162
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
When using labeled substrates with the above high specific radioactivity, their portions inserted into the samples are nanomolar. It evidently
results in a large and unknown isotopic dilution, because the concentration of free amino acids or monosaccharides even in moderately productive natural waters is at the micromolar level (1-IOllmoll-1). Therefore
the cpm (or dpm) bacterial incorporation rates thus determined per individual
sample are meaningless. It is neither the absolute uptake rate (King and
Berman 1984) nor a comparative one, because in different samples the
background contents of corresponding organic compounds can be very variable, especially if the sampling has been done across the heterotrophic gradients: from shore to open sea or on vertical profiles, for example. Thus, the
variability of uptake rates of nanomolar amounts of labeled organic substrates
measured in the individual samples reflects rather the variable isotopic dilution in them, than the difference in the heterotrophic microbial activity in
them.
Being unable to measure absolute uptake rates when using nanomolar
concentrations of labeled substrates, the promoters of this method advocated
another direction for its development and application of the kinetic analysis
of uptake rate (Parsons and Strickland 1962; Wright and Hobbie 1965,1966).
Evidence was obtained that the uptake concentration curves of labeled
organic substrate at such low initial contents in water follow Michaelis-Menten
enzyme kinetics. It provided the possibility to separate their algal and bacterial uptake and evaluate the maximum uptake rates (Vmax), the turnover time
of the pool of a given substance, and even its probable ambiental concentration in natural waters (Sn). This direction of studies again became very popular.
It was found that, while being very approximative, at least some coincidence
exists between these parameters and trophic status of aquatic habitats (Crawford et al. 1973; Sepers 1977); but, in all cases, their values ranged within several
hundred times and often without any visible reason (Hoppe 1978). Often the
uptake data failed to sequence the Michaelis-Menten kinetics at all (Vaccaro
and Jannasch 1967; Hamilton and Preslan 1970; Overbeck 1972; Gocke et al.
1981; Sepers and Van Es 1979; Li 1983; Smith et al. 1984). To date, it is obvious
that the uptake and utilization of organic substrates by heterogeneous
microbial communities cannot be appropriately described by simple enzyme
kinetics. Attempts to correlate the above kinetic parameters with those independently estimated in the continuous cultures of heterotrophic bacteria
completely failed (Van Es and Meyer Reil 1982). This approach to measure
the heterotrophic activity in natural waters has gradually fallen into disuse
because of such drawbacks. Therefore, it is not described here in more
detail.
A somewhat different development in this direction was made when
Hobbie and Crawford (1969) proposed measuring not only incorporated 14C_
label but also the respired part of labeled organic substrate uptaken by
bacterioplankton. However, the proportion between uptake and respired
14C-substrate carbon appeared to be rather constant (Williams and Askew
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
When using labeled substrates with the above high specific radioactivity, their portions inserted into the samples are nanomolar. It evidently
results in a large and unknown isotopic dilution, because the concentration of free amino acids or monosaccharides even in moderately productive natural waters is at the micromolar level (1-IOllmoll-1). Therefore
the cpm (or dpm) bacterial incorporation rates thus determined per individual
sample are meaningless. It is neither the absolute uptake rate (King and
Berman 1984) nor a comparative one, because in different samples the
background contents of corresponding organic compounds can be very variable, especially if the sampling has been done across the heterotrophic gradients: from shore to open sea or on vertical profiles, for example. Thus, the
variability of uptake rates of nanomolar amounts of labeled organic substrates
measured in the individual samples reflects rather the variable isotopic dilution in them, than the difference in the heterotrophic microbial activity in
them.
Being unable to measure absolute uptake rates when using nanomolar
concentrations of labeled substrates, the promoters of this method advocated
another direction for its development and application of the kinetic analysis
of uptake rate (Parsons and Strickland 1962; Wright and Hobbie 1965,1966).
Evidence was obtained that the uptake concentration curves of labeled
organic substrate at such low initial contents in water follow Michaelis-Menten
enzyme kinetics. It provided the possibility to separate their algal and bacterial uptake and evaluate the maximum uptake rates (Vmax), the turnover time
of the pool of a given substance, and even its probable ambiental concentration in natural waters (Sn). This direction of studies again became very popular.
It was found that, while being very approximative, at least some coincidence
exists between these parameters and trophic status of aquatic habitats (Crawford et al. 1973; Sepers 1977); but, in all cases, their values ranged within several
hundred times and often without any visible reason (Hoppe 1978). Often the
uptake data failed to sequence the Michaelis-Menten kinetics at all (Vaccaro
and Jannasch 1967; Hamilton and Preslan 1970; Overbeck 1972; Gocke et al.
1981; Sepers and Van Es 1979; Li 1983; Smith et al. 1984). To date, it is obvious
that the uptake and utilization of organic substrates by heterogeneous
microbial communities cannot be appropriately described by simple enzyme
kinetics. Attempts to correlate the above kinetic parameters with those independently estimated in the continuous cultures of heterotrophic bacteria
completely failed (Van Es and Meyer Reil 1982). This approach to measure
the heterotrophic activity in natural waters has gradually fallen into disuse
because of such drawbacks. Therefore, it is not described here in more
detail.
A somewhat different development in this direction was made when
Hobbie and Crawford (1969) proposed measuring not only incorporated 14C_
label but also the respired part of labeled organic substrate uptaken by
bacterioplankton. However, the proportion between uptake and respired
14C-substrate carbon appeared to be rather constant (Williams and Askew