Determination of Microbial Production
187
geois 1955). In their experiments, these authors obtained values of actual
incorporation of external COz of around 8%. The same level was recorded in
our experiments (Sorokin 1961b, 1965). When being measured in the natural
microbial population using the direct counting method for estimating microbial production, the share of external CO2 in the biomass of bacterioplankton
thus produced was found to be between 4 and 7% (Romanenko 1963, 1964;
Overbeck 1972, 1984). This share is the key value for the calculation of heterotrophic microbial production in the accordance with the Romanenko
method. Its inverse value corresponds to the coefficient K used to calculate
microbial production (Ph) after 14C02-dark uptake estimates: Ph = Ac K mg C
m- 3 day-l, if Ac is dark CO 2 uptake, mg C m- 3 day-1. If, in accordance with Romanenko, this share for natural microbial populations is around 6% then the
value of K will be 16.
The value of this coefficient was thoroughly investigated later with the aid
of more modern methods for direct counting of bacteria using Nucleopore
filters both for microscopic assessment of microbial growth rate and for separation of phytoplankton during 14COz-uptake experiments (Saralov 1988;
Sorokin 1990b). Its most reliable value was found to be around 12. The measurement technique of the bacterial dark uptake in the natural water samples
was corrected taking into account the critical analysis of the method made by
Overbeck and Daley (1973), Li (1982,1984) and others. Their criticisms concerned the following aspects:
1. The external 14C02 in dark may also be assimilated by phytoplankton.
While special experiments done by Romaneko (1964) showed that this
share was small, this factor needed to be accounted for.
2. The coefficient K may change during the growth of bacteria in culture.
However, the ambient microbial community is not a culture. Nevertheless,
during long incubation (over 20h at a temperature above 20°C), the
microflora in the bottle changes and acquires the features of a culture. The
dark CO2 assimilation accelerates in it and the time course curve bends
upward (Fig. 4.7). Therefore, the incubation time during measurement
of 14C02 dark uptake should be selected within the linear part of the
time course curve, especially as the use of liquid scintillation counting
increases by more than an order of values the sensitivity of 14C02 uptake
analyses in comparison with the GM-counters which were in use at
Romaneko's time.
3. The prefiItration of samples through 2-4-f..Im pore size membrane, or later
the Nucleopore filters, in order to eliminate the bulk of phytoplankton, and
to decrease the dark uptake of 14COZ by algae, disturbs in reality the natural
microbial communities, destroys their aggregations, and enhances their subsequent growth (Ferguson et al. 1984). Thus, instead of the expected
decrease, the COz dark uptake often increases in such samples on comparison with intact ones. Therefore the operation of pre filtration is replaced by
postfiltration, also of a fixed sample, after incubation.
187
geois 1955). In their experiments, these authors obtained values of actual
incorporation of external COz of around 8%. The same level was recorded in
our experiments (Sorokin 1961b, 1965). When being measured in the natural
microbial population using the direct counting method for estimating microbial production, the share of external CO2 in the biomass of bacterioplankton
thus produced was found to be between 4 and 7% (Romanenko 1963, 1964;
Overbeck 1972, 1984). This share is the key value for the calculation of heterotrophic microbial production in the accordance with the Romanenko
method. Its inverse value corresponds to the coefficient K used to calculate
microbial production (Ph) after 14C02-dark uptake estimates: Ph = Ac K mg C
m- 3 day-l, if Ac is dark CO 2 uptake, mg C m- 3 day-1. If, in accordance with Romanenko, this share for natural microbial populations is around 6% then the
value of K will be 16.
The value of this coefficient was thoroughly investigated later with the aid
of more modern methods for direct counting of bacteria using Nucleopore
filters both for microscopic assessment of microbial growth rate and for separation of phytoplankton during 14COz-uptake experiments (Saralov 1988;
Sorokin 1990b). Its most reliable value was found to be around 12. The measurement technique of the bacterial dark uptake in the natural water samples
was corrected taking into account the critical analysis of the method made by
Overbeck and Daley (1973), Li (1982,1984) and others. Their criticisms concerned the following aspects:
1. The external 14C02 in dark may also be assimilated by phytoplankton.
While special experiments done by Romaneko (1964) showed that this
share was small, this factor needed to be accounted for.
2. The coefficient K may change during the growth of bacteria in culture.
However, the ambient microbial community is not a culture. Nevertheless,
during long incubation (over 20h at a temperature above 20°C), the
microflora in the bottle changes and acquires the features of a culture. The
dark CO2 assimilation accelerates in it and the time course curve bends
upward (Fig. 4.7). Therefore, the incubation time during measurement
of 14C02 dark uptake should be selected within the linear part of the
time course curve, especially as the use of liquid scintillation counting
increases by more than an order of values the sensitivity of 14C02 uptake
analyses in comparison with the GM-counters which were in use at
Romaneko's time.
3. The prefiItration of samples through 2-4-f..Im pore size membrane, or later
the Nucleopore filters, in order to eliminate the bulk of phytoplankton, and
to decrease the dark uptake of 14COZ by algae, disturbs in reality the natural
microbial communities, destroys their aggregations, and enhances their subsequent growth (Ferguson et al. 1984). Thus, instead of the expected
decrease, the COz dark uptake often increases in such samples on comparison with intact ones. Therefore the operation of pre filtration is replaced by
postfiltration, also of a fixed sample, after incubation.