Rate-Limiting Mass Transfer in Micropollutant Degradation Revealed by Isotope Fractionation in Chemostat

Abstract

Biodegradation of persistent micropollutants like pesticides often slows down at low concentrations (μg/L) in the environment. Mass transfer limitations or physiological adaptation are debated to be responsible. Although promising, evidence from compound-specific isotope fractionation analysis (CSIA) remains unexplored for bacteria adapted to this low concentration regime. We accomplished CSIA for degradation of a persistent pesticide, atrazine, during cultivation of Arthrobacter aurescens TC1 in chemostat under four different dilution rates leading to 82, 62, 45, and 32 μg/L residual atrazine concentrations. Isotope analysis of atrazine in chemostat experiments with whole cells revealed a drastic decrease in isotope fractionation with declining residual substrate concentration from ε(C) = −5.36 ± 0.20‰ at 82 μg/L to ε(C) = −2.32 ± 0.28‰ at 32 μg/L. At 82 μg/L ε(C) represented the full isotope effect of the enzyme reaction. At lower residual concentrations smaller ε(C) indicated that this isotope effect was masked indicating that mass transfer across the cell membrane became rate-limiting. This onset of mass transfer limitation appeared in a narrow concentration range corresponding to about 0.7 μM assimilable carbon. Concomitant changes in cell morphology highlight the opportunity to study the role of this onset of mass transfer limitation on the physiological level in cells adapted to low concentrations.

Isotope Fractionation Pinpoints Membrane Permeability as a Barrier to Atrazine Biodegradation in Gram-negative Polaromonas sp. Nea-C

“…Indeed, the calculated atrazine lipid diffusion coefficient D lip = 1.3 × 10 –17 m 2 /s was, as expected, smaller, but in the same range as D lip recently observed for atrazine in a single lipid bilayer of the Gram-positive A. aurescens TC1. 31 This demonstrates that our modeling approach yields realistic values for k tr allowing us to use k tr to predict a decreasing observed fractionation factor ε* with decreasing atrazine concentrations according to Thullner et al 30 Consistent with our experimental results, at a concentration of 4 mg/L the enzymatic fractionation factor of ε = −5.3 ‰ is already reduced to ε* = −3.5 ‰, and it is predicted to be further reduced to below −3 ‰ already at an atrazine concentration of 1 mg/L.…”

“…29,30 Based on these studies, we recently discovered that cell wall permeation was not relevant for atrazine biodegradation by Arthrobacter aurescens TC1 at high concentrations but became suddenly rate-limiting at low concentrations (low microgram per liter range). 31 This finding is challenged by earlier observations by Meyer et al that even at high concentrations, isotope fractionation in atrazine degradation varied significantly between bacterial strains catalyzing the same reaction. 32 Usually, the isotope fractionation factor is assumed to be characteristic for a specific transformation pathway if the underlying enzyme reaction is identical.…”

High Permeation Rates in Liposome Systems Explain Rapid Glyphosate Biodegradation Associated with Strong Isotope Fractionation

“…[20][21][22][23] Recent work highlights the particular role of pollutant mass transfer into microbial cells as a rate-limiting step for biodegradation, especially at low pollutant concentrations. 24,25 The mass transfer of polar and charged species (e.g., zwitterionic glyphosate 26 ) into bacterial cells is currently assumed to occur by active transport. 27,28 Little is known whether charged molecules can directly permeate the cell membrane as non-polar pollutants do, 29,30 and if so, to what extent the bacterial membrane as diffusion barrier constitutes an even stronger bioavailability limitation for these charged molecules than for non-charged pollutants.…”

“…[51][52][53] As a consequence, the enzymatic isotope fractionation that is observable in solution becomes masked in the presence of mass transfer limitations -i.e., when active transport (or passive membrane permeation) into and out of the cell is the rate-determining step in biodegradation. 25,33 For this study, we used a combined approach to gain insight into the role of passive permeation for biodegradation of the zwitterionic pollutant glyphosate, which carries either one (pH < 6) or two (pH > 6) net negative charges at circumneutral pH. First, an NMR study was conducted to experimentally determine pH-dependent passive membrane permeation of glyphosate in phosphatidylcholine liposomes as model system.…”

“…18,73 We recently demonstrated that mass transfer across the cell membrane becomes rate-limiting for atrazine biodegradation at trace concentrations. 25…”