FEMS Microbiology Letters 20 (1983) 331-335
Published by Elsevier
331
Mass spectrometric measurements of methane and oxygen
utilization by methanotrophic bacteria
(Methane affinity; methane oxidation; Methylosinus trichosporium; growth yield)
Lars J o e r g e n s e n a n d H a n s D e g n
Institute of Biochemisto', Odense University, Campust,ej 55, DK- 5230 Odense M, Denmark
Received 6 July 1983
Accepted 19 July 1983
1. SUMMARY
A mass spectrometer with membrane inlet was
used to measure methane and oxygen utilization
rates at various methane concentrations in Methyl
osinus trichosporium and a locally isolated strain of
a methane-oxidizing coccus (OU-4-1). The apparent K m for methane was found to be 2/~M for
M. trichosporium and 0.8 /~M for strain OU-4-1.
These Kin-values are 10-30 times lower than most
previously reported values. The ratio of oxygen to
methane utilization rates was 1.7 for M. trichosporium and 1.5 for strain OU-4-1 corresponding
to a growth yield of 0.38 and 0.63 g dry weight/g
methane, respectively.
Methane utilization in methanotrophic bacteria
has been studied by various methods involving
oxygen uptake [4-9], methanol formation [10] and
radiotracer technique [11]. However, until now no
method for the direct measurement of dissolved
methane has been applied. We have used a mass
spectrometer with a membrane inlet for the direct
measurement of dissolved methane and oxygen to
study the methane oxidation by methanotrophic
bacteria in an open system. We have previously
used the mass spectrometric technique in studies
of respiration in amoebae [12], and annelids [13],
the Pasteur effect in yeast [14] and nitrogen fixation in cyanobacteria [15].
3. MATERIALS A N D M E T H O D S
2. I N T R O D U C T I O N
3.1. Organism and growth conditions
Methane can be used as sole carbon and energy
source by the methanotrophic bacteria. It is
oxidized to methanol by a monooxygenase which
requires N A D H as reductant and uses molecular
oxygen as the second substrate [1,2]. Methanol is
further oxidized to formaldehyde by a dehydrogenase with a novel prosthetic group (a pyrroloquinoline-quinone) [3]. Formaldehyde is either incorporated as cell material or fully oxidized to
carbon dioxide.
Methylosinus trichosporium OB3b was kindly
provided by Professor H. Dalton, University of
Warwick, Coventry, England. Strain OU-4-1, a
small coccus, was isolated from a pond near Odense
University by a modification of the isolation procedure described by Whittenbury et al. [16]. The
following mineral salt solution was used as the
medium (per liter of distilled water): KNO3, 1 g;
N a z H P O 4, 0.19 g; KH2PO4, 0.15 g; MgSO 4 7 H 2 0 ,
0378-1097/83/$03.00 © 1983 Federation of European Microbiological Societies
332
1 g; CaC12 2H20, 0.04 g; FeSO 4 7H20, 4 mg;
CuSO 4, 1 mg; ZnSO 4 7H20, 0.1 mg; MnC12 4H20,
0.04 mg; HzBO3, 0.3 mg; CoC12 6H20, 0.2 mg;
NiC12 6H20, 0.02 mg; and N a z M o O 4 and 2H20,
0.04 mg. The pH of the medium was adjusted to
6.8. N a 2 H P O 4 and KH2PO 4 were sterilized separately and added to the medium after autoclaving. The bacteria were grown at 28°C in unshaken
100-ml conical flasks containing 50 ml of medium
in an atmosphere consisting of methane, air,
nitrogen and carbon dioxide (approx. 3 0 : 2 0 :
48:2.5).
3.2. Instrumentation
A schematic representation of our experimental
set-up is shown in Fig. 1. The gas mixer for oxygen
and argon was based on calibrated flow resistors
[17]. Methane and argon were mixed by a computer-controlled gas mixer consisting of a threeway magnetic valve which alternated between the
two gases. The mixing ratio was controlled by
regulation of the proportion of time intervals of
opening to each gas supply. This was done by the
help of a microprocessor which converted a serial
ASCII command from the minicomputer into a
switching cycle for the valve. The ratio of time
intervals of the switching cycle could change with
a step of 1 / 1 2 7 and the cycle length was 2 s. The
valve used was from Kuhnke G.m.b.H., Malente,
Holstein, FRG. Methane and oxygen were measured by a quadrupole mass spectrometer (SX 200
with a DPP 16-X microprocessor, VG-Micromass
Ltd., Winsford, UK) fitted with a turbom01ecular
pump (DUO L5A, A. Pfeiffer Vakuumteknik,
Wetzlar G.m.b.H., Asslar, FRG). The inlet to the
CH,., ~ m e g n e t i c
~
[~
q
mass spectrometer was covered with a Teflon
membrane (12.6 jam thick). A minicomputer (Nova
4, Data General Corporation) with four multiplexor lines was used to collect data from the mass
spectrometer, to control the microprocessor connected to the magnetic valve, and to plot reduced
data on a digital plotter (Digi-plot, Watanabe
Instruments Corp., Tokyo, Japan).
