180
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
tivity of thymidine uptake by bacteria, ensured its frequent use. At present, it
is among the basic methods in aquatic microbial ecology; but the correct interpretation of the results of measurements of incorporated radioactivity proved
to be very difficult, and, as time went on, more and more drawbacks became
known in its application. The literature on this problem contains dozens of
papers and reviews (Karl 1986; Robarts and Zohary 1993). Nevertheless, this
method remains very attractive. Indeed, it is, practically speaking, very useful,
especially for monitoring purposes. It will therefore be considered below in
more detail.
Probably because of the above-mentioned problems with the thymidine
method, interest has recently returned to the further development of the 3H_
leucine method to estimate microbial protein synthesis and microbial production. This method was first proposed by Kirchmann et al. (1985), Kirchman
et al. (1985, 1986, 1993), and Kirchman and Hogh (1988). The above-mentioned
authors proposed the use of nanomolar concentrations of 3H-Ieucine with the
application of Michaelis-Menten kinetics for solving the problem of isotopic
dilution. This method, in my opinion and experience has few prospects of
becoming commonly used, as it has even more negative features than the
thymidine one. First of all, the uptake of leucine is not specific only for bacteria. It is, in fact, readily incorporated by other microplankton. Scaling down
to the nanomolar level of concentration in order to decrease its incorporation
by other microplankters cannot actually solve this problem, because even
with the use of low nanomolar leucine concentrations added to the samples,
it remains in any case accessible for picoplanktonic algae and for animal
nanoheterotrophs of the 11m size (my data). The use of nanomolar concentration levels needs the kinetic approach, the applicability of which for diverse
natural microbial populations is doubtful (see Sect. 4.2.3.1). The majority
of aquatic bacteria synthesize most of the leucin they need de novo via the
tricarbonic acid cycle and do not depend upon external sources. The pool of
nonlabeled leucin in natural water samples is rather large and very variable
(Jorgensen 1992). The measurements of isotopic dilution of 3H-Ieucine added
to selected samples are insufficient and from the practical point of view not
safe at the nanomolar level, as with the TDR (Tuomi 1997). These considerations leave little hope for the use of leucine incorporation as a sound method
for measuring in situ microbial production (Fuhrman et al.1986; Bjornsen and
Kuparinen 1991). However, some researchers consider the leucin method as
practically useful for this purpose (Simon and Azam 1989, 1992; Talbot et al.
1997).
The radiophosphorus method with the use 32pO~- (or 33PO~-) as labels is
based on the assumption that most of the phosphorus needed by the bacterioplankton is acquired from the surrounding water as inorganic phosphate
(Sorokin 1985, 1990a), so that the empirical ratio there could be established
between the rates of labeled P04-P incorporation by bacterioplankton and its
production. The positive features of this method are: (1) the possibility to
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
tivity of thymidine uptake by bacteria, ensured its frequent use. At present, it
is among the basic methods in aquatic microbial ecology; but the correct interpretation of the results of measurements of incorporated radioactivity proved
to be very difficult, and, as time went on, more and more drawbacks became
known in its application. The literature on this problem contains dozens of
papers and reviews (Karl 1986; Robarts and Zohary 1993). Nevertheless, this
method remains very attractive. Indeed, it is, practically speaking, very useful,
especially for monitoring purposes. It will therefore be considered below in
more detail.
Probably because of the above-mentioned problems with the thymidine
method, interest has recently returned to the further development of the 3H_
leucine method to estimate microbial protein synthesis and microbial production. This method was first proposed by Kirchmann et al. (1985), Kirchman
et al. (1985, 1986, 1993), and Kirchman and Hogh (1988). The above-mentioned
authors proposed the use of nanomolar concentrations of 3H-Ieucine with the
application of Michaelis-Menten kinetics for solving the problem of isotopic
dilution. This method, in my opinion and experience has few prospects of
becoming commonly used, as it has even more negative features than the
thymidine one. First of all, the uptake of leucine is not specific only for bacteria. It is, in fact, readily incorporated by other microplankton. Scaling down
to the nanomolar level of concentration in order to decrease its incorporation
by other microplankters cannot actually solve this problem, because even
with the use of low nanomolar leucine concentrations added to the samples,
it remains in any case accessible for picoplanktonic algae and for animal
nanoheterotrophs of the 11m size (my data). The use of nanomolar concentration levels needs the kinetic approach, the applicability of which for diverse
natural microbial populations is doubtful (see Sect. 4.2.3.1). The majority
of aquatic bacteria synthesize most of the leucin they need de novo via the
tricarbonic acid cycle and do not depend upon external sources. The pool of
nonlabeled leucin in natural water samples is rather large and very variable
(Jorgensen 1992). The measurements of isotopic dilution of 3H-Ieucine added
to selected samples are insufficient and from the practical point of view not
safe at the nanomolar level, as with the TDR (Tuomi 1997). These considerations leave little hope for the use of leucine incorporation as a sound method
for measuring in situ microbial production (Fuhrman et al.1986; Bjornsen and
Kuparinen 1991). However, some researchers consider the leucin method as
practically useful for this purpose (Simon and Azam 1989, 1992; Talbot et al.
1997).
The radiophosphorus method with the use 32pO~- (or 33PO~-) as labels is
based on the assumption that most of the phosphorus needed by the bacterioplankton is acquired from the surrounding water as inorganic phosphate
(Sorokin 1985, 1990a), so that the empirical ratio there could be established
between the rates of labeled P04-P incorporation by bacterioplankton and its
production. The positive features of this method are: (1) the possibility to