Investigating Mass Transfer Limitations during Biodegradation of Micropollutants with Compound-Specific Isotope Analysis

Abstract

Worldwide, thousands of xenobiotics are discharged into the environment either by accident, e.g. in spills, or on purpose, e.g. when pesticides like atrazine or glyphosate are applied on agricultural fields. Even though most of these chemicals are initially degraded by bacteria, this degradation seems to stall at low concentrations in ground water and surface water. As a consequence, humans are exposed to a large number of these persistent chemical pollutants in drinking water. Two competing paradigms claim that biodegradation is either mass transfer limited or cell physiology limited. While multiple methods, e.g. proteomics, are available to study physiological adaptation, pinpointing mass transfer limitations is challenging, as simple concentration measurements are not sufficient. Therefore, we employed isotope fractionation during biodegradation, a promising concentration independent tool to identify and distinguish different rate determining steps of reactions, to unravel underlying mass transfer limitations during pollutant biodegradation. To mimic oligotrophic conditions where biodegradation seems to stall, we cultivated the atrazine degrader Arthrobacter aurescens TC1 in chemostat with atrazine as the sole carbon and nitrogen source. The dilution rate was varied and we observed a decreasing isotope fractionation factor from ε13C = -5.4 ‰ at 85 µg∙L-1 atrazine down to ε13C = -2.3 ‰ at 33 µg∙L-1 with decreasing residual atrazine concentrations. Thus, we were able to pinpoint a rapid onset of rate limiting mass transfer across the cell envelope when bacteria adapt to oligotrophic conditions and transition to stationary phase at slow growth rates. To further elucidate the role of the cell envelope as barrier to biodegradation, we (i) compared atrazine uptake in Gram-positive Arthrobacter aurescens TC1 and Gram-negative Polaromonas sp. NeaC and (ii) studied glyphosate permeation in liposome models systems and during biodegradation. The intrinsic enzymatic fractionation factor of atrazine hydrolysis by TrzN ε13C = -5.3 ‰ was masked in whole cells of Polaromonas sp. NeaC ε13C = -3.5 ‰, but not in Gram-positive Arthrobacter aurescens TC1. As the atrazine degradation rates were not reduced after inhibition of active transporter, we identified the outer membrane in Gram-negative Polaromonas sp. NeaC as the barrier to atrazine influx. High glyphosate permeation rates in the liposome model system indicate that passive membrane permeation is also an underestimated uptake pathway for charged pollutants like glyphosate. Additionally, that this glyphosate uptake is not rate determining for glyphosate biodegradation was confirmed by strong isotope fractionation during glyphosate biodegradation by a newly isolated degrader strain Ochrobactrum sp. FrEM. To sum up, this thesis not only unravels the role of passive membrane permeation for pollutant degradation but also addresses the environmental implications of rate limiting mass transfer at low concentrations.