9. Microbial Carbon Cycling in Pelagic Ecosystems: Microbial Methods for Ecosystem Scientists
143
ers routinely assume that there is a constant ratio
between measured cell volume and biomass (Watson et al. 1977), or assume that all bacteria have
the same C content per cell (20 fg cell-I). There is
some experimental support for both of these extremely opposite approaches. However, most researchers now assume that there is a power function
relating biomass (m) to volume (V):
m = CVa
(9.2)
where C is the conversion factor (pg C J.lg - 3) between biomass and volume, and a is the scaling
factor. Simon and Azam (1989) suggest a scaling
factor of 0.6, and that C = 0.09VO. 6 .
Growth and Respiration
of Planktonic Bacteria
Bacterial metabolism is important to two different
aspects of the C cycle (see Fig. 9.1). Bacterial respiration converts organic C into inorganic C. The
growth of bacterial cells (bacterial production) converts DOC into new bacterial biomass. Bacteria are
the only organisms that use DOC to a significant
degree, but many higher organisms can consume
bacteria (Pace et al. 1990; Cole 1982; Azam et al.
1983; Sherr and Sherr 1996). Thus, the conversion
of DOC into bacterial biomass represents a potential recovery of organic C that is lost to the rest of
the food web (Paerl 1978).
Bacterial production (BP) is secondary production and is theoretically unconstrained by the inputs
of organic C to the system from primary production
or other sources (Strayer 1988; Scavia 1988;
Jahnke and Craven 1995). Bacterial respiration, on
the other hand, cannot exceed the inputs of organic
C from primary production plus allochthonous
sources (Cole and Pace 1995; Cole et al. 1989).
Bacterial Secondary Production
Bacterial production is most often measured with
one of two techniques, both using radioactive
tracers. By measuring the incorporation of 3H_
thymidine into bacterial DNA, or by measuring the
incorporation of 3H-leucine into bacterial protein,
one can estimate BP. The details for both methods
are available in user-friendly literature (Bell 1993;
Kirchman 1993) and there has been considerable
critical review of the thymidine method in particular (Robarts and Zohary 1993). Both methods depend on the fact that other organisms in the sample
take up little or none of the label at the concentrations added, and that bacteria will take these up.
Both methods use relatively short « 1 hour) incubations under ambient conditions to avoid container
effects. There are a number of steps and potential
pitfalls in getting actual estimates of BP from these
measurements. The substrate needs to be added at
a level that saturates the uptake kinetics. On the
other hand, if the substrate is added at too high a
level one can stimulate catabolic processes. Thus,
the rate of uptake needs to be measured as a function of added substrate to determine the correct
amount to be added. Unlabeled substrate present in
the water or synthesized by the microbe during the
incubation can dilute the specific activity of the
added substrate. This isotope dilution can also be
dealt with kinetically by adding a constant amount
of labeled substrate while varying the amount of
unlabeled substrate (Pollard and Moariaty 1984;
Simon and Azam 1989). Finally, once the absolute
rate of either thymidine or leucine uptake has been
established, it needs to be related to cell growth
with a series of conversion factors. For thymidine,
we need to relate thymidine uptake to DNA synthesis, DNA synthesis to the number of cells produced, and C content per cell to express growth rate
in units of C. For leucine, we need to relate leucine
uptake to protein synthesis, and protein synthesis
to C. (BP is expressed usually as J.lg C liter-I hr- 1
or cells liter-I hr- I ). These conversion factors can
be measured directly, based on theoretical values,
or can be side-stepped by using empirical factors
based on regrowth studies in dilution cultures
(Kirchman et al. 1982a).
In their original forms, for both methods, the labeled bacteria were concentrated initially by filtration. In either method, there can be some nonspecific labeling. That is, some of the 3H from the
added thymidine can be incorporated into molecules other than DNA. Similarly, some of the 3H
from leucine can be incorporated into molecules
other than protein. The nontarget molecules are
therefore washed away by a series of extractions,
and numerous protocols have been developed, especially for the thymidine method (Robarts and Zohary 1993 and references therein).
