Optimising methodology for determining the effect of ocean acidification on bacterial extracellular enzymes

Abstract. To fully understand the impact of ocean acidification on biogeochemical cycles, the response of bacterial extracellular enzymes needs to be considered as they play a central role in the degradation and distribution of labile organic matter. This study investigates the methodology, and potential artefacts involved in determining the response of bacterial extracellular glucosidase and protease to ocean acidification. The effect of pH on artificial fluorophores and substrates was examined, as well as the impact of three different acidification methods. The results indicate that pH has a significant effect on the fluorescence of the artificial fluorophore 4-methylumbeliferone for glucosidase activity, and 7-amino-4-methylcoumarin for protease activity, while artificial aminopeptidase substrate alters the pH of seawater, confirming previous observations. Before use in ocean acidification research these enzyme assay components must be buffered in order to stabilise sample pH. Reduction of coastal seawater pH to 7.8 was shown to increase β-glucosidase activity rapidly (0.5 h), while no significant response was detected for leucine aminopeptidase, highlighting the need for short-term direct effects of pH on enzyme activities. Bubbling with CO₂ gas resulted in higher β-glucosidase activity when compared to acidification using gas-permeable silicon tubing and acidification with HCl. Although bubbling showed variable effects between two experiments conducted at different times of the year. In addition, bacterial cell numbers were 15–40% higher with bubbling relative to seawater acidified with gas-permeable silicon tubing and HCl. Artefacts associated with bubbling may lead to the overestimation of extracellular enzyme activities, and interpretation of the impacts of ocean acidification on organic matter cycling.