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Use of Radioisotopic Methodology in Aquatic Microbial Ecology
units of wet biomass. The specific growth rate h- 1 (/1) may be calculated if the
wet biomass (biovolume, B, mgm- 3 ) of bacterioplankton has been estimated
by direct microscopy (see Sect. 6.2) in a parallel nonlabeled sample incubated
for exactly half the time proposed for the 14C-Iabeled incubations. In this case:
p.
/1 = -4- h - , and the genelation time g = 1//1, h.
2 xB
The above formula for the Ph calculation is valid only in water layers with
a minor possibility of participation methanotophs or chemolithotrophs in the
dark CO2 uptake. In strata where this participation can take place, in the vicinity of redox zones or of the gas seeps, or in the near-bottom layers, the total
bacterial production Ph is created both by heterotrophic bacteria, which
comprise 8% of Ac in bacterial biomass produced and by methanotrophs
and chemolithitrophs with 30 to 100% (Sorokin et a1.1998). Therefore the only
way to arrive at reliable Ph-values in this case is to estimate it as a sum of its
production by the heterotrophic part of the bacterioplankton community (Pht)
and by its autotrophic or semiautotrophic part (Pmc): Pht + P me• The technique
for their separation is described below (Sect. 4.2.2.3). In the case of uncertainty about the possible participation of chemolithotrophs or methanothophs
in the COrdark uptake measured, this is a rather simple way to discover it
and to correspondingly change the mode of Ph calculation. For this it is
sufficient to estimate the ratio between the respiration rate M, mg 0 2 1- 1 and
A e , mg C 1-1 day-l in the water sample taken from the suspected habitat and
prefiltered through the 3-mm Nucleopore filter in the funnel (Fig. 4.8). The M
values are estimated as the oxygen consumption rate in the dark bottles. If the
ratio MI Ac is not less than 80, the possibility of the chemosynthetic CO2 uptake
can be ignored, while if it is significantly less «70) the chemosynthesis in this
sample is a proven matter of fact, and the value Pht and P mc should be estimated separately with the use of different K-coefficients for the P mc calculation (see Sect. 4.2.2.3).
Measurement of bacterial production in the bottom sediments by the
14COrdark uptake method is carried out as follows. The 5-g subsamples of
fresh sediment taken from the upper layer of the grab sample are placed into
the scintillation vials, followed by 15 ml of prefiltered natural water with
known TCOTC content. Formalin is added into one of them to make 2 % final
concentration. A portion of sodium azide is injected into the second to a
final concentration in the whole subsample 8mgml- 1 . The third subsample
remains intact. Then all three subsamples are charged with O.l-ml portions of
working 14C-solution prepared as described above with cpm radioactivity
between 70 and 80 10 6 cpm ml- 1 . During this procedure, which should proceed
in a darkened place after each injection of inhibitors or isotope solutions into
these subsamples, they should be closed with caps and their content should be
mixed by turning two to three times. During this treatment, the subsamples
are placed into a black bag and incubated in the vertical position at the tem-
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
units of wet biomass. The specific growth rate h- 1 (/1) may be calculated if the
wet biomass (biovolume, B, mgm- 3 ) of bacterioplankton has been estimated
by direct microscopy (see Sect. 6.2) in a parallel nonlabeled sample incubated
for exactly half the time proposed for the 14C-Iabeled incubations. In this case:
p.
/1 = -4- h - , and the genelation time g = 1//1, h.
2 xB
The above formula for the Ph calculation is valid only in water layers with
a minor possibility of participation methanotophs or chemolithotrophs in the
dark CO2 uptake. In strata where this participation can take place, in the vicinity of redox zones or of the gas seeps, or in the near-bottom layers, the total
bacterial production Ph is created both by heterotrophic bacteria, which
comprise 8% of Ac in bacterial biomass produced and by methanotrophs
and chemolithitrophs with 30 to 100% (Sorokin et a1.1998). Therefore the only
way to arrive at reliable Ph-values in this case is to estimate it as a sum of its
production by the heterotrophic part of the bacterioplankton community (Pht)
and by its autotrophic or semiautotrophic part (Pmc): Pht + P me• The technique
for their separation is described below (Sect. 4.2.2.3). In the case of uncertainty about the possible participation of chemolithotrophs or methanothophs
in the COrdark uptake measured, this is a rather simple way to discover it
and to correspondingly change the mode of Ph calculation. For this it is
sufficient to estimate the ratio between the respiration rate M, mg 0 2 1- 1 and
A e , mg C 1-1 day-l in the water sample taken from the suspected habitat and
prefiltered through the 3-mm Nucleopore filter in the funnel (Fig. 4.8). The M
values are estimated as the oxygen consumption rate in the dark bottles. If the
ratio MI Ac is not less than 80, the possibility of the chemosynthetic CO2 uptake
can be ignored, while if it is significantly less «70) the chemosynthesis in this
sample is a proven matter of fact, and the value Pht and P mc should be estimated separately with the use of different K-coefficients for the P mc calculation (see Sect. 4.2.2.3).
Measurement of bacterial production in the bottom sediments by the
14COrdark uptake method is carried out as follows. The 5-g subsamples of
fresh sediment taken from the upper layer of the grab sample are placed into
the scintillation vials, followed by 15 ml of prefiltered natural water with
known TCOTC content. Formalin is added into one of them to make 2 % final
concentration. A portion of sodium azide is injected into the second to a
final concentration in the whole subsample 8mgml- 1 . The third subsample
remains intact. Then all three subsamples are charged with O.l-ml portions of
working 14C-solution prepared as described above with cpm radioactivity
between 70 and 80 10 6 cpm ml- 1 . During this procedure, which should proceed
in a darkened place after each injection of inhibitors or isotope solutions into
these subsamples, they should be closed with caps and their content should be
mixed by turning two to three times. During this treatment, the subsamples
are placed into a black bag and incubated in the vertical position at the tem-