A modified pH drift assay for inorganic carbon accumulation and external carbonic anhydrase activity in microalgae

Abstract

The threat of global warming due to CO 2 emissions has stimulated research into carbon sequestration and emissions reduction technologies. Alkaline scrubbing allows CO 2 to be captured as bicarbonate, which can be photochemically fixed by microalgae. The carbon concentrating mechanism (CCM), of which external carbonic anhydrase is a key component, allows the organisms to utilise this bicarbonate. In order to select a suitable strain for this application, a screening tool is required. The current method for determining carbonic anhydrase activity, the Wilbur and Anderson assay, was found to be unsuitable as a screening tool as the associated error was unacceptably large and tests on whole cells were inconclusive. This paper presents the development of a new, whole cell assay to measure inorganic carbon uptake and external carbonic anhydrase activity, based on classical pH drift experiments. Spirulina platensis was successfully used to develop a correlation between the specific carbon uptake (C) and the specific pH change (dpH). The relationship is described by: C (mmol C (g dry algae)-1 h-1) = 0.064 × (dpH). Inhibitor and salt dissociation tests validated the activity and presence of external carbonic anhydrase, and allowed correlation between the Wilbur and Anderson assay and the new whole cell assay. Screening tests were conducted on Spirulina platensis, Scenedesmus sp., Chlorella vulgaris and Dunaliella salina which were found to have carbon uptake rates of 5.76, 5.86, 3.86 and 2.15 mmol C (g dry algae)-1 h-1 respectively. These results corresponded to the species' known bicarbonate utilisation abilities and validated the use of the assay as a screening tool.

Extracellular carbonic anhydrase: Method development and its application to natural seawater

“…Indeed, salt dissociation technique has been established to break ionic interaction between eCA and the membrane from C. reinhardtii (Husic and Quigley ). van Hille et al () applied salt dissociation technique to the cyanobacterium Spirulina platensis and Dunaliela salina , a highly salt‐tolerant cell. van Hille et al () and our results from different culture solutions and natural communities support the efficiency of the salt dissociation technique to extract eCA from other taxa.…”

“…van Hille et al () applied salt dissociation technique to the cyanobacterium Spirulina platensis and Dunaliela salina , a highly salt‐tolerant cell. van Hille et al () and our results from different culture solutions and natural communities support the efficiency of the salt dissociation technique to extract eCA from other taxa. The different levels of eCA in among those species provide compelling evidence for the variability of the carbon acquisition strategy in phytoplankton species.…”

“…For method comparison, we measured eCA activity using the electrometric technique (Wilbur and Anderson ; van Hille et al ). Briefly, the method is based on the time period for the pH to drop from 8.3 to 6.3 after the addition of CO 2 ‐saturated water.…”

“…eCA is found outside the plasmalemma, either in the periplasmic space or attached to the outer cell wall (Kimpel et al ; Coleman and Grossman ). Husic and Quigley () and van Hille et al () found that eCA bound either to the cell or plasma membrane through ionic interaction in various species of the genus chlorophyte and cyanobacteria. eCA is constitutive in most of the marine phytoplankton, whereas the expression was regulated by the concentration of inorganic carbon (Nimer et al ; Elzenga et al ).…”

Ecophysiology matters: linking inorganic carbon acquisition to ecological preference in four species of microalgae (Chlorophyceae)

“…, van Hille et al. ) resulting in the uptake of CO 2 and the recapturing of CO 2 by barriers in the chloroplast (Yamano et al. ).…”