INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
Apr. 1991, p. 267-274
0020-7713/91/020267-08$02.OO/O
Copyright 0 1991, International Union of Microbiological Societies
Vol. 41, No. 2
Characterization of Methanosarcina barkeri MST and 227,
Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-76IT
GLORIA M. MAESTROJUAN' AND DAVID R. BOONE172*
Departments of Environmental Science and Engineering' and Chemical and Biological Science,2
Oregon Graduate Institute, 19600 N . W . von Neumann Drive, Beaverton, Oregon 97006-1999
Members of the genus Methanosarcina are recognized as major aceticlastic methanogens, and several species
which thrive in low-salt, pH-neutral culture medium at mesophilic temperatures have been described.
However, the environmental conditions which support the fastest growth of these species (Methanosarcina
barkeri MST [T = type strain] and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-761T)
have not been reported previously. Although the members of the genus Methanosarcina are widely assumed to
grow best at pH values near neutrality, we found that some strains prefer acidic pH values. M . vacuolata and
the two strains of M. barkeri which we tested were acidophilic when they were grown on H, plus methanol,
growing most rapidly at pH 5 and growing at pH values as low as 4.3. M. mazei grew best at pH values near
neutrality. We found that all of the strains tested grew most rapidly at 37 to 42°C on all of the growth substrates
which we tested. None of the strains was strongly halophilic, although the growth of some strains was slightly
stimulated by small amounts of added NaCI. The catabolic substrates which supported most rapid growth were
H, plus methanol; this combination sometimes allowed growth of a strain under extreme environmental
conditions which prevented growth on other substrates. The cell morphology of all strains was affected by
growth conditions.
Although Methanosarcina species have been the subjects
of extensive metabolic studies, other aspects of the characterization of these organisms do not meet even the minimal
standards for description of methanogenic taxa (9). In this
study we examined the physiology of the following three
mesophilic, aceticlastic species: Methanosarcina barkeri,
Methanosarcina mazei, and Methanosarcina vacuolata.
Methanosarcina acetivorans, the only other mesophilic and
aceticlastic Methanosarcina species, was not included in
this study because it has been well characterized (35).
The original type strain of M . barkeri, isolated by C. G. T.
P. Schnellen (32a), has been lost. In 1966, Bryant isolated
strain MS (lo), which has been adopted as the type strain (4).
Although this strain is one of the most extensively studied
methanogens, previous studies have not included definitive
determinations of its responses to environmental conditions
such as pH, temperature, and salinity. Strain 227 (25) is
another extensively studied strain which DNA hybridization
studies (37) indicate is a member of M. barkeri.
Methanogenic cocci were first described by Groenewege
(12) in 1920 and were later observed by Kluyver and van Niel
(17). These cocci were physiologically similar to M . barkeri,
but because of morphological differences between the cocci
and M . barkeri, Kluyver and van Niel proposed a new genus
(Methanococcus) for the cocci. Later Barker (6) studied a
purified, nonaxenic culture and named it Methanococcus
mazei. Pure cultures of this species were not available until
more than 40 years later. Zhilina and Zavarzin (44) also
studied an aceticlastic enrichment culture which contained
morphological forms similar to those described by Barker as
Methanococcus mazei. Although it is not clear from the
report of Zhilina and Zavarzin (44) whether an axenic culture
was obtained, an axenic culture (referred to as Methanosarcina biotype 3) was later deposited in the Oregon Collection of Methanogens as strain OCM 92 (= 2-558 = DSM
* Corresponding author.
