174
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
sensitivity of this method is about 2 orders more than the dark bottle oxygen
method and also because the 14C-method is at least three times less timeconsuming.
4.3.2 Practical Implementation
1. Decomposition rate and turnover time of individual specific radiolabeled
organic substances
Above, it was also pointed out that there is little reason to measure these parameters for ordinary low molecular substances like sugars or amino acids while
the technique has been adopted from a large experience of such measurements. In practice, commercially available individual hydrocarbons, detergents,
phenol having the specific 14C-radioactivity between 10 to SOmCimmol-\ as
well as the substances prepared by the researchers themselves from the
labeled plant material, were employed as the specific labeled substrates.
Among the latter are lignocellulose, lignin, cellulose, or humic acids. To
prepare the lignocelluloses, the plants are grown in solutions containing their
14C-Iabeled presecutors such as phenylalanine, tyrosine, glucose, or cinnamic
acid (Benner et al. 1984). Kirk et al. (197S) prepared synthetic 14C-Iabeled
lignin. To prepare the mixture of humic acids, a macrophyte is grown in water
with 14C-bicarbonate (see Sect. 3.3). Then the wet plant is crushed in the mortar
and the slurry with water is contaminated with silt. After 3-S months, the
humic acids formed are extracted and purified in accordance with the technique used in soil chemistry. The substances poorly miscible with water, like
hydrocarbons, can be mixed with fine sand devoid of organic matter right in
the experimental bottle before filling it with the sample. The particulated material like cellulose or lignocellulose is added to the samples as fine suspension.
The experimental vessels in this case are periodically turned to resuspend the
particles in the water column. The labeled humic acids are added to the
samples being dissolved in O.OS N KOH.
If the estimation involves numerous samples collected during the spatial
sampling missions, the corrisponding 40-S0ml subsamples are accomplished
into 100-lS0-ml serum bottles with silicon rubber stoppers, to which the glass
or transparent plastic cups of l-2-ml capacity are attached with the aid of a
sharp-ended handle (Fig. 4.S). The subsamples of water or the mud-water suspension are charged with 0.2-0.S /-lCi of the labeled substrates. The stopper
(together with the cup) is fixed on the place. The bottle thus closed is incubated with the sample for a time preselected by previous experimentation. For
substances like phenol or hydrocarbons, it might be O.S-l day, for the lignocellulose or cellulose 2-4 days, and for humic acids 1 week and more. Before
the end of incubation, 0.3-0.S ml of ethanolamine solution is injected into the
cup with the aid of a syringe and then 1 ml of 1 N H 2S04 is injected into the
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
sensitivity of this method is about 2 orders more than the dark bottle oxygen
method and also because the 14C-method is at least three times less timeconsuming.
4.3.2 Practical Implementation
1. Decomposition rate and turnover time of individual specific radiolabeled
organic substances
Above, it was also pointed out that there is little reason to measure these parameters for ordinary low molecular substances like sugars or amino acids while
the technique has been adopted from a large experience of such measurements. In practice, commercially available individual hydrocarbons, detergents,
phenol having the specific 14C-radioactivity between 10 to SOmCimmol-\ as
well as the substances prepared by the researchers themselves from the
labeled plant material, were employed as the specific labeled substrates.
Among the latter are lignocellulose, lignin, cellulose, or humic acids. To
prepare the lignocelluloses, the plants are grown in solutions containing their
14C-Iabeled presecutors such as phenylalanine, tyrosine, glucose, or cinnamic
acid (Benner et al. 1984). Kirk et al. (197S) prepared synthetic 14C-Iabeled
lignin. To prepare the mixture of humic acids, a macrophyte is grown in water
with 14C-bicarbonate (see Sect. 3.3). Then the wet plant is crushed in the mortar
and the slurry with water is contaminated with silt. After 3-S months, the
humic acids formed are extracted and purified in accordance with the technique used in soil chemistry. The substances poorly miscible with water, like
hydrocarbons, can be mixed with fine sand devoid of organic matter right in
the experimental bottle before filling it with the sample. The particulated material like cellulose or lignocellulose is added to the samples as fine suspension.
The experimental vessels in this case are periodically turned to resuspend the
particles in the water column. The labeled humic acids are added to the
samples being dissolved in O.OS N KOH.
If the estimation involves numerous samples collected during the spatial
sampling missions, the corrisponding 40-S0ml subsamples are accomplished
into 100-lS0-ml serum bottles with silicon rubber stoppers, to which the glass
or transparent plastic cups of l-2-ml capacity are attached with the aid of a
sharp-ended handle (Fig. 4.S). The subsamples of water or the mud-water suspension are charged with 0.2-0.S /-lCi of the labeled substrates. The stopper
(together with the cup) is fixed on the place. The bottle thus closed is incubated with the sample for a time preselected by previous experimentation. For
substances like phenol or hydrocarbons, it might be O.S-l day, for the lignocellulose or cellulose 2-4 days, and for humic acids 1 week and more. Before
the end of incubation, 0.3-0.S ml of ethanolamine solution is injected into the
cup with the aid of a syringe and then 1 ml of 1 N H 2S04 is injected into the