FEMS Microbiology Letters 109 (1993) 283-288
© 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00
Published by Elsevier
283
FEMSLE 05440
Tetraether type polar lipids increase
after logarithmic growth phase
of Methanobacterium thermoautotrophicum
in compensation for the decrease of diether lipids
Hiroyuki Morii and Yosuke Koga
Department of Chemistry, University of Occupational and Environmental Health, Kitakyushu, Japan
(Received 5 January 1993; revision received 8 March 1993; accepted 11 March 1993)
Abstract: The ratios of tetraether to diether type lipids in the total lipid during cell growth in batch cultures of Methanobacterium
thermoautotrophicum AH (DSM 1053) were examined. The proportion of tetraether type lipids to the total lipid was about 80%
during the log phase, and at the onset of the transient phase it began to rise up to about 93%. It was kept almost constant at that
level throughout the stationary phase. The polar lipid composition changed with the age of the cell culture. The proportions of all
the diether type polar lipids were lower and the levels of all tetraether type polar lipids were higher in the stationary phase than in
the log phase. On the other hand, the composition of polar head groups, irrespective of the core lipids, was nearly constant in both
growth phases measured so far despite the change in core lipid composition.
Key words: Archaebacteria; Ether lipid; Methanogenic bacteria
Introduction
Methanobacterium thermoautotrophicum possesses two kinds o f c o r e lipids: d i p h y t a n y l g l y c e r o l
d i e t h e r ( a r c h a e o l ) a n d d i b i p h y t a n e d i y l diglycerol
t e t r a e t h e r ( c a l d a r c h a e o l ) . T h e r e have b e e n several investigations o f c o r e lipid c o m p o s i t i o n o f
t h e o r g a n i s m [1-4]. R e c e n t l y , K r a m e r a n d S a u e r
[5] r e p o r t e d , for t h e first time, t h a t the d i e t h e r
Correspondence to: H. Morii, Department of Chemistry, University of Occupational and Environmental Health, 1-1
Iseigaoka, Yahatanishi-ku, Kitakyushu, Fukuoka 807, Japan.
type lipid ( d i e t h e r lipid) to t e t r a e t h e r t y p e lipid
( t e t r a e t h e r lipid) r a t i o i n c r e a s e d in t h e early log
p h a s e o f g r o w t h o f M. thermoautotrophicum
( A T C C 29183). B e c a u s e t h e possibility exists o f
f r a c t i o n a l e x t r a c t i o n o f lipids f r o m t h e cells d u e
to low e x t r a c t i o n yield d u e to t h e n e u t r a l solvent,
w e a t t e m p t e d to r e - e x a m i n e t h e c h a n g e of die t h e r lipid to t e t r a e t h e r lipid r a t i o d u r i n g growth
o f this o r g a n i s m by using an i m p r o v e d e x t r a c t i o n
m e t h o d . This p a p e r r e p o r t s on t h e results a n d
p o l a r lipid c o m p o s i t i o n s o f M. thermoautotrophicum in t h e log a n d s t a t i o n a r y p h a s e s . T h e
n o m e n c l a t u r e o f a r c h a e b a c t e r i a l lipids p r o p o s e d
by N i s h i h a r a et al. [6] is u s e d in this text.
284
Materials and Methods
tracted three times with petroleum ether. The
hydrocarbons were measured by gas-liquid chromatography (GLC) as described previously [9]
except at a temperature increasing from 140 to
340°C at a rate of 20°C m i n - 1. n-Hexacosane was
used as an internal standard. The proportion of
hydrocarbons was expressed in weight% unless
stated otherwise. The polar lipid composition was
measured as described previously [3] based essentially on phosphorus a n d / o r sugar measurements
of the spots on a TLC chromatogram, and expressed in mol%.
M. thermoautotrophicum AH (DSM 1053) was
obtained from the Deutsche Sammlung von
Mikroorganismen und Zellkulturen G m b H
(Braunschweig). The bacteria were grown at 65°C
in a 15-1 fermenter containing 5 1 of medium 2 as
described by Balch et al. [7] except that 0.1 mg
1- t NiCI 2 was added, while sparging with
H 2 / C O 2 (4: 1) at a flow rate of 2.5 1 min -t, and
N a 2 S . 9 H 2 0 solution was added every 12 h to
bring the concentration to 0.3 g I-1. Total lipid of
cells was extracted by the method of Nishihara
and Koga [3] with 5% TCA-acidified solvent.
To estimate the proportion of diether and
tetraether lipid, hydrocarbon chains were prepared from the total lipid by HI cleavage of the
ether linkages followed by LiAlH 4 reduction as
described by Kates et al. [8] simplified as follows.