2244). We observed this culture growing on methanol in MS
medium and never saw cysts or multicellular aggregates
larger than two cells. The predominant morphology was
individual cocci (unpublished data). Thus, if cells similar to
the cells of this strain were present in Barker's enrichment
cultures, other multicellular methanogenic pseudosarcinae
must also have been present. This strain appears to conform
neither to Barker's description of Methanococcus mazei nor
to Mah's later description of Methanococcus mazei S-6T (T
= type strain) (23) in its morphology. In order to obtain an
axenic culture of Methanococcus mazei, Mah (23) performed the enrichment protocol of Barker (6) and isolated
the first axenic culture of a methanogen (strain S-6T) which
exhibited a life cycle and included morphological forms like
those described earlier by Barker (6). Strain 5-6 was designated the type strain of Methanococcus mazei, and later this
species was transferred to the genus Methanosarcina as
Methanosarcina mazei (24).
Zhilina (43) also isolated a gas-vacuolated Methanosarcina strain, and she and Zavarzin (46) named this organism Methanosarcina vacuolata. These authors suggested
that this species should include vacuolated Methanosarcina
strains, although the phylogenetic significance of vacuoles
has not been established.
Members of M . barkeri, M . mazei, and M . vacuolata can
be distinguished from other bacteria by the following three
characteristics: (i) they can produce methane from trimethylamine, from methanol, and from H, plus CO, or acetate; (ii)
they appear as either coccoid cells, pseudosarcinae, or
aggregates (i.e., biotype 3, 2, or 1 in the system of Zhilina
[42]); and (iii) they grow well in nonsaline medium (C0.2
osM) at mesophilic temperatures. Although it is not very
difficult to determine whether an organism belongs within
this group of three species, it is much more difficult to assign
it to a particular species because of a lack of distinguishing
characteristics.
Determination of the environmental conditions which support most rapid growth is an important part of the charac-
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268
INT. J. SYST.BACTERIOL.
MAESTROJUAN AND BOONE
terization of new species of methanogens (9). Such determinations are usually made by keeping all environmental
conditions except one constant and measuring the specific
growth rates at various values of a single parameter, for
instance pH. Such an experiment determines the pH value
which allows most rapid growth (often called the optimum
pH) under the conditions of the assay. However, it is
unlikely that the effects of environmental growth conditions
(such as pH, temperature, and salinity) are independent, so,
for instance, one range of pH values may support the most
rapid growth at one temperature and a different range of pH
values may support the fastest growth at a second temperature. The present study was initiated to determine whether
the optimal conditions for growth are mutually dependent
and to further characterize M . barkeri MST and 227, M .
mazei S-6T, and M . vacuolutu Z-761T.
(Portions of the results were reported at the 1990 Annual
Meeting of the American Society for Microbiology, Anaheim, Calif.)
MATERIALS AND METHODS
Bacterial strains and culture methods. The following bacterial strains were obtained from the Oregon Collection of
Methanogens (OCM). M . barkeri MST (= OCM 38T = DSM
800T)and 227 (= OCM 35 = DSM 1538), M. rnazei S-6T (=
OCM 26T = DSM 2053T), M. vacuoluta Z-761T (= OCM 85T
= DSM 1232T), and Methanosarcina sp. strain 2-558 (=
OCM 92 = DSM 2244 = biotype 3). Strains were cultivated
by using the anaerobic techniques of Hungate (15) with
syringes and needles.
The basal culture medium was MS medium (7), which
contained yeast extract, peptones, minerals, and coenzyme
M and sulfide as reducing agents and was used with a
bicarbonate buffer system in equilibrium with a CO,. gas
phase (pH 6.5). After the medium was dispensed into individual vessels, it was sterilized by autoclaving. The pH of
the medium was adjusted by replacing the headspace gas in
the vessels with N, or an N,-CO, mixture (a 7:3 N,-CO,
mixture resulted in a pH of 7.2, and 100% N, resulted in a pH
of 8.2) and by adding NaOH or HCl solutions to obtain pH
values higher than 8.2 or lower than 6.5. The pH usually
varied less than 0.1 to 0.2 pH unit during growth except in
some poorly buffered media (those with pH values less than
6). The growth rates in poorly buffered media were determined from the early portions of the growth curves, when
the pH values varied less than 0.2 pH unit.