143
ers routinely assume that there is a constant ratio
between measured cell volume and biomass (Watson et al. 1977), or assume that all bacteria have
the same C content per cell (20 fg cell-I). There is
some experimental support for both of these extremely opposite approaches. However, most researchers now assume that there is a power function
relating biomass (m) to volume (V):
m = CVa
(9.2)
where C is the conversion factor (pg C J.lg - 3) between biomass and volume, and a is the scaling
factor. Simon and Azam (1989) suggest a scaling
factor of 0.6, and that C = 0.09VO. 6 .
Growth and Respiration
of Planktonic Bacteria
Bacterial metabolism is important to two different
aspects of the C cycle (see Fig. 9.1). Bacterial respiration converts organic C into inorganic C. The
growth of bacterial cells (bacterial production) converts DOC into new bacterial biomass. Bacteria are
the only organisms that use DOC to a significant
degree, but many higher organisms can consume
bacteria (Pace et al. 1990; Cole 1982; Azam et al.
1983; Sherr and Sherr 1996). Thus, the conversion
of DOC into bacterial biomass represents a potential recovery of organic C that is lost to the rest of
the food web (Paerl 1978).
Bacterial production (BP) is secondary production and is theoretically unconstrained by the inputs
of organic C to the system from primary production
or other sources (Strayer 1988; Scavia 1988;
Jahnke and Craven 1995). Bacterial respiration, on
the other hand, cannot exceed the inputs of organic
C from primary production plus allochthonous
sources (Cole and Pace 1995; Cole et al. 1989).
Bacterial Secondary Production
Bacterial production is most often measured with
one of two techniques, both using radioactive
tracers. By measuring the incorporation of 3H_
thymidine into bacterial DNA, or by measuring the
incorporation of 3H-leucine into bacterial protein,
one can estimate BP. The details for both methods
are available in user-friendly literature (Bell 1993;
Kirchman 1993) and there has been considerable
critical review of the thymidine method in particular (Robarts and Zohary 1993). Both methods depend on the fact that other organisms in the sample
take up little or none of the label at the concentrations added, and that bacteria will take these up.
Both methods use relatively short « 1 hour) incubations under ambient conditions to avoid container
effects. There are a number of steps and potential
pitfalls in getting actual estimates of BP from these
measurements. The substrate needs to be added at
a level that saturates the uptake kinetics. On the
other hand, if the substrate is added at too high a
level one can stimulate catabolic processes. Thus,
the rate of uptake needs to be measured as a function of added substrate to determine the correct
amount to be added. Unlabeled substrate present in
the water or synthesized by the microbe during the
incubation can dilute the specific activity of the
added substrate. This isotope dilution can also be
dealt with kinetically by adding a constant amount
of labeled substrate while varying the amount of
unlabeled substrate (Pollard and Moariaty 1984;
Simon and Azam 1989). Finally, once the absolute
rate of either thymidine or leucine uptake has been
established, it needs to be related to cell growth
with a series of conversion factors. For thymidine,
we need to relate thymidine uptake to DNA synthesis, DNA synthesis to the number of cells produced, and C content per cell to express growth rate
in units of C. For leucine, we need to relate leucine
uptake to protein synthesis, and protein synthesis
to C. (BP is expressed usually as J.lg C liter-I hr- 1
or cells liter-I hr- I ). These conversion factors can
be measured directly, based on theoretical values,
or can be side-stepped by using empirical factors
based on regrowth studies in dilution cultures
(Kirchman et al. 1982a).
In their original forms, for both methods, the labeled bacteria were concentrated initially by filtration. In either method, there can be some nonspecific labeling. That is, some of the 3H from the
added thymidine can be incorporated into molecules other than DNA. Similarly, some of the 3H
from leucine can be incorporated into molecules
other than protein. The nontarget molecules are
therefore washed away by a series of extractions,
and numerous protocols have been developed, especially for the thymidine method (Robarts and Zohary 1993 and references therein).