Ethanol-extractable substrate pools and the incorporation of thymidine, L-leucine, and other substrates by bacterioplankton

Bacterioplankton productivity measurements based on [methyl-3H]-thymidine (TdR) or L-[3,4,5-3H]leucine (L-leu) incorporation typically depend on cold trichloroacetic acid (TCA) precipitation to separate 3H uptake from incorporation. An additional rinse with cold 80% ethanol (EtOH) removed an average of 22 (L-leu) and 32% (TdR) of 3H "incorporated" by San Francisco Bay samples and decreased the between-duplicate difference by a factor of 3.5. Similar results were obtained with samples from Tomales Bay, Calif., and Palmer Station, Antarctica. Varying amounts of cold TCA insoluble radiolabel from six other substrates were removed by the EtOH rinse. Regression analysis showed relationships between the effect of the EtOH rinse and a group of environmental variables and derived parameters. The percentage of 3H removed was generally independent of filter type; however, there were often large differences in the amount of 3H retained by Millipore versus Nuclepore or Poretics filters. The results strongly suggest that an EtOH rinse or other organic extraction should be included in protocols to determine incorporation of radiolabeled substrates into macromolecules. Furthermore, sequestering low molecular weight substrates in some sort of lipid-bound pool may represent a general storage mechanism employed by bacterioplankton. Key words: bacterioplankton, production, San Francisco Bay, filtration, incorporation.