Soluble catabolic substrates were added from sterile,
anoxic stock solutions to final concentrations of 50 mM, and
H, was added by pressurizing the preparation with pure H,
(final partial pressure, 101 kPa). All cultures containing
added H, were incubated on a shaker. The culture vessels
were 121-ml serum vials which contained 20 ml of culture
medium.
Specific growth rates were determined during growth from
the amount of methane formed, which served as an estimate
of the amount of biomass present. Because Methanosarcina
strains often grow as aggregates, other methods for estimating biomass are unsatisfactory; the presence of various sizes
of clumps prevents the use of turbidity as a measure of
biomass, and other methods which quantify biomass in
liquid samples (such as protein analysis or measurement of
dry weight) are inaccurate because it is impossible to obtain
small, representative samples of cell suspensions when small
numbers of large clumps are present (16, 22, 41). Assuming
that the growth yield is constant during the growth of each
culture, the amount of methane that accumulates in a culture
vessel is proportional to the increase in biomass, but this
measure neglects the biomass added as inoculum (29).
Therefore, we added the amount of methane formed by the
inoculum during its growth to the amount of methane present
in the culture vessel at each time point to obtain a value
proportional to total biomass. The specific growth rate was
estimated by performing a least-squares analysis of the
logarithm of this value during the period of exponential
growth. Inocula were grown under conditions similar to the
test conditions. The inoculum volumes were 5% (for cultures
growing as individual cells or very small clumps) or 10% (for
cultures growing as aggregates) of the culture volumes;
smaller inocula gave inconsistent results, perhaps because it
was impossible to obtain representative inocula when cells
were present as small numbers of large clumps.
Analytical methods. The methane in gas samples was
separated by gas chromatography in a column of activated
charcoal and was quantified by flame ionization detection.
Samples at the pressure of the culture vessel were collected
in a 10-pl loop (Fig. 1). Thus, the peak areas of methane
were proportional to the partial pressure, rather than to the
mole fraction (as when a sample is equalized to atmospheric
pressure). When the valve was in the load position, each
odd-numbered port was connected to the even-numbered
port whose number was one higher (port 1 to port 2, port 3
to port 4, port 5 to port 6, and port 7 to port 8). The gas
sample was collected via a needle through the stopper of the
culture vessel (connected to port 3 of the valve via 1.6-mm
[1/16-in.] stainless steel tubing). Thus, the sample flowed
through the valve (from port 3 to port 4), swept through the
10-pl loop to port 7, and then flowed to port 8 and into a
previously evacuated, sealed 0.5-ml space. Within a few
seconds pressures equalized so that the portion of the
sample remaining in the loop was at the pressure of the
culture vessel. During this time, carrier gas flowed through
the valve (from port 6 to port 5) and to the column of the gas
chromatograph. To analyze the sample in the loop, the valve
was turned to the inject position, which connected each
odd-numbered port to the even-numbered port whose number was one lower (port 1 to port 8, port 3 to port 2, port 5
to port 4, and port 7 to port 6). Thus, the carrier gas now
flowed through the valve (from port 6 to port 7), through the
sample loop, again through the valve (from port 4 to port 5),
and then to the column. The entire sample contained in the
loop was thus placed in the flow path to the chromatograph
and analyzed. Meanwhile (while the valve was in the inject
position), a vacuum pump connected to port 1 drew a
vacuum on the 0.5-ml space, which was necessary to draw
the subsequent sample through the loop. While the valve
was in the inject position, the sample collection line was
connected to port 2, which was sealed to block the flow. We
found that this method for analysis of methane gave peak
areas that were proportional to methane partial pressures at
partial pressures between 10 Pa and 200 kPa.
