Carbon sequestration by recombinant carbonic anhydrase in Ralstonia eutropha

Introduction and Objectives: Emission of greenhouse gases and plastic wastes are among the most important environmental concerns. The purpose of this study was to use the bacterium Ralstonia eutropha as the host for recombinant carbonic anhydrase enzyme. The enzyme is used for carbon sequestration. The by-product of this sequestration is PHB (poly hydroxy butyrate) which can potentially be used as biodegradable plastic. Materials and Methods: Carbonic anhydrase gene (CA) was inserted in expression vector and transformed into Ralstonia eutropha. Then PHB production was assayed by FT-IR and GC-MS methods in recombinant and non-recombinant bacteria in different concentrations of LB medium in the presence and absence of CO2. Results: The accuracy of cloning was confirmed by PCR using specific primers for the internal region of CA gene. Results of FT-IR indicated that extracted bacterial biopolymer spectrum was aligned with the standard PHB spectrum. Production of PHB in LB medium increased 31.94 percent in recombinant bacteria in presence of CO2. These increases were 18.44 and 34.44 percent compared to non-recombinant bacteria in presence and absence of CO2, respectively. In 0.5 LB medium, the recombinant bacterium produced 26.57 percent PHB in presence of CO2 more than in its absence. Non-recombinant bacterium in this medium produced 18.30 and 30.63 percent PHB less than the recombinant cell in presence and absence of CO2, respectively. Conclusion: The quantity of PHB production using GC-MS revealed that CA gene expression was effective for increasing PHB production in recombinant R. eutropha. The recombinant bacterium could produce PHB in the presence of CO2 by using a simple medium.

Keywords:

Carbonic anhydrase , Carbon sequestration , Ralstonia eutropha