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Soil biodegradable mulch films composed of the polyester polybutylene adipate-co-terephthalate (PBAT) are being increasingly used in agriculture. Analytical methods to quantify PBAT in field soils are needed to assess its soil occurrence and fate. Here, we report an analytical method for PBAT in soils that couples Soxhlet extraction or accelerated solvent extraction (ASE) with quantitative protonnuclear magnetic resonance (q-1H NMR) spectroscopy detection. The 1H NMR peak areas of aromatic PBAT protons increased linearly with PBAT concentrations dissolved in deuterated chloroform (CDCl3), demonstrating accurate quantitation of PBAT by q-1H NMR. Spike-recovery experiments involving PBAT addition to model sorbents and soils showed increased PBAT extraction efficiencies into chloroform (CHCl3) with methanol (MeOH) as cosolvent, consistent with MeOH competitively displacing PBAT from H-bond donating sites on mineral surfaces. Systematic variations in solvent composition and temperatures in ASE revealed quantitative PBAT extraction from soil with 90/10 volume % CHCl3/MeOH at 110–120 °C. Both Soxhlet extraction and ASE resulted in the complete recovery of PBAT added to a total of seven agricultural soils covering a range of physicochemical properties, independent of whether PBAT was added to soils dissolved in CHCl3, as film, or as particles. Recovery was also complete for PBAT added to soil in the form of a commercial soil biodegradable mulch film with coextractable polylactic acid (PLA). The presented analytical method enables accurate quantification and biodegradation monitoring of PBAT in agricultural field soils.

To optimize removal of organic micropollutants from the water cycle, understanding the processes during activated sludge treatment is essential. In this study, we hypothesize that aliphatic amines, which are highly abundant among organic micropollutants, are partly removed from the water phase in activated sludge through ion trapping in protozoa. In ion trapping, which has been extensively investigated in medical research, the neutral species of amine-containing compounds diffuse through the cell membrane and further into acidic vesicles present in eukaryotic cells such as protozoa. There they become trapped because diffusion of the positively charged species formed in the acidic vesicles is strongly hindered. We tested our hypothesis with two experiments. First, we studied the distribution of the fluorescent amine acridine orange in activated sludge by confocal fluorescence imaging. We observed intense fluorescence in distinct compartments of the protozoa, but not in the bacterial biomass. Second, we investigated the distribution of 12 amine-containing and eight control micropollutants in both regular activated sludge and sludge where the protozoa had been inactivated. In contrast to most control compounds, the amine-containing micropollutants displayed a distinctly different behavior in the noninhibited sludge compared to the inhibited one: (i) more removal from the liquid phase; (ii) deviation from first-order kinetics for the removal from the liquid phase; and (iii) higher amounts in the solid phase. These results provide strong evidence that ion trapping in protozoa occurs and that it is an important removal mechanism for amine-containing micropollutants in batch experiments with activated sludge that has so far gone unnoticed. We expect that our findings will trigger further investigations on the importance of this process in full-scale wastewater treatment systems, including its relevance for accumulation of ammonium.

2,6-Dichlorobenzamide (BAM) is an omnipresent micropollutant in European groundwaters. Aminobacter niigataensisMSH1 is a prime candidate for biologically treating BAM-contaminated groundwater since this organism is capable of utilizing BAM as a carbon and energy source. However, detailed information on the BAM degradation kinetics by MSH1 at trace concentrations is lacking, while this knowledge is required for predicting and optimizing the degradation process. Contaminating assimilable organic carbon (AOC) in media makes the biodegradation experiment a mixed-substrate assay and hampers exploration of pollutant degradation at trace concentrations. In this study, we examined how the BAM concentration affects MSH1 growth and BAM substrate utilization kinetics in a AOC-restricted background to avoid mixed-substrate conditions. Conventional Monod kinetic models were unable to predict kinetic parameters at low concentrations from kinetics determined at high concentrations. Growth yields on BAM were concentration-dependent and decreased substantially at trace concentrations; i.e., growth of MSH1 diminished until undetectable levels at BAM concentrations below 217 μg-C/L. Nevertheless, BAM degradation continued. Decreasing growth yields at lower BAM concentrations might relate to physiological adaptations to low substrate availability or decreased expression of downstream steps of the BAM catabolic pathway beyond 2,6-dichlorobenzoic acid (2,6-DCBA) that ultimately leads to Krebs cycle intermediates for growth and energy conservation.

2,6-Dichlorobenzamide (BAM) is a major groundwater micropollutant posing problems for drinking water treatment plants (DWTPs) that depend on groundwater intake. Aminobactersp. MSH1 uses BAM as the sole source of carbon, nitrogen, and energy and is considered a prime biocatalyst for groundwater bioremediation in DWTPs. Its use in bioremediation requires knowledge of its BAM-catabolic pathway, which is currently restricted to the amidase BbdA converting BAM into 2,6-dichlorobenzoic acid (2,6-DCBA) and the monooxygenase BbdD transforming 2,6-DCBA into 2,6-dichloro-3-hydroxybenzoic acid. Here, we show that the 2,6-DCBA catabolic pathway is unique and differs substantially from catabolism of other chlorobenzoates. BbdD catalyzes a second hydroxylation, forming 2,6-dichloro-3,5-dihydroxybenzoic acid. Subsequently, glutathione-dependent dehalogenases (BbdI and BbdE) catalyze the thiolytic removal of the first chlorine. The remaining chlorine is then removed hydrolytically by a dehalogenase of the α/β hydrolase superfamily (BbdC). BbdC is the first enzyme in that superfamily associated with dehalogenation of chlorinated aromatics and appears to represent a new subtype within the α/β hydrolase dehalogenases. The activity of BbdC yields a unique trihydroxylated aromatic intermediate for ring cleavage that is performed by an extradiol dioxygenase (BbdF) producing 2,4,6-trioxoheptanedioic acid, which is likely converted to Krebs cycle intermediates by BbdG.