FEMS MicrobiologyEcology73 (1990) 221-224
Published by Elsevier
221
FEMSEC 00253
Methanogenic bacteria as a key factor involved in changes
of town gas stored in an underground reservoir
P. Smigfih 1, M. Greksfik 1, j. Kozfinkovfi 1 F. Buzek z, V. O n d e r k a 3 a n d I. Wolf ~
institute of Animal Physiology, Slovak Academy of Sciences, lvanka pri Dunoji, CzeehoMovokia~
2 Geological Survey. Prague, Czechoslovakia. and 3 Institute of Geological Engineering, Brno, Czechoslovakia
Rctmived21 July 1989
Revision received 19 October 1989
Accepted 24 October1989
Key words: Methanogenic bacteria; Town gas; Underground gas reservoir
1. SUMMARY
The microbial population present in stratal
water withdrawn from an underground town gas
reservoir transformed tt 2 and CO 2, which are the
obligatory constituents of town gas, to methane.
By a common procedure one type of mesophilic
Gram-positive methanogenic bacteria was enriched. The bacteria grew and produced methane
with H 2 + CO 2 only. This finding suggests that
methanogenic bacteria present in the underground
town gas reservoir could be responsible for the
consumption of hydrogen and carbon dioxide from
the stored town gas, causing a diminution of the
gas volume and an enrichment of the gas with
methane. An isotopic analysis of the stored town
gas, in which 6t3C for methane around -80%0
was found, supports this suggestion.
Correspondence to: P. ~migLh, Institute o[ Animal Physiotogy,
Slovak Academy of Sciences, 900 28 IvarJga pri Dunaji,
Czechoslovakia.
2. I N T R O D U C T I O N
The study of microbial ecology of the terrestrial
subsurface led to reevaluation of our opinions on
the region beneath the surface of the earth [1].
Identification of different microorganisms in deep
subsurface environments [2,3] and new sampling
methods [4] have opened the way to the understanding of microbial life in this kind of allobiosphere [5]. Methanogenic bacteria also have been
found to be members of bacterial communities in
some underground environments [1,6-10]. In a
water-saturated structure of an underground reservoir of town gas containing hydrogen and
carbon dioxide, a diminution of stored town gas
was observed. Since methanogenic bacteria exhibit
extreme habitat diversity [1I] and most of them
can grow with hydrogen and carbon dioxide as
their sole carbon and energy source [12], we regard
them as one of the chief agents for the changes
observed.
Here we report a search for methanogenic
bacteria in this environment, their enrichment,
partial characterization, and their potential contribution to the observed town gas volume diminution including an enrichment of the stored gas
with methane.
0168-6496/90/$03.50 © 1990 Federation of European Microbiological~k~eties
222
3. MATERIALS AND METHODS
3.1. Site description and sampling,
The underground reservoir of town gas had
been artificially created in water-saturated strata
of an anticlinal structure near Lobodic¢,
Czechoslovakia. The main storage strata of the
reservoir are heterogenous, formed by Miocene
rock-sand, gravel and sandstone. Samples of underground stratal water were collected from wells
located near the water-gas contact phase. The
wells equipped with valves were integral parts of
the town gas reservoir. A special sampler 'Subsurface Sampler Model 60' (Leutert, F.R.G.) was
used for sampling. The samples were withdrawn
from 400 to 500 m depth. The construction of the
subsurface sampler allowed to perfoxTn sampling
in a manner that prevented contamination of sampies from surface microorganisms and oxygen.
3.2. Enrichment
The original samples of the stratal water (5 ml)
for enridtment were anaerobically transferred to
50-ml volumes of anaerobic cultivating medium
No. 2 prepared according to Balch et at. [12] using
the Hungate technique as modified by Bryant [t3],
pressurized to 150 kPa with hydrogen and carbon
dioxide mixture in the ratio 4:1, and incubated on
a gyratory shaker at 37 and 60 °C for 2-4 weeks.
Bacterial suspensions yielding methane were
transferred ~veral times to new medium and
cultivated as described above. After several such
successive transfers, specific antibiotics (penicillin
G 500, cycloserine 10 and kanamycin 100 pg per
nd) were included into the growth medium to
minimize growth of non-methanogenic microorganisms. This procedure was repeated twice.
After sub-cultivation of these bacteria in antibiotic-free medium, only one type of methane-producing cells was obtained.
3. 3. A nalytical methods
Methane formation by bacterial suspensions
was measured as described before [14]. The
13C/12C ratios of CO t samples, expressed as 813C,
were analysed with a Finningan MAT 251 Mass
Spectrometer (Finningan, Cincinnati, OH, U.S.A.)
in which PDP carbonate was used as a standard.
