Algal heterotrophy: effects of gentamycin and cycloheximide on bacteriat and algal heterotrophic and photosynthetic activities
The ability of many planktonic algae to use particulate and/or dissolved organic carbon directly by phagotrophy or osmotrophy in laboratory cultures is well documented (DROOP, 1974; NEILSON and LEWIN, 1974; HELLEBUST and LEWIN, 1977; BIRD and KALFF, 1986). In axenic cultures, numerous microalgae grow in the dark with micromolar concentrations of diverse organic nutrients as their sole sources of carbon and energy (RIVKIN and PUTT, 1987). However, to demonstrate algal heterotrophy in the field, it is necessary to differentiate between bacterial and algal activities. In the course of this study, we tested the effect of an antibacterial substance (gentamycin) and of a metabolic inhibitor of eukaryotic cells (cycloheximide) on algal and bacterial cultures, and also on lake water samples submitted to differential filtration. The effect of these inhibitors was tested both at the overall heterotrophic and photosynthetic activities level and the level of the incorporation of radiolabeled tracers in macromolecules and low molecular weight compounds. Gentamycin was tested on bacteria and on an axenic culture of the diatom Melosira italica subsp. subarctica, the dominant species of the spring phyto-planktonic bloom of many temperate lakes. Bacterial culture was obtained by filtration of a senescent culture of Melosira through a 0.45 μm pore-size membrane. During exponential growth, gentamycin (40 μg.ml-1) was added to different flasks containing 100 ml of culture, 30 mn after gentamycin addition, Na H14CO3 (12μCi/100 ml) was introduced into the flasks. In each case, two replicates were incubated in the light and two in the dark for 0.5, 1, 2, 4, 6, 8, 12 and 24 hours. After incubation, aliquots were collected on a filtration membrane (0.45 μm) and the radioactivity was measured using an LKB liquid scintillation counter to estimate photosynthetic activity. Incorporation of inorganic carbon into macromolecules was measured using the procedure described by LANCELOT and MATHOT (1985), which allows the separation of lipids, polysaccharides, proteins and low molecular weight compounds (e.g. amino acids, organic acids and monosaccharides) by virtue of their relative solubilities in different extraction solvents : - lipids were extracted with a 2/1 (v/v) chloroform-methanol mixture; - low molecular weight compounds were extracted with hot ethanol; - proteins were precipated with TCA at elevated temperature; this also separated them from polysaccharides. Nucleic acids were recovered with the polysaccharides fraction. Results are given as a percentage of total radioactivity. The same method, as that described for photosynthetic incorporation, was used to measure incorporation of glucose 3H (19 nmoles.l-1) and for the allocation of radioactive marker in macromolecules. To assess the effect of cycloheximide, the same procedure as that described for gentamycin was used. However, due to the lack of references, three cycloheximide concentrations were tested = 50, 100 and 150 μg.ml-1, and aliquots were collected on a 0.2 μm pore-size membrane. Field samples were taken in Lake Pavin, an oligomesotrophic French lake, during the sedimentation of Melosira italica subsp. subarctica, the prevailing species of the spring bloom. The effects of gentamycin on photosynthetic and heterotrophic incorporations were tested with the came procedure as that described for cultures. However, radioactivity was measured for different size tractions : 0.2-0.45 μm, 0.45-5 μm and 5-160 μm, after incubations for 0.5, 1, 3, 6, 12 and 24 hours. Phytoplanktonic cells were counted on a Wild M40 inverted microscope and bacterial enumeration was realized in epifluorescence microscopy after staining with acridine orange (HOBBIE el al., 1977). Results show that gentamycin used at 40 μg.ml-1 inhibits significantly but not completely the activity of the bacterial culture (mean inhibition percent = 67 %). Gentamycin efficiency increases with incubation time, the inhibition reaching 81 % after 24 h. At the same time, the secondary effects of gentamycin on heterotrophic and photosynthetic activities of Melosira italica subsp. subarctica in culture were only tolerable with short incubation times (< 4 h), when the percents of inhibition were respectively 13.6 and 12.2%. On the other band, cycloheximide produced no significant effect on photosynthetic and heterotrophic activities of Melosira italica in culture, the percent of inhibition always remaining below 6.5 %. The use of gentamycin in natural samples reduced bacterial interference with algal heterotrophic activily measurements. The percent of inhibition caused by gentamycin was high (< 76 %) In the small-size fraction where the bacterial biomass predominate on the phytoplanktonic one. Lastly, in all samples, we could demonstrate that gentamycin modifies the allocation of inorganic carbon and radioactivity in macromolecules. The incorporation in proteins is significantly reduced essentially to benefit of the incorporation in low molecular weight compounds.