184
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
by aquatic microorganisms no longer creates any technical problems since the
introduction of the 14COz-methodology by E. Steemann Nielsen. The only
problem is to estimate a reliable coefficient to calculate bacterial production
from the 14COZ uptake values thus received, e.g., to estimate the average share
of exogenous COz-carbon in the biomass of a given microbial population. The
first attempt at practical implementation of such a possibility ,,,as carried out
by Prof. S. Kuzetsov in 1951, whom I assisted at that time as postgraduate
student. Later, in 1953-1955, using this methodology, I studied chemosynthesis in earth-bottom anoxic layers containing methane and hydrogen, which
were formed in water under the ice in the Rybinsk reservoir, and found
that the chemosynthetic production of microbial biomass in these layers
allowed the survival of abundant zooplankton in its former river beds, providing food during the winter period (Sorokin 1955b, 1958c, 1965). High rates
of chemosynthetic production by bacteria were also observed at that time in
the Black Sea and in meromictic lakes (Sorokin 1964a, 1965, 1970b). During
these studies, I found that a significant uptake of 14COz proceeds in the dark
also in upper water layers outside the redox zones, which cannot be attributed
to chemosynthetic bacteria because in these layers the energy sources for
chemosynthesis were absent; so it could only be a result of uptake by heterotrophic bacteria.
By that time it was also known that heterotrophic bacteria use exogenous
COz for biosynthesis (Lebedev 1921; Wood and Werkman 1936; Kornberg
1966). From the first approach it seemed nonsensical because the heterotrophic cells oxidizing organic matter produce a much COz internally. Also,
why should they expend energy driving external COz inside their cells? Later,
it became clear that bacteria accomplish hydrolysis and oxidation of organic
molecules for obtaining energy outside the cell in their cell membrane. The
simple organic molecules needed for their biosynthesis, as well as the CO2 molecules, are transported inside the cell with the participation of the enzymatic
transport system through the special channels. The most striking examples
confirming this scheme I have observed myself when I studied the share of
external CO2 in the cells of bacteria with different types of carbon nutrition.
A new species of bacteria isolated, which used formate as a single substrate
for growth: Bacterium formoxidans. Then I discovered that this bacterium,
when growing on 14C-Iabeled formate as single organic substrate in cylinders
through which the stream of air + 5% CO2 gas mixture was bubbling, was producing practically unlabeled biomass. This means that Bacterium formoxidans
does not use 14C02 at all for biosynthesis, which it produces during the oxidation of the labeled formate (Sorokin 1961b). I observed the same phenomenon when I studied the carbon metabolism in the anaerobic sulfate-reducing
bacterium Desulfovibro desulfuricans, which oxidizes lactate to acetate anaerobically to obtain energy. However, when being grown on whole-chain labeled
lactate, this bacterium does not use the labeled acetate formed during lactate
oxidation for its biosynthesis, but incorporates the exogenous unlabeled
acetate from the medium (Sorokin 1966).
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
by aquatic microorganisms no longer creates any technical problems since the
introduction of the 14COz-methodology by E. Steemann Nielsen. The only
problem is to estimate a reliable coefficient to calculate bacterial production
from the 14COZ uptake values thus received, e.g., to estimate the average share
of exogenous COz-carbon in the biomass of a given microbial population. The
first attempt at practical implementation of such a possibility ,,,as carried out
by Prof. S. Kuzetsov in 1951, whom I assisted at that time as postgraduate
student. Later, in 1953-1955, using this methodology, I studied chemosynthesis in earth-bottom anoxic layers containing methane and hydrogen, which
were formed in water under the ice in the Rybinsk reservoir, and found
that the chemosynthetic production of microbial biomass in these layers
allowed the survival of abundant zooplankton in its former river beds, providing food during the winter period (Sorokin 1955b, 1958c, 1965). High rates
of chemosynthetic production by bacteria were also observed at that time in
the Black Sea and in meromictic lakes (Sorokin 1964a, 1965, 1970b). During
these studies, I found that a significant uptake of 14COz proceeds in the dark
also in upper water layers outside the redox zones, which cannot be attributed
to chemosynthetic bacteria because in these layers the energy sources for
chemosynthesis were absent; so it could only be a result of uptake by heterotrophic bacteria.
By that time it was also known that heterotrophic bacteria use exogenous
COz for biosynthesis (Lebedev 1921; Wood and Werkman 1936; Kornberg
1966). From the first approach it seemed nonsensical because the heterotrophic cells oxidizing organic matter produce a much COz internally. Also,
why should they expend energy driving external COz inside their cells? Later,
it became clear that bacteria accomplish hydrolysis and oxidation of organic
molecules for obtaining energy outside the cell in their cell membrane. The
simple organic molecules needed for their biosynthesis, as well as the CO2 molecules, are transported inside the cell with the participation of the enzymatic
transport system through the special channels. The most striking examples
confirming this scheme I have observed myself when I studied the share of
external CO2 in the cells of bacteria with different types of carbon nutrition.
A new species of bacteria isolated, which used formate as a single substrate
for growth: Bacterium formoxidans. Then I discovered that this bacterium,
when growing on 14C-Iabeled formate as single organic substrate in cylinders
through which the stream of air + 5% CO2 gas mixture was bubbling, was producing practically unlabeled biomass. This means that Bacterium formoxidans
does not use 14C02 at all for biosynthesis, which it produces during the oxidation of the labeled formate (Sorokin 1961b). I observed the same phenomenon when I studied the carbon metabolism in the anaerobic sulfate-reducing
bacterium Desulfovibro desulfuricans, which oxidizes lactate to acetate anaerobically to obtain energy. However, when being grown on whole-chain labeled
lactate, this bacterium does not use the labeled acetate formed during lactate
oxidation for its biosynthesis, but incorporates the exogenous unlabeled
acetate from the medium (Sorokin 1966).