Lipid (1 mg) was heated at 115°C with 55% HI (1
ml) for 20 h in a Teflon-lined screw-capped test
tube. After the resultant alkyl iodides were extracted three times with petroleum ether, they
were reduced to the corresponding hydrocarbons
with 7 mg of LiAlH 4, 1 ml of diethyl ether and
two drops of chloroform in a Teflon-lined screwcapped test tube for 1 h at 100°C under continuous stirring in an aluminum block heating bath
with a magnetic stirrer (Dry-block multi stirrer
DM-8, Scinics, Japan) and a small Teflon-coated
stirring rod. The excess hydride was decomposed
by dropwise addition of 1 ml water, after which 3
ml of 2 M HCI was added to the mixture. From
this mixture the resultant hydrocarbons were ex-
Results
Although Hedrick et al. [4] described that
preparation of hydrocarbons by HI cleavage of
ether bonds and L i A l n 4 reduction was not quantitative as a method for the determination of core
lipid composition, we employed this method after
re-examining it. Phytanyl and biphytanediyl chains
represent diether and tetraether type polar lipids,
respectively, in M. therrnoautotrophicum. Known
amounts of gentiobiosyl caldarchaetidylinositol
and archaetidylinositol were mixed and then the
hydrocarbon chains were prepared from the lipid
mixture. Table 1 shows that C20 and C40 hydrocarbon chains were detected with almost identical
recoveries (80%) in two mixtures with different
compositions. Because the hydrocarbon chains
prepared from M. thermoautotrophicum lipids
were exclusively composed of C20-phytanyl and
C40-biphytanediyl chains (more than 99%), this
Table 1
Recovery of hydrocarbon chains during preparation by HI cleavage and LiAIH 4 reduction method from the lipid mixtures of
gentiobiosyl caldarchaetidylinositol (GCI) and archaetidylinositol (AI)
Expt.
No.
Lipids mixed
at the ratio of (mol%)
Hydrocarbon chains
detected (mol%)
Recovery
of hydrocarbon chains (%)
GCI a AI a
C40H82 b C20H42 b
C40H82
from GCI
C20H42
from AI
70:
53:
82
81
78
81
1
69:
2
53:
31
47
30
47
Gentiobiosyl caldarchaetidylinositol and archaetidylinositol were purified from the total lipids of M. thermoautotrophicum by
TLC and were mixed at two different ratios. Mol% was estimated by the determination of phosphorus in the lipids.
b Hydrocarbon chains were prepared from the mixture of gentiobiosyl caldarchaetidylinositol and archaetidylinositol, and
measured by GLC as described in Materials and Methods.
a
285
result confirmed that the H I - L i A I H 4 method
could readily be used for the determination of
core lipid composition even if the individual recovery was not 100%.
Figure 1 shows a growth curve and the proportion of tetraether lipid to total lipid during the
growth. In spite of the variation of tetraether
lipid proportion depending on batches of cultures, the proportion of tetraether lipids was kept
almost constant (about 80%) during the log phase
until 22 h in each culture, and then rose up to
around 93% between 22 h and 61 h (the transient
phase). When the cells entered the stationary
phase (at 61 h), the tetraether lipid proportion
was levelled off and remained unchanged at about
93% afterwards.
The recoveries of total lipid by the TCA-acid
extraction were 47.5 ___0.5 mg g - 1 dry ceils in the
log phase (n = 2) and 33.5 + 0.5 mg g-1 dry cells
in the late stationary phase (n = 2). On the other
hand, the proportions of lipid to protein in the
cells in these growth phases were almost the
same: 67 and 62 mg lipid g-~ protein in the log
and the late stationary phases, respectively. When
the lipid was extracted with a neutral solvent by
the usual Bligh and Dyer method, only 3 mg lipid
g-1 dry cells was obtained in the late stationary
phase. I t was 1 / 1 0 of the TCA-acid extract. Autoclaving cells before lipid extraction, which was
employed by Kramer and Sauer [5], showed little
improvement of recovery of lipid. The proportion
of tetraether lipid in the neutral solvent extract
from the log phase cells (day 1) was 11%, which
was remarkably lower than that (80%) of the
TCA-acid extract.
Polar lipid compositions of the organism in the
log and stationary phases were shown in Table 2.
During the course of growth from the log phase
to the stationary phase, the proportions of all the
diether type polar lipids were lowered while those
of all the tetraether type polar lipids rose. The
compositional change shown in Table 2 con-
Table 2
Compositions of polar lipids extracted from M. thermoautotrophicum cells in the log phase (day 1) and in the late stationary phase
(day 5) by TCA-acid extraction
Lipid
mol%
Log phase
( A 6 6 0 = 1.0; n = 2)
Late stationary phase
( A 6 6 0 = 3.0; n = 2)
Diether iipids
Archaetidylethanolamine
Gentiobiosyl archaeol
Archaetidic acid
Archaetidylserine
Archaetidylinositol
4.2 ± 0.7
9.9 + 1.4
0.9 + 0.1
9.1 ± 0.2
15.7 ± 1.8
1.4 + 0.3
5.5 + 0.9
0
2.8 ± 0.4
3.1 ± 0.5
Subtotal
39.6 ± 1.1
12.8 ± 2.1
Caldarchaetidylethanolamine
Gentiobiosyl caldarchaeol
Caldarchaetidic acid
Caldarchaetidylserine
Caldarchaetidylinositol
Gentiobiosyl caldarchaetidylethanolamine
Gentiobiosyl caldarchaetidylserine
Gentiobiosyl caldarchaetidylinositol
0
0.4±0.1
1.0±0
0
0
3.2±0.2
4.0±0.7
45.1±2.3
Trace
3.6±0.6
2.5±0.3
Trace
0.5±0.1
5.0±0.3
4.5±0.2
65.1±3.9
Subtotal
53.6±1.7
81.1±3.0
6.8±0.6
6.3±0.9
Tetraether lipids
Others
286
100"
73% in the log phase and 93% in the stationary
phase. These values were consistent with the hydrocarbon proportion in each phase shown in
Fig. 1.