RESULTS AND DISCUSSION
pH optima and effects of catabolic substrates. The Methanosarcina strains generally grew faster at pH values below 7
than at pH values above 7 (Fig. 2). Depending on the
catabolic substrate, these strains grew rapidly at pH values
below 5. We found that the choice of catabolic substrate
usually had only a minor effect on the optimal pH for growth,
except that growth on methanol plus H, was most rapid at
decidedly low pH values. More rapid growth on H, plus
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CHARACTERIZATION OF METHANOSARCINA STRAINS
VOL. 41, 1991
A
To sample
collection
w
w
To column
LOAD
Sample loop
B
To sample
collection
INJECT
7
7
'
Sample loop
FIG. 1. Valve connections for analysis of gas samples. This
apparatus collected a 10-pl sample of gas maintained at the pressure
of the sample bottle, thus allowing direct analysis of partial pressures of gases. (A) Valve in the load position. The dark lines
between numbered ports indicate which ports were connected. (B)
Connections made by the valve in the inject position.
methanol than on methanol alone has been observed previously (ll), but we found that at higher pH values M . barkeri
MST and M . rnazei S-6T grew faster on methanol in the
absence of H,. The finding that Methanosarcina strains are
slightly acidophilic confirms data obtained by Hutten et al.
(16) but contrasts with the results of several other reports
(lo, 45, 46). It also is consistent with our unpublished
findings that Methanosarcina morphotypes predominate in
acetate-containing enrichment cultures maintained below
pH 7 and Methanothrix morphotypes predominate in cultures maintained above pH 7. The low pH optima of Methanosarcina strains may also have caused difficulties in
enriching Methanosarcina cultures from digestors (25).
M . rnazei S-tjTgrew well on H, plus CO, in this study and
in a study performed by Harris (13), in contrast to the results
of previous studies in which workers used strain S-6T (23,24)
and M . mazei MC3 (38). This discrepancy may have been
due to changes which may have occurred in the culture or to
269
some difference in our culture medium. M . barkeri MST and
227 also grew well on H, plus CO, in complex medium, but
M . vacuolata Z-761T grew poorly, as suggested by Zhilina
and Zavarzin (46). The inability of M . vacuolata to grow
under such conditions may be an important distinguishing
characteristic and supports the view of Zhilina and Zavarzin
(46) that this bacterium is a distinct species. However, the
ability to catabolize H, plus CO, may not be a stable
characteristic for Methanosarcina strains or may depend on
the culture medium or other conditions. For instance, M .
rnazei S-6T was originally reported to be unable to use H,
plus CO, rapidly, but we found that it could.
We had expected growth rates on methanol to be similar to
growth rates on trimethylamine because the catabolic pathways should differ only in the initial step of methyl transfer
(14). However, growth rates on trimethylamine generally
were slower. In most cases the ranges of pH values which
supported growth were similar regardless of whether methanol or trimethylamine was the substrate; the exception was
M . rnazei S-6T, which in media having pH values of more
than 7 grew much faster on methanol than on trimethylamine. It is possible that growth on trimethylamine was
inhibited by ammonia formed during its catabolism, but such
an inhibition might then have been evident in batch cultures,
in which the amount of ammonia increases during growth.
We observed no decreases in the growth rates of batch
cultures on trimethylamine until the catabolic substrate was
exhausted. These data generally confirm findings from other
studies of M . barkeri 227 ( 5 , 11, 25) and M . rnazei S-tjT,
MC3, and LYC (23, 38, 40). Unlike Hutten et al. (16), we
found no acidification of culture medium during growth on
methanol (or significant alkalinization during growth on
acetate), perhaps because our medium was more strongly
buffered.
Our data were generally consistent with those of previously published studies, but there were some differences
between our data and those of Hutten et al. (16); most
notably, we observed good growth of M . barkeri MST on H,
plus CO, (as did Ferguson and Mah [ll]), whereas Hutten et
al. found (16) that growth on this substrate was poor (only
twice as fast as growth on acetate).
