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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 (e(C) = −1.8 ± 0.3‰) was observed and significantly smaller in atrazine biodegradation by Rhizobium sp. CX-Z than that expected in acid hydrolysis (e(C) = −4.8 ± 0.4‰) and hydrolysis by the pure enzyme TrzN (e(C) = −5.0 ± 0.2‰). However, isotope fractionation was restored in membrane-free cells of Rhizobium sp. CX-Z (e(C) = −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−1, 0.09 ± 0.03 h−1) was observed to be statistically less than in the control group (0.23 ± 0.02 h−1, 0.33 ± 0.02 h−1), 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.

Compound-specific stable isotope analysis of micropollutants has become an established method for the qualitative and quantitative assessment of biodegradation in the field. However, many of environmental factors may have an influence on the observed isotope fractionation. Herein, we investigate the impact of substrate concentration on the observed enrichment factor derived from Rayleigh plot of batch laboratory experiments conducted to measure the atrazine carbon isotope fractionation of Rhizobium sp. CX-Z subjected to the different initial concentration level of atrazine. The Rayleigh plot (changes in bulk concentration vs. isotopic composition) derived from batch experiments shown divergence from the linear relation towards the end of degradation, confirming bioavailability of atrazine changed along with the decay of substrate concentration, consequently, influenced the isotope fractionation and lowered the observed enrichment factor. When microbial degradation is coupled to a mass transfer step limiting the bioavailability of substrate, the observed enrichment factor displays a dependence on initial atrazine concentration. Observed enrichment factors (e) (absolute value) derived from the low concentration (i.e. 9.5 μM) are below 3.5‰ to the value of −5.4‰ determined at high bioavailability (membrane-free cells). The observed enrichment factor depended significantly on the atrazine concentration, indicating the concentration level and the bioavailability of a substrate in realistic environments should be considered during the assessment of microbial degradation or in situ bioremediation based on compound-specific stable isotope analysis (CSIA) method.