Comparison of three techniques for administering radiolabeled substrates to sediments for trophic studies: Incorporation by microbes

Abstract

Three principal methods have been used to administer substrates to sediments: injection, porewater replacement, and slurry. Here we assess how each of these techniques affects incorporation of radiolabels into macromolecules of marine sedimentary microbes. Eighty-five cores of intertidal sand were collected in a randomized-block, factorial design. One set of cores received(14)C-bicarbonate/(3)H-thymidine and was incubated in the light; another set received(14)C-acetate/(3)H-thymidine and was incubated in the dark. Following a 5-hour incubation, sediments were analyzed for incorporation of radiolabel into lipid fractions (neutral, glyco-, and polar) and DNA. The three methods of isotope administration were also applied to cores subsequently analyzed for polar lipid phosphates and phospholipid fatty-acid (PLFA) profiles. In general, incorporation was greatest when injections were made, consistent with the prediction that incorporation would decrease as specific activity of the radiolabeled substrate was diminished by dilution. The ratio of(14)C from acetate incorporated into polar and glycolipid fractions indicated that a significant disturbance accompanied the porewater and slurry techniques. Substantial amounts of(3)H were recovered in the neutral-lipid fraction, indicating that thymidine was catabolized by sedimentary microbes and tritiated products were incorporated by eukaryotes. There were no significant differences in PLFA profiles or estimates of microbial biomass among methods or controls. Incorporation of(3)H into DNA was similar with all combinations of methods and radiocarbon substrates.(14)C was extensively incorporated into DNA, indicating that photoautotrophs and heterotrophs utilized radiocarbon from bicarbonate and acetate, respectively, for de novo synthesis of DNA. Injection is suggested as the method of choice, as it presents more flexibility in its application than porewater replacement and disturbs the consortia of gradients in sediments to a significantly lesser degree than porewater replacement and slurry.