Abstract: The removal and response patterns of copper (Cu) and zinc (Zn) by the algal-bacterial co-culture system remain unclear. In this study, Chlorella vulgaris was co-cultured with Agrobacterium sp. W01p at a ratio of 2∶1 and exposed to single or combined Cu (0.1, 0.5 mg·L^-1) and Zn (1.0, 5.0 mg·L^-1) treatments for four days. Chlorophyll, dry weight, components of extracellular polymeric substances, superoxide dismutase activity, and Cu/Zn removal rates were measured to compare the differences in responses and removal of those two metals between the co-culture and the monoculture of alga. The results showed that, under the single treatment of Cu (0.1, 0.5 mg·L^-1) or single Zn (1.0, 5.0 mg·L^-1), the chlorophyll content in the algae-bacteria co-culture was 1.1-2.8 times that in monoculture of C. vulgaris. Under the combined Cu and Zn treatments (0.1Cu+1.0Zn, 0.5Cu+1.0Zn, 0.1Cu+5.0Zn), the chlorophyll content in the algae-bacteria co-culture was increased by 1.1-4.1 times compared with that in single C. vulgaris system. Compared with the monoculture system, the removal rate of Cu and Zn by the co-culture increased by 7.4%-16.0%. The inhibition rate of chlorophyll synthesis in Chlorella by Cu-Zn combined stress was significantly higher than that in single Cu or Zn treatments (P<0.05). The combined treatment of Zn (1.0, 5.0 mg·L^-1) and Cu (0.1 mg·L^-1) increased the Cu removal by the co-culture system from 39.3% to 60.2% and 68.3%, respectively. The combined treatment of Cu (0.1, 0.5 mg·L^-1) and Zn (1.0 mg·L^-1) decreased the Zn removal from 89.1% to 56.1% and 40.8%, respectively. Co-culture with Agrobacterium sp. W01p significantly increased the chlorophyll content of C. vulgaris to 1.1-4.1 times that of the monoculture of alga (P<0.05), and increased the total amount of extracellular polymeric substances to 1.1-2.4 times that of the single alga (P<0.05). The superoxide dismutase activity of the algal-bacterial co-culture system increased by 18.4%-215.9% compared with the alga alone under Cu and Zn treatments, confirming the stress alleviation through antioxidant enzymes. Our results indicated that the algal-bacterial co-culture system had stronger Cu and Zn tolerance and removal ability than monoculture algal system. The coexistence of Cu and Zn favored the removal of Cu but not Zn. Increased secretion of extracellular polymeric substances, up-regulation of antioxidant enzyme activities, and specific binding of functional groups may explain the enhanced capacity of the algal co-culture system to tolerate and remove Cu and Zn.

Key words: Chlorella vulgaris, Agrobacterium sp., co-culture, algae-bacteria interaction