3.3. Cell mass determination
Total carbon was measured with a Beckman
model 915 total organic carbon analyser. Analyses
were made on whole cell cultures after centrifugation (5000 × g, 10 min) and resuspension of the
pellet in CO2-depleted water. Total cell carbon is
equivalent to 47% of bacterial dry weight provided
the cell formula C4HvO2.2N0.75[18].
3.4. Methods
The open system technique has been described
in detail by Degn et al. [19]. It consists of a rapidly
stirred liquid sample in contact with a continually
renewed gas phase. In our experiments the gas
phase consisted of methane, oxygen and argon.
The oxygen and methane tensions were varied
independently by the two gas mixers. The diffusion of a gas between the gas phase and the liquid
sample is proportional to the difference in the
tension in the gas phase (T6) and the tension in
the liquid sample (TL). In a steady-state situation
where the tensions of the two phases are constant,
the rate of gas uptake can be calculated from
where K is a constant depending on the
gas-liquid interface, the volume of the
rate of stirring and the temperature.
ments where we have used a linearly
gradient of methane in the liquid phase
gas uptake was calculated from
V = K ( T G - TL)
Fig. 1. Schematicrepresentationof the measuring system.
(1)
V = K ( T G - - TL)
dTG
dt
area of the
liquid, the
In experiincreasing
the rate of
(2)
The concentrations of the dissolved gases were
determined from their solubilities. The solubility
used was 1100/IM for oxygen [20] and 1165 /~M
333
for methane [21], both values at 1 atm partial
pressure and 30°C.
Oxygen and methane were measured at m / e
= 32 and m / e = 16, respectively. All experiments
were carried out at 30°C in a 20 mM K-phosphate
medium, pH 7.0, with 5 mM MgCI 2.
-- 6 O
45
/
>
30
m
15
4. RESULTS
Fig. 2 shows the methane and oxygen concentrations in a sample of M. trichosporiurn. The
methane content of the gas phase was changed
stepwise as indicated by the dotted line. The oxygen
content of the gas was replaced by argon at the
arrow. The steady-state methane and oxygen utilization rates in the bacterial suspension were
calculated from equation (1) and Lineweaver-Burk
plots were made on the basis of the rate determinations (Fig. 3). Both plots show straight
lines which intercept at the x-axis. Thus both
o x y g e n and m e t h a n e utilization follows
Michaelis-Menten kinetics with respect to its dependence on the methane concentration in the
sample. The apparent K m for methane was 2 ~tM
and the ratio of oxygen to methane utilization
rates was 1.7.
100
80
50
- - -\. _ ~
........
x
J
i
.5
.S
0
i
I/[METHRNE]
1.5
2
I I / ~ HI
F i g . 3. Double reciprocal plot of methane utilization ( O ) and
oxygen utilization ( x ) a s a f u n c t i o n o f methane concentration.
The data were obtained from the experiment shown in F i g . 2 as
described in M A T E R I A L S
AND METHODS.
The computer-controlled experimental set-up
(Fig. 1) could be used to make a linear methane
gradient in the sample by a continuous adjustment
of the methane content of the gas phase. Fig. 4
shows such an experiment with a sample of strain
OU-4-1. Methane and oxygen utilization rates were
calculated from equation (2) and plotted as double
reciprocal plots. By this linear gradient method
Lineweaver-Burk plots with many measuring
points could be obtained in less than an hour, as
we do not have to wait for a steady state between
40
i
-x
:3.
v
x
.-
IE
i
- -
6O
Ar
3o
c~
taJ
co
--2.5
¢-4
2O ~
.......
20
10
5
__r---
0
~
'. . . .
I
0
I
30
2.5
rain
F i g . 2. Steady-state measurement of methane and oxygen utilization at different methane and oxygen concentrations by a
sample of Methylosinus trichosporium. The oxygen content o f
the gas phase was changed from 7.5% to 0% at the point
indicated by the arrow. • . . . . . , methane content of the gas
phase; - - ,
methane content of the sample; . . . . . .
, oxygen
content of the sample. The cell content of the sample was 100
mg organic carbon per liter.
1/[METHANE]
I1/#M1
F i g . 4. Double reciprocal plot of methane utilization ( O ) and
oxygen utilization ( x ) a s a f u n c t i o n o f methane concentration
in a sample of strain OU-4-1. The cell content of the sample
was 220 mg organic carbon per liter.
