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To determine whether the bicarbonate in karst limestone soil could be used as a new carbon resource for biomass production by the catalysis of carbonic anhydrase (CA), a simulative karst drought stress experiment was designed and performed. Three plants used for biofuel energy, Orychophragmus violaceus L. (Ov), Brassica juncea L. (Bj), and Euphorbia lathyris L. (El), were grown under simulated karst drought stress. In response to drought stress, the photosynthesis of the three energy plants was inhibited, but their CA activity increased. The hypothesis was confirmed by plant physiological and stable isotope techniques. The obtained results showed that plant biomass was produced with atmospheric CO2 as well as bicarbonate under drought stress. Bicarbonate use was proportional to the CA activity of the plants. With high CA activity over a long period, El had the highest proportional bicarbonate use compared to Ov and Bj, reaching 26.95%. Additionally, a new method is proposed for the screening of plants grown for energy in karst habitats.