The growth rates of cultures grown on acetate were
generally about 10-fold lower than the growth rates of
cultures grown on methanol or on methanol plus H, and
about 4- to 5-fold lower than the growth rates of cultures
grown on trimethylamine. The maximum specific growth
rate which we measured for M . rnazei S-6T grown on acetate
was about 0.013 h-' (doubling time, 51 h), in contrast to a
previous report (23) of a doubling time of 16.6 h. Also,
Peinemann et al. (28) reported more rapid growth of M .
barkeri Fusaro than we found for M . barkeri MST or 227
growing on acetate, and Yang and Okos (41) found growth
rates that were about twofold higher than our growth rates.
These differences may be attributed to differences in culture
media. For instance, our medium did not contain sludge
supernatant, whose presence could have allowed the more
rapid growth found by Mah (23). Furthermore, if inoculumproduced methane was not included in the calculations, the
specific growth rates may have been overestimated, especially when they were based on time points early in the
growth curve. The higher rates of Yang and Okos (41) may
also be attributed to differences in methods of calculation.
The growth rates which we measured are consistent with the
growth rates of other aceticlastic methanosarcinae and with
the inability of acetate-degrading methanosarcinae significantly to colonize the rumen, which has a solids retention
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INT. J. SYST.BACTERIOL.
MAESTROJUAN AND BOONE
270
A
0.15
-a-
0*05/
0
Methand, MS medium
+ H 9 MS medium
Hydrogen, MS medium
Hydro en, mineral medium
TrimetRyimine, MS mediurr
-+-Acetate,
MS medium
- - - Acetate, mineral medium
-t- Methanol
*-
-----0.10
-.-
Methamsarcinit barked MS
-$-
0.010
1Methanosarcha&ked
227
Methamsarcha barked 227
0.10
0
t
Methanosarchamazei S-6
1 Metbanosarcha vacuo/afa2-761
FIG. 2. Specific growth rates of Methanosarcina cultures at various pH values.
time of about 1 day. Methanosarcina strains enriched and
isolated from the rumen (26) may have been growing there
on methyl compounds, such as methanol released from
pectins.
Growth in mineral medium and mineral medium containing
acetate as a catabolic substrate. Growth with H, plus CO, as
the substrate was generally slower in mineral medium than in
complex medium (MS medium) (Fig. 2B). Cultures of M.
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VOL. 41,1991
CHARACTERIZATION OF METHANOSARCZNA STRAINS
vacuolata grew poorly in such media. When inoculated into
mineral medium containing H, plus CO,, these cultures
produced methane for a short time and stopped after about
30 to 40% of the expected methane was formed. Then these
cultures were transferred to fresh mineral medium containing H, plus CO,, and they again produced a similar amount
of methane and stopped.
All four Methanosarcina strains grew in mineral medium
to which acetate had been added as the sole catabolic
substrate (Fig. 2B). Growth on acetate as a catabolic substrate was generally slower in medium containing no other
organic compounds, except in the case of M . barkeri 227,
which usually grew as rapidly in both media. Smith and Mah
(33, 34) also reported rapid growth of strain 227 in mineral
medium containing added acetate. However, at pH values of
7 and above, growth was significantly faster in complex
medium, suggesting that some anabolic enzymes necessary
for growth with acetate as a sole carbon and energy source
do not function well at higher pH values. Our finding that M.
vacuolata growing on acetate was stimulated in complex
media contrasts with the findings of a previous report (43).
Likewise, during growth on H, plus CO,, the relative effects
of pH on growth rate were similar in complex and mineral
medium, except in the case of M . barkeri 227. The growth
range of M. barkeri 227 extended to lower pH values in
complex medium than in mineral medium. M . mazei S-6T
grew in mineral medium containing added acetate, but when
a logarithmically growing culture in MS medium containing
acetate was inoculated into mineral medium, a long lag phase
(2 to 3 months) occurred. This lag may be compared with
previously observed lags of starved cells (8), because in this
case the inoculum was growing logarithmically with acetate
as a substrate. Subsequent transfers resulted in much shorter
lag phases before logarithmic growth resumed.