In spite of the striking change of polar lipid
composition, the composition of polar head
groups, irrespective of the core lipids, was maintained almost constant throughout the growth
cycle (Table 3).
a
Go-
i.
I.--
70
i
.
0
,
~n
,
,
•
40
,
eO
.
i
80
.
i
100
•
120
10
b
0
Discussion
20
e)
~
80
11111
120
Time ( h i
Fig. 1. The proportion of tetraether type lipids to the total
lipid (a) and the growth curve (b) of M. thermoautotrophicum.
The curves represent the average values (n = 5) except the
tetraether lipid proportion data at 0 h (n = 2).
firmed the increase of tetraether lipids proportion detected by hydrocarbon chain ratio during
the transient phase. Roughly assuming that the
average molecular mass of tetraether polar lipids
is twice that of diether polar lipids, it was calculated from the mol% shown in Table 2 that
weight% of the total tetraether polar lipids was
Table 3
Compositions of polar head groups of polar lipids extracted
from M. thermoautotrophicum cells in the log phase and the
late stationary phase
Polar head groups
Phosphate (monoester)
Phosphoethanolamine
Phosphoserine
Phosphoinositol
Gentiobiose
mol%
Log phase
1.3
5.0
9.0
41.7
42.9
Late stationary
phase
1.5
3.8
4.3
40.7
49.6
The results were calculated from the data of Table 2.
The results presented here were contradictory
t o the results of K r a m e r and Sauer [5]. The
differences of strains and the growth conditions
may be part of possible causes of the discrepancy.
The main and more probable reason of the contradiction lies, however, in the differences in
methods of lipid extraction. We had already developed indispensable methods for methanogen
lipid analysis in 1987, one of which is the extraction method of lipid [3]. Kramer and Sauer [5]
extracted lipid (1.6%) by the usual Bligh and
Dyer method with a neutral solvent. The neutral
extraction gives not only low recovery of lipids
but also causes fractional extraction, that is, the
method shows almost quantitative recovery in diether lipids but lower recovery in tetraether lipids
[3]. However, we extracted three times the amount
of lipid (4.8%) of dry mass from the log phase
cells by using TCA-acid extraction. The lower
value (3.4%) of lipid per cell in the late stationary
phase seems to reflect the truly low content of
the cellular lipid but seems not to be the result of
low recovery in extraction, because the proportions of lipid to protein in the cell were held at an
almost constant level throughout the growth
phases. This suggests that fractionation of lipids
did not occur upon extraction.
A n o t h e r cause of contradiction between
Kramer and Sauer [5] and us might be the method
of core lipid preparation (acid methanolysis). We
have discussed the incomplete removal of the
polar head groups from ether type aminophospholipids [3,10]. We estimated core lipid composition from the determination of isoprenoid chains
of lipids because of simplicity, accuracy, sensitiv-
287
ity and availability of GLC. The core lipid ratio
obtained was satisfactory because of the identical
recovery of C20 and C40 chains.
We hypothesize that the tetraether type polar
lipids are synthesized from two diether type polar
lipid precursors because of the structural regularities of diether and tetraether type polar lipids of
Methanospirillum hungatei [11], M. thermoautotrophicum [9] and Methanobrevibacter arboriphilicus [12]. The almost constant composition
of polar head groups is compatible with the above
hypothesis of biosynthesis of tetraether type polar
lipid.
T h e reason why the proportion of tetraether
lipid rises in the transient phase remains unknown. In the transient phase, the cell growth
seems to be restricted by the limitation of H 2
supply. Tetraether lipid synthesis might be accelerated by the limitation of H 2.
The lipid composition of batch-cultured ceils
reported here did not confirm the lipid composition of the cells of the same species cultured
semi-continuously [3]. Particularly, the contents
of caldarchaetidylethanolamine, caldarchaetidylserine and caldarchaetidylinositol in batch cultures were much lower and the content of gentiobiosyl caldarchaetidylinositol was much higher
than those of the semi-continuously cultured cells.
A long-term semi-continuous cultivation (more
than 1 year) might have caused the strain to alter
the lipid metabolism. Unfortunately, after the
previous study had been completed, the 'strain' of
the prolonged semi-continuous culture that
showed the reported lipid composition had not
been maintained. A new active culture of M.
thermoautotrophicum AH was obtained from
D S M and used throughout this study.
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