mixed
microbial population had a broader pH response. Under in situ
conditionsin McLean Bog peat (pH , 4.5, 4–20 8C), rates of methano-
genesis by this organism would be considerably limited.
In summary, we have isolated a novel acidiphilic methanogen that
is part of an indigenous population adapted to conditions in a
methanogenic peat bog, a habitat that is not only low in pH, but also
low in concentrations of mineral ions such as sodium
17
. 6A8 is clearly
a member of a novel genus in the order Methanomicrobiales, and
based on its preliminary characterization, we propose the name
‘Candidatus Methanoregula boonei’, describing the morphology of
the thin rod (from the Latin regula, meaning slat) and in honour of
the late D. Boone, whose pioneering research on anaerobes included
studies on extremophilic methanogens growing at low temperatures
25
or pH
24
.
METHODS
Culture growth. Culture 6A8 was grown in anaerobic PM1 medium
7
buffered to
,pH 5.1 by the organic buffer Homopipes (pK
a
¼ 4.7 at 28 8C). Methane
production by cultures was quantified by gas chromatography
17
.
Molecular and phylogenetic analyses. 16S rRNA gene clone libraries were
generated from 6A8 DNA by PCR amplification using primers specific to
methanoarchaea (1Af(-1) and 1100Ar) or Bacteria (27f and 1492r), or universal
archaeal primers (1Af and ArchLSU47) spanning the 16S rRNA gene, the
internal transcribed spacer (ITS) region, and part of the 23S rRNA gene. Cloned
PCR amplicons were screened by restriction endonuclease digestion, and
representative clones were sequenced at Cornell’s Bioresource Center and were
analysed using ARB and PHYLIP phylogenetic packages.
Fluorescence microscopy. Fluorescence in situ hybridization (FISH) was per-
formed using fixed cells on black polycarbonate membranes
20
. The species-
specific Cy3-labelled oligonucleotide probe 6A8 644 was designed using the ARB
software package. Probe specificity was verified by failure to hybridize with
Escherichia coli or M. hungatei JF1 cells. Stained cells were viewed using a Nikon
Eclipse E600 microscope equipped with a Hamamatsu CCD digital camera, or
an Olympus BX61 microscope equipped with a Cooke SensiCam camera and
SlideBook software.
A more detailed description of methods is provided in Supplementary
Information.
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