Effect of salinity on growth rate. The inhibitory effect of
added NaCl was observed in all of the cultures examined,
when either trimethylamine or methanol was the substrate
(Fig. 3). Exceptions to this general observation were M.
barkeri 227 and M . vacuolata Z-761T grown on methanol,
which showed very small increases in growth rate when the
concentration of Na+ was increased from 0.1 to 0.125 M
(Fig. 3). All of the strains in this study had been enriched and
isolated from anaerobic digestors, but all of them grew on
trimethylamine in media with salinities near the salinity of
the ocean. M. vacuolata Z-761T was the most sensitive to
salinities above 0.5 M N a f .
It is unlikely that members of these four Methanosarcina
species are the predominant trimethylamine degraders in
marine environments. Trimethylamine-using methanogens
isolated from sulfate-containing marine sources are obligately
halophilic (9); furthermore, these marine strains differ phylogenetically from the strains used in our study in that the
marine strains cannot use acetate. Sulfate-reducing bacteria
may outcompete Methanosarcina strains for acetate and H,
when sulfate is in excess, so in most marine environments
trimethylamine is the only available methanogenic substrate.
M . acetivorans is the only marine methanogen which can
catabolize acetate, but this methanogen was isolated from a
kelp bed where sulfate probably was depleted (35).
MS medium contains 2 g of yeast extract per liter, and the
glycine betaine from this quantity of yeast extract may have
been taken up by the methanogens and used as a compatible
solute, enhancing the ability of the methanogens to tolerate
salinity (30).
Effect of temperature on growth rate. Figure 4 shows that
all four of the strains tested were mesophiles, growing most
o.ol 1
271
+
-
Methanol, MS medium
Trimethylamine, MS medlurr
t
0*01
0 0
-
0
5
Methanosacha vacuo/BIcB2-761
0.01
FIG. 3. Specific growth rates of Methanosarcina cultures growing at different salinities.
rapidly at 37 to 42°C. Generally, the relative effects of
temperature were independent of the catabolic substrate or
other culture conditions. For example, M . barkeri MST and
M . mazei S-6T grew fastest at 40 to 42"C, regardless of the
substrate. For other cultures, growth conditions appeared to
shift the temperature optima slightly, but these small shifts
may not be significant. For instance, M . vacuolata Z-761T and
M . barkeri 227 grew more rapidly at 42 than at 37°C when the
pH was 6.5, but at a less optimal pH value (pH 7.2) the
organisms grew more slowly at 42 than at 37°C. This finding
contrasts with other reports (45,46) for M. vacuolata Z-761T.
Effects of growth conditions on morphology. The following
four different Methanosarcina forms have been described:
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~
INT. J. SYST. BACTERIOL.
MAESTROJUAN AND BOONE
272
0.15
0.10
-
Mehmmm&.mq
d MS
:Methanol
M medium
Methanol: ME medium, pH 7.2
-0- Trimethylamins, MS medium
Acetab, MS medium
0.05
0
0.10
5 0.05
8
$
fRi
0
0.15
0.10
0.05
0
0.10
0.05
0
FIG. 4. Specific growth rates of Methunosavcina cultures growing at different temperatures.
individual cells surrounded by only a protein cell wall,
microcysts (31,44), and small and large aggregates of cells or
pseudosarcinae (13, 21, 23, 31, 32, 42, 44). The morphology
of the cells could usually be determined by simply looking at
our cultures. Turbid cultures consisted mainly of small
aggregates or individual cells; in contrast, large aggregates
(when present) could be seen with the unaided eye. Cultures
containing such aggregates usually were devoid of turbidity.
These gross observations were confirmed by microscopic
examination.
