Abstract
Growth rate-dependent variations in intracellular free amino acid and protein composition were characterized and compared to their
14C content as a function of time in NH
-
4 -limited continuous cultures of the marine chlorophyte alga,
Nannochloris atomis Butcher (Strain GSBNANNO). The relative abundances of cellular carbon in free amino acids and protein were positively correlated with relative growth rate (
μ
μ
max
). There was also a positive correlation between relative growth rate and the proportion of cellular nitrogen in free amino acids, but not in protein. Significant changes in the composition of the intracellular free amino acid pool accompanied the growth rate-related decline in pool size. The percentage of intracellular free amino acid carbon contained in glutamate increased from 9 to 53% and the glutamine/glutamate carbon pool ratio decreased as relative growth rate decreased. The percentage of intracellular free amino acid carbon contained in proline decreased from 76% to 21 %, while small increases were observed for glycine + alanine, valine, and beta-aminobutyrate as relative growth rate decreased.
Equilibration of the intracellular free amino acid pool with inorganic
14C was incomplete even after incubation for 25% of the generation time. Equilibration rates of glutamate, glutamine, glycine + alanine, and valine were positively correlated with growth rate. Incomplete equilibration led to underestimates of free amino acid carbon pool sizes based on
14C content. However, because the saturation rates of glutamate and glutamine were similar, their ratio of
14C pools could be used as an index of the growth rate-dependent changes in the ratio of their cellular carbon pools.
Isotope disequilibrium resulted in an initially higher relative abundance of
14C in alcohol soluble low molecular weight material than the corresponding relative abundance of cellular carbon as determined from
14C distributions in equilibrium-labeled cells. Most of the short term time-dependent variation in the distribution of
14C in low molecular weight material was associated with the free amino acids. For incubations up to 25% of the generation time, the distribution of
14C in protein was lower than the equilibrium distribution. Discrepancies between short term distributions of
14C and cellular carbon distributions diminished if free amino acids and protein were considered together as a unit of cellular material. Growth rate-dependent changes in distributions of cellular carbon in free amino acids + protein were reflected by changes in the corresponding
14C distributions.
The techniques described provide the ability to specifically assay growth rate-dependent variations in algal metabolism and biochemical composition without the interference of detritus and non-algal biomass.