Isotope fractionation in atrazine degradation reveals rate-limiting, energy-dependent transport across the cell membrane of gram-negative rhizobium sp. CX-Z.

In the biological mass transfer of organic contaminants like atrazine, the cellular membrane limits bioavailability of pesticides. We aimed to illustrate the roles of cellular membrane physiology and substrate uptake (e.g., passive diffusion and energy-dependent transport) on the limitations of bioavailability in atrazine biodegradation by Gram-negative strain Rhizobium sp. CX-Z. Compound-specific stable isotope analysis revealed energy-dependent transport across cellular membrane led to bioavailability limitations in atrazine biotransformation. Carbon isotope fractionation (ε = -1.8 ± 0.3‰) was observed and significantly smaller in atrazine biodegradation by Rhizobium sp. CX-Z than that expected in acid hydrolysis (ε = -4.8 ± 0.4‰) and hydrolysis by the pure enzyme TrzN (ε = -5.0 ± 0.2‰). However, isotope fractionation was restored in membrane-free cells of Rhizobium sp. CX-Z (ε = -5.4 ± 0.2‰) where no cellular membrane limits substrate uptake. When respiratory chain was inhibited by rotenone, the pseudo-first order kinetic rate constants (0.08 ± 0.03 h, 0.09 ± 0.03 h) was observed to be statistically less than in the control group (0.23 ± 0.02 h, 0.33 ± 0.02 h), demonstrating that energy-dependent transport dominated atrazine transfer across the cellular membrane. Therefore, our results revealed energy-dependent transport across cellular membrane existing in Gram-negative strain Rhizobium sp. CX-Z determines bioavailability of atrazine in biotransformation process even at high concentration.