INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
July 1986, p. 380-382
0020-7713/86/030380-03$02.W/O
Copyright 0 1986, International Union of Microbiological Societies
Vol. 36, No. 3
Methanobacterium alcaliphilum sp. nov. an H2-Utilizing
Methanogen That Grows at High pH Values
SUCHADA WORAKIT,lt DAVID R. BOONE,l ROBERT A. MAH,l* MOHIY-ELDIN ABDEL-SAMIE,2
AND M. M . EL-HALWAG12
Environmental and Occupational Health Sciences, School of Public Health, University of California, Los Angeles,
California 90024‘ and National Research Centre, Dokki, Cairo, Egypt2
Four strains of alkaliphilic methanogens (strains WeN1, WeN2, WeN3, and WeN4T [T = type strain])
previously enriched and isolated from sediments of four low-salt, high-pH (pH 8.3 to 9.3) lakes in the Wadi el
Natrun of Egypt were further characterized. These organisms were H2-oxidizing, CO2-reducing, rod-shaped
bacteria which grew best at high pH values (strains WeN1, WeN2, and WeN4T preferred pH 8.4 and grew at
pH values up to 9.9; strain WeN3 grew best at pH 7.8 and grew at pH values up to 9.2). Each strain required
sulfide as a sulfur source and was resistant to elevated concentrations (growth rates in the presence of 0.5%
NazS * 9H20 were one-third to one-half the maximum observed rates). The guanine-plus-cytosinecontent of the
deoxyribonucleic acid of strain WeN4T was 57 mol%. A new species of methanogen, Methanobacterium
alcaliphilum, is described; strain WeN4 (= DSM 3387) is the type strain.
crimp closures and autoclaved. The final pH was 8.4. Additions of soluble substrates were made from sterile, O2-free
stock solutions. H2 was added immediately after inoculation
by pressurization of the atmosphere in the tubes to 170 kPa
(the total H2 partial pressure was 68 kPa). The medium used
for enrichment (Boone et al., in press) was made by decreasing the Trypticase peptone and yeast extract concentrations
to 0.5 g of each per liter and by omitting L-cysteine hydrochloride. Media of different pH values were prepared by
adding 02-free, sterile 1 M solutions of HCl or NaOH to
tubes.
Culture techniques. In this study and a previous one
(Boone et al., in press) the culture techniques of Hungate (4)
were used, with some of the modifications described by
Balch and Wolfe (1).Cultures were incubated at 37°C; those
with added H2 were incubated in a shaker. As H2 was used,
it was periodically replaced by repressurizing the tubes with
a mixture of H2 and C02 (4:l).
The effect of the pH value of the medium on growth was
determined (Boone et al., in press) by measuring the specific
growth rate. The culture medium was initially adjusted to
various pH values, and growth was monitored during the
early phase when the pH of the medium increased by no
more than 0.2 pH unit. Media adjusted to pH values above
8.3 were prepared with 50 mM 2-cyclohexylaminoethanesulfonate buffer.
Analytical methods. Optical density was determined with a
Spectronic 70 spectrophotometer (Bausch 8z Lomb, Inc.,
Rochester, N.Y.). Methane was analyzed by gas chromatography with thermal conductivity detection. Growth rates
were determined from the exponential increase in the sum of
inoculum-produced methane and the methane accumulated
in culture vessels (6). Often these growth rates were corroborated by optical density measurements; these latter growth
rates were always similar to those calculated from methane
production but were less precise. A Zeiss Universal Research microscope was used for epifluorescence microscopy. Deoxyribonucleic acid was isolated by the method of
Marmur ( 5 ) and was analyzed by the buoyant density
method with CsCl gradients (7).
Radioisotopic techniques. To determine the ability of cells
to assimilate acetate, 0.54 pCi of [U-14C]acetateand 15 pmol
of unlabeled acetate were added per 5 ml of medium (the
Most previously studied methanogens were isolated from
habitats with pH values near neutrality, and such pH values
support the best growth of these microbes. Recently, several
alkaliphilic strains were isolated, including the thermophilic
organism “Methanobacterium thermoalcaliphilicum” (3)
and four mesophilic isolates from the Wadi el Natrun in
Egypt (D. R. Boone, S . Worakit, I. M. Mathrani, and R. A.