334
each gas shift. Control experiments showed that
K m and Vmax values obtained by this method resembled those found by the steady-state method.
The apparent K m for methane by strain OU-4-1
was found to be 0.8 /zM. The ratio of oxygen to
methane utilization rates was constant at all
methane concentrations in the sample and was
found to be 1.5.
Enriched cultures of methane-oxidizing bacteria
from different Danish lakes were also tested by the
linear gradient method. The apparent K m values
for methane of these cultures were between 0.5
and 2/zM (not shown).
5. D I S C U S S I O N
The ratio of oxygen to methane utilization rates
was found to be 1.7 for M. trichosporium and 1.5
for strain OU-4-1. A partial oxidation of methane
to methanol would give a ratio of 1 whereas a
complete oxidation to carbon dioxide would give a
ratio of 2. Some of the methane carbon is used for
the synthesis of cell material. If we assume that all
methane is either converted to cell material or
completely oxidized to carbon dioxide the equation for methane and oxygen utilization would be
CH 4 +
4 - 22325a 02 ~ aCH1.7500.55N0.2
+ (1 - a) CO 2 +
4 - 1.75a
2
H20
(3)
The growth yield, a, is then found by
a
4-2X
2.325
(4)
where X is the oxygen utilization rate divided by
the methane utilization rate. The ratio of oxygen
to methane utilization rates found for M. trichosporium and strain OU-4-1 are compatible with a
growth yield of 0.38 g dry w e i g h t / g methane and
0.65 g dry w e i g h t / g methane, respectively. A maxi m u m growth yield of 0.63 g dry w e i g h t / g methane
has been reported for M. trichosporium whereas
values between 0.31 and 1.01 g dry w e i g h t / g
methane have been reported for Methylococcus
capsulatus, depending on the growth conditions
[221.
Table 1
Reported values of apparent K m for methane
Organism
Km,
~M
Method a
Reference
(a)
This work
Methylosinu.s" trichosporium
OB3b
2
Mett~vlosinus trichosporium
OB3b
Methane monooxygenase
from Mettg,losinu~
trichosporium OB3b
Methylococcus spp.
Pseudomonas spp.
Strain OU-4-1
Methane oxidizing
bacterium
Mixed cultures
Mixed culture
45-48
(b)
9
66
32, 40
15, 26
0.8
(c)
(d)
(d)
(a)
10
5, 6
7, 8
This work
4.7
44, 19
1.73
(e)
(d)
(f)
11
5,4
23
a Methane oxidation rate was calculated from: (a) methane
and oxygen uptake measured by a mass spectrometer: (b)
oxygen uptake measured by gas-liquid chromatography: (c)
methanol formation measured by gas-liquid chromatography:
(d) oxygen uptake measured by an oxygen electrode: (e)
radiotracer technique; (f) steady-state growth rate in a continuous culture at several methane concentrations.
Reported Kin-values for methane by different
methanotrophic bacteria are shown in Table 1.
Most of these Km-values are higher than the values
we have found in this investigation. They have
been obtained without direct measurement of
methane concentration in the sample. In most
cases a small volume of a solution saturated with
methane was added to the sample and then the
oxygen uptake was measured. A different method
was used by L a m b and Garver [23]. They calculated steady-state methane concentrations in a
continuous culture of methane-oxidizing bacteria
at several partial pressures of gaseous methane. In
these calculations allowance was made for methane
oxidation by the culture. They found the Kin-value
to be 1.73 /xM which agrees well with our values.
The high Km-values reported in other papers (Table 1) are probably due to a too high estimate of
the methane concentration in the sample. The
oxidation of methane by the bacterial culture will
lead to an appreciable reduction of the dissolved
methane concentration, especially at low methane
concentrations. We suggest that all methanotrophic
335
b a c t e r i a h a v e a K m for m e t h a n e of a b o u t 1 # M . A
K i n - v a l u e o f 1 ~tM m e a n s t h a t m e t h a n e m o n o o x y g e n a s e is a l m o s t as w e l l - a d a p t e d to m e t h a n e as
c y t o c h r o m e c o x i d a s e is to o x y g e n , a n d t h a t n e a r l y
all m e t h a n e to w h i c h a c u l t u r e is e x p o s e d c a n be
used. T h e last p o i n t is of s o m e i m p o r t a n c e if
m e t h a n e o x i d i z i n g b a c t e r i a are to b e u s e d for
single cell p r o t e i n p r o d u c t i o n .
ACKNOWLEDGEMENTS
W e t h a n k Dr. R.P. C o x f o r r e a d i n g o f the
m a n u s c r i p t . T h i s w o r k was s u p p o r t e d b y the
D a n i s h S c i e n c e R e s e a r c h C o u n c i l , g r a n t 113495.
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