Modeling of Contaminant Biodegradation and Compound-Specific Isotope Fractionation in Chemostats at Low Dilution Rates.

We present a framework to model microbial transformations in chemostats and retentostats under transient or quasi-steady state conditions. The model accounts for transformation-induced isotope fractionation and mass-transfer across the cell membrane. It also verifies that the isotope fractionation ϵ can be evaluated as the difference of substrate-specific isotope ratios between inflow and outflow. We explicitly considered that the dropwise feeding of substrate into the reactor at very low dilution rates leads to transient behavior of concentrations and transformation rates and use this information to validate conditions under which a quasi-steady state treatment is justified. We demonstrate the practicality of the code by modeling a chemostat experiment of atrazine degradation at low dilution/growth rates by the strain Arthrobacter aurescens TC1. Our results shed light on the interplay of processes that control biodegradation and isotope fractionation of contaminants at low (µg/L) concentration levels. With the help of the model, an estimate of the mass-transfer coefficient of atrazine through the cell membrane was achieved (0.0025 s-1).

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

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.

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

Bacterial uptake of charged organic pollutants such as the widely used herbicide glyphosate is typically attributed to active transporters, whereas passive membrane permeation as an uptake pathway is usually neglected. For 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) liposomes, the pH-dependent apparent membrane permeation coefficients ( Papp) of glyphosate, determined by nuclear magnetic resonance (NMR) spectroscopy, varied from Papp (pH 7.0) = 3.7 (±0.3) × 10-7 m·s-1 to Papp (pH 4.1) = 4.2 (±0.1) × 10-6 m·s-1. The magnitude of this surprisingly rapid membrane permeation depended on glyphosate speciation and was, at circumneutral pH, in the range of polar, noncharged molecules. These findings point to passive membrane permeation as a potential uptake pathway during glyphosate biodegradation. To test this hypothesis, a Gram-negative glyphosate degrader, Ochrobactrum sp. FrEM, was isolated from glyphosate-treated soil and glyphosate permeation rates inferred from the liposome model system were compared to bacterial degradation rates. Estimated maximum permeation rates were, indeed, 2 orders of magnitude higher than degradation rates of glyphosate. In addition, biodegradation of millimolar glyphosate concentrations gave rise to pronounced carbon isotope fractionation with an apparent kinetic isotope effect, AKIEcarbon, of 1.014 ± 0.003. This value lies in the range typical of non-masked enzymatic isotope fractionation demonstrating that glyphosate biodegradation was not subject to mass transfer limitations and glyphosate exchange across the cell membrane was rapid relative to enzymatic turnover.

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

Biodegradation of persistent pesticides like atrazine often stalls at low concentrations in the environment. While mass transfer does not limit atrazine degradation by the Gram-positive Arthrobacter aurescens TC1 at high concentrations (>1 mg/L), evidence of bioavailability limitations is emerging at trace concentrations (<0.1 mg/L). To assess the bioavailability constraints on biodegradation, the roles of cell wall physiology and transporters remain imperfectly understood. Here, compound-specific isotope analysis (CSIA) demonstrates that cell wall physiology (i.e., the difference between Gram-negative and Gram-positive bacteria) imposes mass transfer limitations in atrazine biodegradation even at high concentrations. Atrazine biodegradation by Gram-negative Polaromonas sp. Nea-C caused significantly less isotope fractionation (ε(C) = -3.5 ‰) than expected for hydrolysis by the enzyme TrzN (ε(C) = -5.0 ‰) and observed in Gram-positive Arthrobacter aurescens TC1 (ε(C) = -5.4 ‰). Isotope fractionation was recovered in cell-free extracts (ε(C) = -5.3 ‰) where no cell envelope restricted pollutant uptake. When active transport was inhibited with cyanide, atrazine degradation rates remained constant demonstrating that atrazine mass transfer across the cell envelope does not depend on active transport but is a consequence of passive cell wall permeation. Taken together, our results identify the cell envelope of the Gram-negative bacterium Polaromonas sp. Nea-C as a relevant barrier for atrazine biodegradation.

Single molecule kinetics of horseradish peroxidase exposed in large arrays of femtoliter-sized fused silica chambers.

Large arrays of femtoliter-sized chambers were etched into the surface of fused silica slides to enclose and observe hundreds of single horseradish peroxidase (HRP) molecules in parallel. Individual molecules of HRP oxidize the fluorogenic substrate Amplex Red to fluorescent resorufin in separate chambers, which was monitored by fluorescence microscopy. Photooxidation of Amplex Red and photobleaching of resorufin have previously limited the analysis of HRP in femtoliter arrays. We have strongly reduced these effects by optimizing the fluorescence excitation and detection scheme to yield accurate single molecule substrate turnover rates. We demonstrate the presence of long-lived kinetic states of single HRP molecules that are individually different for each molecule in the array. The large number of molecules investigated in parallel provides excellent statistics on the activity distribution in the enzyme population, which is similar to that reported for other enzymes such as ß-galactosidase. We have further confirmed that the product formation of HRP in femtoliter chambers is 10-fold lower than that in the bulk solution due to the particular two-step redox reaction mechanism of HRP.