Master's Thesis (Open Access)
Master of Science (M.S.)
Moss Landing Marine Laboratories
Chlorophyll a serves as the routine proxy for most estimates of phytoplankton biomass in limnology and oceanography. It is well known that enzymatic chlorophyllase activity breaks chlorophyll a into the degradation product chlorophyllide a (chlide a). This degradation could result in potentially large underestimates of the true chlorophyll a concentration when the analysis is made by modern chromatographic methods. The goal of this project was to determine proper protocol in the methodological elimination of artifactual chlorophyllide a formed during extraction of chlorophyll pigments through the application of a microwave-assisted solvent extraction technique. This study aimed to answer the question: Is chlorophyllide a a fact or artifact? Previous literature has suggested that chromatographically analyzed chlorophyllide a might be either an artifact of the extraction process or an in situ indicator of senescent, physiologically compromised phytoplankton due to environmental stressors. This study addresses this decades-old problem for chlorophyll analysis. The microwave technique described inhibits chlorophyllase enzymatic activity, preventing the artifactual production of chlorophyllide a. The heat of the microwave technique not only denatures the chlorophllyase enzyme activity, but also evaporates filter-retained water (known to promote enzyme activity in solvents such as acetone and methanol). This technique results in a sizeable increase in the yield of extracted chlorophyll a that was up to four times greater than the routine protocol of cold temperature solvent-soak technique using 90% acetone. Microwave-assisted extraction methodology consistently yielded higher concentrations of total chlorophyll a-like pigments than commonly employed solvent-soak technique used in long term sampling programs such as JGOFS, HOTS and BATS. The result of this study provides a method that 1) eliminates artifactual chlorophyllide a production and 2) increases the extraction yield of photosynthetic pigments in phytoplankton, including both chlorophylls and carotenoids. This new technique may have significant implications for estimates of oceanic primary production.
Helmann, Sierra Claire, "Chlorophyllide a: Fact or Artifact - Resolution of the Chlorophyllide a Problem in the Routine Measurement of Planktonic Chlorophyll a" (2019). Capstone Projects and Master's Theses. 652.