Several hypotheses have been advanced to explain aggregation and disaggregation as survival strategies. Sowers and
co-workers suggested (35) that aggregates are a survival
stage for M . mazei. If so, then it may be beneficial for the
organism to form aggregates when culture conditions shift
dramatically and to revert to growth as individual cells when
conditions are agreeable or when the cells have adapted to
the new conditions. This hypothesis is supported by the data
presented below. In contrast, Robinson (31) has suggested
that disaggregation is a mechanism of dispersal and that M .
mazei responds to unfavorable conditions by disaggregation
and release of individual cells. Zhilina and Zavarzin have
described thick-walled microcysts which may be a survival
structure (44). Krzycki et al. (18) have suggested that aggregates may conserve H,, which is given off by Methanosarcina strains during growth on acetate, with neighboring
cells taking up produced H,. However, Boone et al. (8) have
shown that H, pressures below 100 Pa have little effect on
aceticlastic cultures.
(i) M . barkeri. M . barkeri MST usually grew as small
aggregates of cells which settled to the bottom of the culture
vessels but could be dispersed easily to produce a uniform
turbidity. We never found single cells of M . barkeri which
were sensitive to lysis by sodium dodecyl sulfate. However,
during the slow growth which occurred in mineral medium
(H2 plus CO, as the catabolic substrate), the aggregates were
considerably larger and were often visible to the naked eye,
although they were not as large as the aggregates of M .
mazei. M . barkeri MST in MS medium containing a high
concentration (60 mM) of trimethylamine grew as a dense
culture of very small aggregates (only two to eight cells per
clump). M . barkeri Fusaro growing on methanol also produces aggregates when conditions are poor (32), and M .
barkeri UBS grows as aggregates in defined medium (32).
(ii) M . mazei. Like Harris (13), we found that M . rnazei
S-6' could disaggregate to individual coccoid cells during
growth in liquid medium and that individual cells (but not
aggregates) lysed when they were treated with sodium
dodecyl sulfate (9). Previous reports (23, 38) have indicated
that cells are resistant to lysis. Although we grew and
examined cultures of M . m a x i under many different conditions and found changes in the morphology of cells, we were
not able to determine definitively what conditions led to
growth as aggregates or individual cells. Methanosarcina
thermophila TM-lT disaggregates and grows as individual
cells in marine media (36), but we found no correlation
between the formation of aggregates by M . mazei and the
salinity of the culture medium. In general, we observed that
slow-growing cultures (i.e., cultures under extreme conditions relative to the conditions that supported the maximal
growth rate) were more likely to grow as aggregates. However, often one subculture had a different morphology than
another subculture of the same strain under identical conditions. We were unable to find a pattern to these inconsistencies, but it seemed that cultures which had been subjected to
recent shifts in growth conditions were more likely to grow
as aggregates. This was especially true when the shift was to
a less favorable medium, as when cells were transferred
from another catabolic substrate to acetate (8, 25, 33). When
the growth conditions of cultures containing individual cells
were changed profoundly, a long lag phase often ensued,
with cells sinking to the bottom of the vial. This sediment
often became viscous, and after several weeks growth
slowly resumed as a few visible aggregates formed. Gas
production was very slow at first and increased logarithmically, suggesting that a small fraction of the original inoculum had recovered and begun to grow. A similar phenomenon was reported previously (40) for the recovery cultures of
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VOL. 41, 1991
CHARACTERIZATION OF METHANOSARCINA STRAINS
YE,T
YE
T
Betaine
273
YE ash
FIG. 5 . Effects of culture conditions on the morphology of M . mazei S-6=. Inocula were grown in MS medium containing H, plus CO,; these
cells were coccoid. After transfer to mineral media containing various additions (still with H, plus CO, as the catabolic substrate), the cells began
to grow as aggregates. Specific growth rates were measured after adaptation of the cultures to the test conditions, and the numbers of transfers
required before the morphology reverted to coccoid cells are shown. Abbreviations for additions to mineral medium: YE, 2 g of yeast extract
per liter; T, 2 g of Trypticase peptones per liter; Vitamins, 10 mi of a vitamin solution (39) per liter; CoM, 0.5 g of coenzyme M per liter; Ac,
0.5 mM sodium acetate; betaine, 5 mM glycine betaine; YE ash, ash (SSOOC, 1 h) from a preparation containing 2 g of yeast extract per liter.
aggregates from single Methanosarcina cells. Such cells
usually continued to grow as aggregates when they were
transferred to fresh medium under identical conditions.
