Modification of the 3H-leucine centrifugation method for determining bacterial protein synthesis in freshwater samples

The validity of the H-3-leucine centrifugation method for determining bacterial secondary production in oligotrophic and eutrophic fresh- and seawater samples was examined. For freshwater samples, we found that the established protocol developed by Smith & Azam (1992) led to significantly lower values (up to 57%) than a novel protocol presented here, where bacterial proteins are precipitated under acidic conditions (trichloroacetic acid) at 4 degrees C with a humic extract and solubilizing DNA and RNA at 100 degrees C for 30 min. For seawater samples, no difference was found when an ethanolic washing step was included in the novel protocol. We also used different salt solutions instead of humic extract; these both act as co-precipitants for the precipitation of the proteins. An unbuffered 3.5 % (final cone.) NaCl solution was found to be highly effective and gave consistent results and lower blank values. Incorporation rates obtained with our protocol showed good agreement with the commonly used filtration method. Therefore, we argue that for freshwater samples an NaCl or humic extract addition is necessary for an efficient precipitation of the proteins when the centrifugation method for determining bacterial secondary production via H-3-leucine incorporation is applied.