The effects of CO2 levels (0.03, 0.5, 1, 2 and 5%) on growth and antioxidant responses in 51 bioreactor (working volume 41) root suspension cultures of Echinacea angustifolia were studied. CO2 caused reduction in growth, malondialdehyde content (MDA), lipoxygenase (LOX, EC 1.13.11.13) activity and superoxide anion (O-2(-)) accumulation along with high total phenols accumulation and pH levels. Maximal increases (37%) in total phenols were obtained at 1% CO2 compared to the control. An increase in total glutathione and total ascorbate (ASC+DHA), accompanied with enhanced ascorbate-glutathione cycle enzymes were observed in CO2-treated roots, which played an important role for the detoxification of harmful substances. CO2 also induced an increase in glutathione peroxidase (GPx, EC 1.11.1.9) and glutathione-S-transferase (GST, EC 2.5.18) activities. After native polyacrylamide gel electrophoresis (PAGE) analysis, three superoxide dismutase (SOD, EC 1.15.1.1) isoenzymes were detected. Increase in SOD and CAT (EC 1.11.16) activities observed at 0.5% CO2-treated roots and induced SOD activity seemed to be mainly due to Mn-SOD. However, both SOD and CAT activities were inhibited at 2 and 5% CO2 but reached similar to control value. Three APX (EC 1.11.1.11) and three guaiacol peroxidase (G-POD, EC 1.11.1.7) isoenzymes were strongly detected, an increase in APX and G-POD activities suggests increased scavenging of ROS, indicating the tolerance to CO2. These results indicated that limited oxidative damage as shown by lower MDA level, low LOX activity and low O-2(-) accumulation may be due to the induced activities of antioxidant enzymes and non-enzymatic antioxidant. It is therefore, suggested that roots cultured in a bioreactor could protect themselves from CO2 by altering the defense systems. (c) 2006 Elsevier Inc. All rights reserved.