The addition of CO2 to algal culture systems can increase algal biomass effectively. Generally, gas bubbling is used to increase CO2 levels in culture systems; however, it is difficult to quantitatively operate to control the concentration using this method. In this study, we tested the usability of CO2 hydrate for phytoplankton culture. Specifically, green algae Pseudokirchneriella subcapitata were cultured in COMBO medium that contained dissolved CO2 hydrate, after which its effects were evaluated. The experiment was conducted according to a general bioassay procedure (OECD TG201). CO2 promoted algae growth effectively (about 2-fold relative to the control), and the decrease in pH due to dissolution of the CO2 in water recovered soon because of photosynthesis. Since the CO2 hydrate method can control a CO2 concentration easily and quantitatively, it is expected to be useful in future applications. (C) 2014 Elsevier Ltd. All rights reserved.