Radiolabeled tracer method
Template:BreadcrumbsSecondaryProduction
| Bacterial production |
|---|
| Approach: radiolabeled tracer incorporation |
| Context: incubation, lab |
| Spatial scale: point sample |
| Temporal scale: hours to days |
| Units: mol incorporated tracer L-1 h-1 |
| Community captured: bulk, size-fractionated |
| Co-measurements: cell abundance |
Method Overview
Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly 3H-leucine (into protein) or 3H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate[1].
The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.
Scale of measurement
As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.
Data generated
The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d-1) can be derived.
Units & currency
Units are mol incorporated tracer L-1 h-1.
Sample size
Replicate small-volume subsamples (1–5 mL) are typically processed per station.
Repositories & databases
Limitations
Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.
Example Applications & Protocols
Classic examples
- Kirchman (2001) Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments [1]
Recent applications
- Kirchman et al. (2009) Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean [2]
Common calculations/conversions
- Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
- Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 1018 cells mol-1).
References
- ↑ 1.0 1.1 Kirchman, D. L. (2001). Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. Methods in Microbiology, 30, 227–237. https://doi.org/10.1016/S0580-9517(01)30047-8
- ↑ Kirchman, D. L., Hill, V., Cottrell, M. T., Gradinger, R., Malmstrom, R. R., & Parker, A. (2009). Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean. Deep-Sea Research Part II, 56(17), 1237–1248. https://doi.org/10.1016/j.dsr2.2008.10.018