Study of Microbial Methane Oxidation Using 14C
223
of methane-oxidizing bacteria in the water column and in the upper layer of
bottom sediments with the aid of 14COZ as a label, (3) measuring the absolute
rates of methane oxidation in water columns and in upper layer of sediments
with the use of 14CH4 gas as the label, and (4) investigation of the probable
role of methane-oxidizing microfiora in the production of food for aquatic
animals, using of 14COZ or l 1 CH4 as labels. The raJioisotopic kchniyue for the
study of ambient methane oxidizing microfiora and its activity was developed
accounting for the most specific feature of the methane-oxidizing bacteria, that
they involve a significant amount of external COz in their cell synthesis. Its
share in their biomass in natural populations is usually close to 30%, while in
cultures it can reach 50-70% (Sorokin 1961b, 1964b; Malashenko et al. 1978;
Romanovskaya et al. 1980, see Table 4.4). Grown on methane, in methaneoxidizing bacteria over 85% of the carbon oxidizes to COz, and 10 to 15% use
their biomass for synthesis. Therefore to measure their production, 14COZ can
be used as a label, which is much more convenient than 14C-Iabeled methane
gas. Similarly, 14COZ may be used also to measure the relative activity of the
microbial populations oxidizing methane in water columns, which greatly
simplifies resolution of the above problems. The labeled methane gas 14CH4 is
used mainly for measuring in situ rates of oxidation in water basins.
5.2.2 Practical Procedures
1. Preparation of 14CH4 gas working stocks
The working solution of 14C-carbonate with a radioactivity of about 2010 6 cpm
ml- 1 (-1011 Ci ml- 1 ) can be used to study the spatial distribution of methaneoxidizing bacteria in water basins. Its method of preparation is described
above (see Sect. 2.3.2.1). To study in situ rates of methane oxidation the 14CH4
working gas stock should be prepared having a specific radioactivity of
about 0.511 Ci cm- 3 • To prepare it, first the labeled methane should be extracted
from its batch turned upside down in water with the aid of a syringe supplied
with a fine silicon tube. It is then transferred into a serum bottle overturned
in water, which contains the necessary volume of nonlabeled methane to
achieve the above-mentioned specific radioactivity of about 0.211 Ci cm 3 in
accordance with the total radioactivity contained in the original 14CH4 batch
purchased from the supplier. Then the bottle is closed with the serum silicon
rubber stopper arranged as shown in Fig. 5.1. The amount of methane necessary for field work is stored in a little serum bottle arranged in the same way.
Required volumes are extracted for this bottle with a syringe through the
serum cork. The volume of gas removed is replaced by 10% NaCI solution
through the tube. In the same way, the stock of nonlabeled methane can be
prepared for injection into the bottles in experiments where 14COZ serves as
a label.
223
of methane-oxidizing bacteria in the water column and in the upper layer of
bottom sediments with the aid of 14COZ as a label, (3) measuring the absolute
rates of methane oxidation in water columns and in upper layer of sediments
with the use of 14CH4 gas as the label, and (4) investigation of the probable
role of methane-oxidizing microfiora in the production of food for aquatic
animals, using of 14COZ or l 1 CH4 as labels. The raJioisotopic kchniyue for the
study of ambient methane oxidizing microfiora and its activity was developed
accounting for the most specific feature of the methane-oxidizing bacteria, that
they involve a significant amount of external COz in their cell synthesis. Its
share in their biomass in natural populations is usually close to 30%, while in
cultures it can reach 50-70% (Sorokin 1961b, 1964b; Malashenko et al. 1978;
Romanovskaya et al. 1980, see Table 4.4). Grown on methane, in methaneoxidizing bacteria over 85% of the carbon oxidizes to COz, and 10 to 15% use
their biomass for synthesis. Therefore to measure their production, 14COZ can
be used as a label, which is much more convenient than 14C-Iabeled methane
gas. Similarly, 14COZ may be used also to measure the relative activity of the
microbial populations oxidizing methane in water columns, which greatly
simplifies resolution of the above problems. The labeled methane gas 14CH4 is
used mainly for measuring in situ rates of oxidation in water basins.
5.2.2 Practical Procedures
1. Preparation of 14CH4 gas working stocks
The working solution of 14C-carbonate with a radioactivity of about 2010 6 cpm
ml- 1 (-1011 Ci ml- 1 ) can be used to study the spatial distribution of methaneoxidizing bacteria in water basins. Its method of preparation is described
above (see Sect. 2.3.2.1). To study in situ rates of methane oxidation the 14CH4
working gas stock should be prepared having a specific radioactivity of
about 0.511 Ci cm- 3 • To prepare it, first the labeled methane should be extracted
from its batch turned upside down in water with the aid of a syringe supplied
with a fine silicon tube. It is then transferred into a serum bottle overturned
in water, which contains the necessary volume of nonlabeled methane to
achieve the above-mentioned specific radioactivity of about 0.211 Ci cm 3 in
accordance with the total radioactivity contained in the original 14CH4 batch
purchased from the supplier. Then the bottle is closed with the serum silicon
rubber stopper arranged as shown in Fig. 5.1. The amount of methane necessary for field work is stored in a little serum bottle arranged in the same way.
Required volumes are extracted for this bottle with a syringe through the
serum cork. The volume of gas removed is replaced by 10% NaCI solution
through the tube. In the same way, the stock of nonlabeled methane can be
prepared for injection into the bottles in experiments where 14COZ serves as
a label.