196
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
through the tube going down to the bottom of the experimental bottle. The
bottle is then hermetically closed with a stopper, trying not to provoke the formation of air bubbles, and placed into a black bag. One extra sample from the
redox zone should be taken to replenish water in bottles after the injection of
the working solution of 14C-carbonate. The bigger the bottles, the easier it is
to preserve the ambient redox potential in them. A brief description of all
three above ways of separation of chemosynthetic and heterotrophic 14C02
dark uptake is given below.
1. Use of respiration: CO2 uptake ratio
The experiment is conducted as described in the above experiments for the
control of possible chemosynthetic CO2 uptake (see Sect. 4.4.2.2.2), and the
ratio MI Ac is calculated, where M is the respiration as mg 0 21- 1 day, and Ac is
the dark CO2 uptake as mgCl- 1 day-I. If this ratio is <80, chemosynthetic 14C02
assimilation takes place. In this case, only the heterotrophic dark CO2 assimilation (Ach) is first calculated as follows: ACh = M12 /-lg C 1-1, where 12 is the
amount of CO2 fixed by heterotrophic bacterioplankton per 1 mg of O 2 consumed during respiration, /-lg C. Then the chemosynthetic CO2 (Aca) uptake
will be: Aca = Ac - A ch' The total bacterial production contributed by both heterotrophic and autotrophic microbial populations (Ph) in this case can be calculated as follows: Ph = (Ach X 12) + (Aca x 2.5) x 10 3 mg C m- 3 day-I in the
samples taken in layers with predomination of methanotrophs, e.g., in oxygendeficient strata, where the free H 2S is practically absent. In the samples taken
from the redox-zone layer situated at the 02/H2S interface, where the
lithotrophic thiobacilli are the main consumers of exogenous CO2, the value
of Ph could be estimated as: Ph = (12 x A ch ) + (Aca x 1.2).
2. Use of Na-azide
This technique has also been described above for the separation of Ach and
Aca in bottom sediments. In the water column the same approach could be
used, e.g., three bottles are subsampled from each sample with the corresponding precautions recording the redox potential: one intact, one with 8 mg
I-I of Na-azide added, and one the dead control. The rest of the procedure is
the same as described above for Ac estimation in water columns. All the calculations of Ach and Aca are similar to those given above for the bottom sediment, while the calculations of Ph is similar to those described above (1).
3. Use of iodoacetamide (lAM) 5 mmoll- I
This inhibits chemosynthetic CO2 assimilation. With the aid of this inhibitor,
it is possible to estimate chemosynthesis also in bottom sediments (Tobin and
Anthony 1978).
4. Separation of ACh and Aca by measuring the relative activity of heterotrophic bacteria
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
through the tube going down to the bottom of the experimental bottle. The
bottle is then hermetically closed with a stopper, trying not to provoke the formation of air bubbles, and placed into a black bag. One extra sample from the
redox zone should be taken to replenish water in bottles after the injection of
the working solution of 14C-carbonate. The bigger the bottles, the easier it is
to preserve the ambient redox potential in them. A brief description of all
three above ways of separation of chemosynthetic and heterotrophic 14C02
dark uptake is given below.
1. Use of respiration: CO2 uptake ratio
The experiment is conducted as described in the above experiments for the
control of possible chemosynthetic CO2 uptake (see Sect. 4.4.2.2.2), and the
ratio MI Ac is calculated, where M is the respiration as mg 0 21- 1 day, and Ac is
the dark CO2 uptake as mgCl- 1 day-I. If this ratio is <80, chemosynthetic 14C02
assimilation takes place. In this case, only the heterotrophic dark CO2 assimilation (Ach) is first calculated as follows: ACh = M12 /-lg C 1-1, where 12 is the
amount of CO2 fixed by heterotrophic bacterioplankton per 1 mg of O 2 consumed during respiration, /-lg C. Then the chemosynthetic CO2 (Aca) uptake
will be: Aca = Ac - A ch' The total bacterial production contributed by both heterotrophic and autotrophic microbial populations (Ph) in this case can be calculated as follows: Ph = (Ach X 12) + (Aca x 2.5) x 10 3 mg C m- 3 day-I in the
samples taken in layers with predomination of methanotrophs, e.g., in oxygendeficient strata, where the free H 2S is practically absent. In the samples taken
from the redox-zone layer situated at the 02/H2S interface, where the
lithotrophic thiobacilli are the main consumers of exogenous CO2, the value
of Ph could be estimated as: Ph = (12 x A ch ) + (Aca x 1.2).
2. Use of Na-azide
This technique has also been described above for the separation of Ach and
Aca in bottom sediments. In the water column the same approach could be
used, e.g., three bottles are subsampled from each sample with the corresponding precautions recording the redox potential: one intact, one with 8 mg
I-I of Na-azide added, and one the dead control. The rest of the procedure is
the same as described above for Ac estimation in water columns. All the calculations of Ach and Aca are similar to those given above for the bottom sediment, while the calculations of Ph is similar to those described above (1).
3. Use of iodoacetamide (lAM) 5 mmoll- I
This inhibits chemosynthetic CO2 assimilation. With the aid of this inhibitor,
it is possible to estimate chemosynthesis also in bottom sediments (Tobin and
Anthony 1978).
4. Separation of ACh and Aca by measuring the relative activity of heterotrophic bacteria