After several transfers, the cells often disaggregated spontaneously (as previously described [40]) and grew as single
cells. When M . mazei S-6* growing on H, plus CO, as the
catabolic substrate was shifted from complex (MS) medium
to less complete medium (such as mineral medium), cells
grew as aggregates. Sometimes cells continued to grow as
aggregates for as many as 10 transfers and then disaggregated into individual cells (Fig. 5). In general, cultures
growing under better conditions took less time (fewer transfers) to revert to individual cells than did cultures growing
under extreme conditions (Fig. 5).
M . mazei LYC disaggregates when the pH is shifted from
6 to 7 (21), and likewise we found that M . mazei S-6T
disaggregated more rapidly at pH values closer to its optimum pH (data not shown).
(iii) M . vacuolata. In contrast to previous findings (43, 43,
we found that M . vacuolata Z-761T changed during growth
from small to large aggregates. This strain grew as small
clumps which appeared as a uniform turbidity during rapid
growth, such as when it was grown on methanol at pH 6.
Then, when growth was complete or almost complete, larger
aggregates visible to the unaided eye occurred as a sediment
at the bottom of the culture vessel. Also, small aggregates of
M . vacuolata Z-761T grew into larger aggregates when the
culture was shifted from complex medium to defined medium (i.e., mineral medium containing acetate added as the
catabolic substrate). This may be analogous to the shift of M .
mazei S-ST from individual cells to large aggregates under
similar conditions.
Taxonomy. (i) Differentiating M . mazei from M . barkeri and
M . vacuolata. When M . m a x i was transferred to the genus
Methanosarcina, its differentiation from M . barkeri was
based mainly on the inability of M . mazei to catabolize H,
plus CO, rapidly (24). However, we found that in our media
M . mazei S-6Trapidly catabolizes H, plus CO,, so this is not
a characteristic by which these species can be differentiated.
Also, M . vacuolata, which is known to be closely related to
M . barkeri ( 4 , 46), uses H, plus CO, only poorly.
The major characteristics that distinguish M . mazei from
the other two species appear to be morphology and pH
range. M . mazei often forms individual coccoid units which
are susceptible to lysis by sodium dodecyl sulfate, whereas
M . barkeri and M . vacuolata rarely grow as individual cells,
and when they do, these cells remain resistant to lysis.
Physiologically, M . mazei prefers higher pH values than M .
barkeri and M . vacuolata do.
(ii) Differentiating M . vacuolata and M . barkeri. M . vacuolata is closely related to M . barkeri, with 61% sequence
similarity between the type strains (46). M . barkeri cell
membranes contain no squalenes, but those of M . vacuolata
do (20,27). Gas vesicles occur in M . vacuolata but not in M .
barkeri. However, the taxonomic usefulness of gas vesicles
in methanogens is questionable because it is not a stable
characteristic (23a). At least two other gas-vacuolated Methanosarcina strains have been reported (strain W [4] and
strain FR-1 [l-3]), and if these strains were found to form a
phylogenetically cohesive group with M . vacuolata Z-761T,
it would support the view that M . vacuolata is a species
separate from M . barkeri.
ACKNOWLEDGMENTS
This study was supported by contract 5086-260-1303E from the
Gas Research Institute as part of a joint program on methane from
biomass sponsored by the Gas Research Institute and the University
of Florida Institute of Food and Agricultural Sciences and by the
Consejo Superior de Investigaciones Cientificas, Spain.
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