Mah, Syst. Appl. Microbiol., in press).
We report here further characterization of the four mesophilic strains, which belong to a new species of bacterium,
Methanobacterium alcaliphilum sp. nov.
MATERIALS AND METHODS
Inocula and media. Sediment samples were collected from
four Egyptian lakes of the Wadi el Natrun (Boone et al., in
press). The culture media used contained (per liter of deionized water) 10 g of NaHC03, 2.0 g of yeast extract, 2.0 g of
Trypticase peptone, 1 g of NH4C1, 0.5 g of L-cysteine
hydrochloride, 0.1 g of Mg2C1 . 6H20, 0.02 g of CaC12, 0.4 g
of K2HP04, 1 mg of resazurin, 0.1 mg of H2Se03, 1 mg of
MnC12 - 4H20, 1 mg of F e S 0 4 7H20, 1.5 mg of
CoCl2 - 6H20, 1 mg of ZnC12, 0.1 mg of H3B03,0.1 mg of
NaMo04.2H20, 0.2 mg of CuC12. 2H20, 0.2 mg of
NiS04 - 6H20,0.4 mg of A1C13 - 6H20, and 5 mg of disodium
ethylenediaminetetraacetic acid dihydrate. The gas phase
was 100% N2. All ingredients except NaHC03, yeast extract, Trypticase peptone, and L-cysteine hydrochloride
were dissolved in 1,020 ml of deionized water, which was
boiled under a stream of 02-free nitrogen. Trypticase
peptone, yeast extract, ,and L-cysteine hydrochloride were
added; for solid media, 18 g of purified agar was also added.
Boiling was continued until the volume was reduced to 1,000
ml. The medium was cooled, and 02-free NaHC03 was
added. The NaHC03 was rendered 02-free by storage in an
anaerobic chamber or in a sealed tube flushed with N2 gas.
The medium was distributed to serum tubes, which were
sealed with butyl rubber stoppers secured with aluminum
* Corresponding author.
t Present address: Biological
Sciences Division, The Office of
Atomic Energy for Peace, Thanon Viphavadee Rungsit, Bangkhen,
Bangkok 10900, Thailand.
380
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VOL.36, 1986
0.10
METHANOBACTERIUM ALCALIPHILUM SP. NOV.
177
n
T-
I
c
Y
a
0
5
Sulfide (g/l)
10
FIG. 1. Effect of sulfide concentration on the growth rates of
isolates from the Wadi el Natrun. Symbols: a, strain WeN1; H,
strain WeN2; A, strain WeN3; V, strain WeN4=. p., Specific growth
rate.
concentration of acetate in the medium was 3 mM; the
specific activity was 36 mCi/mol). Late-log-phase cultures
were harvested by filtration through 0.45-p.m Metricel membrane filters (Gelmah Sciences, Inc., Ann Arbor, Mich.).
The filters were washed three times with 10 ml of 5%
trichloroacetic acid and then with 10 ml of water and 10 ml of
3 mM sodium acetate. The filters were dried to a constant
weight in a vacuum oven at 105°C. Uninoculated-medium
controls were treated in the same way. The dry weight of
cells was determined and corrected for the small loss in
weight of the control filters. The radioactivity on the filters
was determined by counting in scintillation vials with 15 ml
of Aquasol-2 (New England Nuclear Corp., Boston, Mass.).
The radioactivity trapped on the filters was used as a
measure of the acetate incorporated into cells, and the
percentage of incorporation was calculated as the weight of
acetic acid incorporated per unit of dry weight of cells.
RESULTS AND DISCUSSION
The four strains used in this study were isolated and
partially characterized as described elsewhere (Boone et al.,
in press). Strains WeNl (= DSM 3457), WeN2 (= DSM
3458), WeN3 (= DSM 3459), and WeN4T (= DSM 3387T)(T
= type strain) were maintained by weekly transfers of 2 ml of
culture into 20 ml of liquid medium in serum vials. Microscopic examination of wet mounts revealed no motility.
