Effect of long-term high CO2 environment on gene expression of Phaeodactylum tricornutum. Effect of long-term high CO2 environment on gene expression of Phaeodactylum tricornutum

P. tricornutum (Bacillariophyta, Pennatae, NEPCC640) was obtained from the Algal Center of the Institute of Oceanology of the Chinese Academy of Sciences. The cells were cultured in a modified f/2 medium (Guillard, 1975) at 20 +/- 1C, and illuminated with 120 umol photon m-2 s-1 under a 12:12 light: dark cycle. Flasks were shaken by the researchers twice a day at the fixed times. Experiments were conducted in triplicate in 3L sterilized and acid-washed Erlenmeyer flask containing 2L medium. The equipment used in this study is similar to the ones used in previous ocean acidification research (Fu et al., 2007; Hutchins et al., 2007; Wu et al., 2010). Prior to inoculation, the mediums were treated by different CO2 concentrations. The low CO2 medium was bubbled with ambient air of about 400 ppmv (low CO2, LC) and the high CO2 medium was bubbled with pre-mixed air-CO2 mixtures (1000 ppmv; high CO2, HC) from a plant growth CO2 chamber (HP400G-D, Ruihua Instrument & Equipment Ltd, Wuhan, China) with a variation of less than 5%. Semi-continuous cultures were used to maintain the pH stability during P. tricornutum growth in the present study, All the cultures were diluted to 1x104 cells mL-1 with fresh medium and pre-acclimated to the desired CO2 level every 24 h to maintain an exponential growth phase and minimize pH fluctuations of the cells. Cultures were harvested after 8 months of semi-continuous incubation. Significant differences between the carbonate systems in different cultures.

三角褐指藻（P. tricornutum）隶属于硅藻门（Bacillariophyta）、羽纹纲（Pennatae），菌株编号NEPCC640，购自中国科学院海洋研究所藻类中心。本研究采用改良型f/2培养基（Guillard，1975）进行细胞培养，培养温度设置为20±1℃，光照强度为120 μmol光子·米⁻²·秒⁻¹，光暗周期为12:12。研究人员每日于固定时段摇晃培养瓶两次。实验采用经灭菌及酸洗处理的3L锥形瓶，每瓶装入2L培养基，每组设置3次生物学重复。本研究使用的实验设备与既往海洋酸化相关研究（Fu等，2007；Hutchins等，2007；Wu等，2010）所用设备一致。接种前，需对培养基施加不同CO₂浓度处理：低CO₂组（LC）通入约400ppmv的常压空气，高CO₂组（HC）则通入由武汉瑞华仪器设备有限公司植物生长CO₂培养箱（HP400G-D）配制的预混合空气-CO₂混合气（1000ppmv），混合气浓度波动幅度小于5%。本研究采用半连续培养体系以维持三角褐指藻生长过程中的pH稳定性：所有培养物每日均使用新鲜培养基稀释至1×10⁴ 个细胞·毫升⁻¹，并预适应目标CO₂浓度，以维持指数生长阶段并最小化细胞的pH波动。半连续培养8个月后收获培养物。不同培养体系的碳酸盐系统存在显著差异。