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Assessing the Influence of Environmental pH on Algal Physiology Using a Novel Culture System Public Deposited

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  • Since the Industrial Revolution, surface ocean pH has declined due to the input of anthropogenically derived carbon dioxide, termed ocean acidification. Examinations of phytoplankton physiology in the face of changing pH are becoming more important as anthropogenically-driven pH decreases in the surface ocean progress (termed ocean acidification). Previous research has shown that phytoplankton response to acidification are highly variable, with some taxa showing improvement and some showing marked deterioration. The ability to maintain homeostasis of intracellular pH is an important adaptation for phytoplankton to continue to thrive under changing conditions; increased energy production has been shown to mitigate the negative effects of acidification. This dissertation examines the effect of steady state and changing pH environments on the internal pH, esterase activity, and photosynthetic efficiency (the latter two parameters are involved with energy production or utilization) of the marine phytoplankton species, Isochrysis galbana. To accomplish this, a novel pHstat system was developed for the culture of marine phytoplankton, capable of maintaining both steady state and dynamic pH environments autonomously over extended periods. The pHstat system was used to grow phytoplankton at three steady state pH levels (pH 7.5, 8.0, and 8.5), as well under two separate dynamic conditions. The dynamic conditions were differentiated by coupling or decoupling light cycles from their natural relationship with pH (i.e., pH increases during the day due to photosynthetic uptake of inorganic carbon and decreases at night due to carbon addition through cellular respiration) in an effort to determine if intracellular pH changes were driven by changes in external pH or by changes in internal pools of dissolved inorganic carbon (DIC) availability due to carbon fixation and cellular respiration. To determine intracellular pH using a rapid, reliable method, I explored the efficacy of the fluorescent intracellular pH indicator for phytoplankton work, SNARF, and its suitability for use in phytoplankton cultures. Three methods of fluorescence detection (fluorescence spectroscopy, flow cytometry, and laser scanning microscopy) were compared as methods of fluorescence detection. Fluorescence detection was found to be dependent on the loading concentration of the fluorophore for flow cytometry; higher sensitivities were achieved using fluorescence spectroscopy and microscopy, which enabled the use of lower concentrations of the dye. Because of the greater flexibility in choice of excitation and emission wavelengths, fluorescence spectroscopy was found to be the superior method for pH measurement, with lower percent error and more reliable calculations of pHi. However, the use of flow cytometry with SNARF has the added advantage of providing viability data for individual cells, as SNARF only accumulates within live, intact cells. Using the novel culture system and the pH sensitive dye, SNARF, I determined that intracellular pH changes in I. galbana fluctuated in response to changing external pH, not light:dark cycles. Esterase activity was highest in cells with the lowest photosynthetic efficiency, suggesting a link between esterase activity and energy production/demand, as cells can use esterases to access energy stored in the form of lipids. This work presents new links between cellular energy balance in phytoplankton and environmental pH response, providing valuable data regarding microalgal adaptation to changing ocean pH as ocean acidification progresses.
  • Available for download at: https://doi.org/10.6083/M44B30VV
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