Acetate incorporation. Acetate was not required for
growth and did not relieve the requirement for Trypticase
peptone and yeast extract. We repeated earlier tests (Boone
et al., in press) of acetate assimilation in the presence of
yeast extract and 3 mM acetate and found that strain WeNl
incorporated 7.0% of its cell carbon from acetic acid; strain
WeN2 incorporated 8.5%, strain WeN3 incorporated 0.9%,
and strain WeN4T incorporated 11%.
Sulfide requirement and toxicity. Figure 1 shows the effect
of sulfide concentration on the growth rate of cells. In
medium without sulfide (but containing cysteine, Trypticase
peptone, and yeast extract as potential sulfur sources), all
strains grew after the first transfer. However, after growth
was completed, the cultures did not grow in fresh media
without added sulfide (5%, vol/vol, transfer), indicating that
381
sulfur sources present as medium components could not
replace sulfide.
Taxonomy. The morphology and substrate range of the
four strains studied place them in the genus Methanobacterium. However, these four strains differ from previously described species of the genus Methanobacterium
by their lack of immunological cross-reactions, their high pH
range for growth, and the high guanine-plus-cytosine contents of their deoxyribonucleic acids (Boone et al., in press).
Unlike other species of Methanobacterium, our four strains
stained gram negative; two other isolates (“Methanobacterium thermoaggregans” [2] and l ‘Methanobacterium
thermoalcaliphilum’ ’ [3]) appear to be gram-negative bacteria which belong to the genus Methanobacterium. We propose a new species, Methanobacterium alcaliphilum, for our
isolates, with strain WeN4 as the type strain.
There were significant differences between strain WeN3
and strains WeN1, WeN2, and WeN4T, including a slightly
lower temperature optimum (Boone et al., in press), a lower
pH optimum (Boone et al., in press), a lower rate of acetate
incorporation, and an ability to grow for several transfers in
mineral medium (Boone et al., in press). However, the
similarities between strain WeN3 and the other strains
indicate that at present the description of M . alcaliphilum
should be broad enough to include all four strains.
We propose the species description given below.
Methanobacterium alcaliphilum sp. nov. (al.ca.li.phi1’uni.
M.L. alcali alkali [from Arabic a1 end; qaliy soda ash]; Gr.
adj. philum loving; M.L. adj. alcaliphilum liking alkaline
media). Long rods, 0.5 to 0.6 by 2 to 25 Fm, occurring
individually or in pairs, more rarely in short chains or
filaments. Gram negative. Nonmotile. Colonies grow to 1.0
to 1.5 mm in diameter. Colonies are yellowish to cream
colored, smooth, opaque, raised, convex, and circular with
entire margins. Subsurface colonies are cream colored, small
(diameter, 0.2 mm) irregular spheroids. HZ-COZ is the sole
substrate for growth and methanogenesis. Growth factors
present in Trypticase peptone or yeast extract are required
for growth.
Optimal growth occurs between pH 8.1 and 9.1 and near
37°C.
The quanine-plus-cytosine content of the deoxyribonucleic acid is 57 mol%.
The type strain is strain WeN4 (= DSM 3387).
ACKNOWLEDGMENTS
We thank Indra M. Mathrani for analyzing the deoxyribonucleic
acid.
This research was supported in part by research grant DE-AT0380ER10684 from the U.S. Department of Energy. A fellowship
provided by the Division of Technical Assistance and Co-operation
of the International Atomic Energy Agency supported S.W. This
work was part of a coordinated program of research under sponsorship of the International Atomic Energy Agency, Vienna, Austria.
LITERATURE CITED
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cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic
acid (HS-CoM)-dependent growth of Methanobacterium
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2. Blotevogel, K.-H., and U. Fischer. 1985. Isolation and characterization of a new thermophilic and autotrophic methane producing
bacterium: Methanobacterium thermoaggregans spec. nov.
Arch. Microbiol. 142:21&222.
3. Blotevogel, K.-H., U. Fischer, M. Mocha, and S. Jannsen. 1985.
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382
WORAKIT ET AL.
INT. J. SYST.BACTERIOL.
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