Before measurement, town gas samples containing
CO2, CO, and methane were passed through liquid
nitrogen traps to remove CO: which was subsequently used for isotopic analysis. Methane and
carbon monoxide were separated from gas mixtures by gas chromatography and, after separation, converted to CO2 by combustion at 950 o C
on columns filled with CuO. The redox potential
( E h ) of the samples was measured in a simple
apparatus for measuring the E h of anaerobic media
[15], using a platinum r~lox electrode No. 96-78
connected to an Ionanalyzcr Modal 901 (Orion
Research, Cambridge, U.S.A.).
3.4. Microscopy
A Jena-Lumar Microscope (G.D.R.) equipped
for epifluorescence and with appropriate filters
was used for direct ob,~ervation of bacterial fluorescence.
3.5. Chemicals
All chemicals were of analytical grade purity
and purchased mostly from Lachema (Brno,
C.S.S.R.), except for antibiotics which were obtained from Svrva (Heidelberg, F.1LG.). The gases
hydrogen, carbon dioxide and argon in which
oxygen was removed on a column containing
copper particles at 350°C were purchased from
Chemika (Bratislava, C.S.S.R.).
4. RESULTS AND DISCUSSION
Observed changes in volume (losses of 10 to
20~) and composition (see Table 1) of town gas
stored in an underground reservoir (pressure
wound 4 MPa, temperature between 25 and 45 ° C)
have so far not been attributed to microbial activity. Therefore, we examined whether the microbial
population of the stratal water in the reservoir
performed the observed transformation.
Samples of underground stratal water were
characterized as a slightly opalescent, yellowish
and not very turbid liquid, pH 6.5 to 7.0, exhibiting an oxidoreduction potential around - 330 inV.
Collected samples exerted a population density of
methanogen-like microorganisms about 10 ~ to 104
223
Table l
Chemical composition * of town gas before (Input) and after
storage (Output) for 7 months in the underground reservoir
Component
Input
(vol %)
Output
(vol IS)
CH4
C~ H 4
C 2H s
C~-hydrocarbons
C4-hydrucarbons
CO
CO 2
N2
H2
21.90
0.05
0.36
0.08
0.01
9.00
11.67
2~50
54.00
40.00
0.01
0.52
0.16
0.02
3.30
8.78
8.60
37.00
* The data were kindly provided by the analytical laboratory
of the Reservoir Gasworks at Lobodice.
cells per ml as observed by direct epifluorescence
microscopy.
To imitate partly the conditions in the reservoir, sample, s of the stratal water were aseptically inoculated into sterile media of the following
composition: (i) original stratal water; (ii) original
stratal water containing 10% of gently powdered
rocks obtained from the underlying paleozoie
structure of the reservoir composed of amphibolites and phyllites; (iii) 10% of the rock powder in
distilled water. As can be seen from Fig. 1, a
microbial population cultivated at 37°C with hydrogen and carbon dioxide transformed hydrogen
and carbon dioxide to methane in all med~.z used.
The reck powder suspended in the stratal water
was the most suitable medium for methane production. This result indicated that methanogenic
bacteria were part of the bacterial community of
the reservoir and could be responsible for the
diminution of the stored gas volume and for its
enrichment in methane content.
Enrichments with the samples incubated at
3 7 " C yielded methane-forming cultures containing rods which fluoresced at 420 nm. No methanogenie bacteria were enriched in the samples incubated at 60°C. The enriched bacteria were
Gram-positive non-motile rods with a tendency to
form aggregates but not filaments. They grew and
produced methane with hydrogen and carbon dioxide, but not with acetate, formate, methanol,
trimethylamine or monomethylarnine. The temperature optimum for growth and methane produetion was between 37 and 40°C, the optimal
pH value was 6.5-7.0.
To verify oar suggestion that methanogenie
bacteria can play an important role in the transformation of the stored town gas, isotopic analysis
of the gas was performed. Mass spectrometry allows to differentiate methane of biological origin
from other processes on the basis of the ~3C/t2C
ratio I16-18]. The ~3C value of methane in the
town gas stored in the reservoir was around - 8 0 ~ ,
while ~13Cct~" of the town gas before being pressed
into the reservoir was in average -34.5%0. This
result indicates that the methane formed during
storage of the town gas in the underground reservoir was of biological origin.
The obtained results suggest that methanogenie
bacteria present in the underground town gas reservoir can take part in the consumption of some
constituents - - hydrogen and carbon dioxide - of the town gas in that reservoir. This biological
transformation is associated with gas volume diminution and with an enrichment of the starting
gas with methane, and represents a serious economic and technological problem.
'70
//o. / f
31
o
2O
II
I
50
100
150
200
250
Time (h)
Fig, l, Methane formation from H 2 + C O ~ by the stratalwater
bacterial population in the following media: ~ - original
stratat water; o - original stratal waler containing 10'$ of
gently powdered rocks obtained from underlying palco'zoic
structure of the reservoir; • - 10% of the rock powder in
distilled water. Cultivation temperature 37 e C.
224
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