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Enzymes as indices of growth rate and nitrate metabolism in marine phytoplankton Berges, John Alexander

Abstract

Determining the in situ rates of growth and nitrogen incorporation of marine phytoplankton is critical to understanding energy transfer and nutrient and carbon cycling in the world’s oceans. To overcome the limitations in current methods of estimating biological rates (i.e. incubations under unrealistic conditions, or inadequate estimates of spatial and temporal variability) the use of enzyme activity measurements was examined. Because enzymes are functional proteins that adapt to suit prevailing conditions, enzyme levels may provide an integrated index of in situ rates of phytoplankton metabolism. Nucleoside diphosphate kinase (NDPK) an enzyme which directs cellular energy towards biosynthesis was examined as an index of specific growth rate (μ) in the diatom Thalassiosira pseudonana grown under light limitation, NDPK activity was significantly, but wealdy correlated with μ . Activity per cell rose at high μ , but also increased at very low μ . Although of limited value as a predictive index by itself, NDPK may be useful in conjunction with measurements of ATP concentration, or adenylate turnover rates. Nitrate reductase (NR), an enzyme specific for nitrate assimilation may be used in calculating rates of nitrate incorporation (μN) and thus new production, but previous measurements of NR have not matched μN. A new assay using bovine serum albumin to protect the enzyme from proteases was developed that gave close agreement with μN in light limited cultures of T. pseudonana and Skeletonema costatum. The relationship also held for T. pseudonana during transitions in irradiance, under nitrate limitation (although NR exceeded μN at low μ ), during growth on light-dark cycles, different light spectra, in the presence of ammonium, and during nitrate starvation. In each case, NR accurately predicted μN. NR was closely related to nitrate incorporation rates in three additional diatom species, but for other taxa, particularly the Dinophyceae, NR underestimated μN. Preliminary field experiments were conducted in Monterey Bay, California during a diatom bloom. μN predicted from NR measurements always equalled or exceeded rates estimated by other methods, including ¹⁵N incorporation. Appendices to the thesis compare and validate different protein assays in marine phytoplankton, provide details of a computer program to automate and collect enzyme kinetic data from a spectrophotometer, and compare methods of fitting rectangular hyperbolae to a variety of oceanographic data.

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