Uptake, Metabolism, and Accumulation of Tire Wear Particle-Derived Compounds in Lettuce.

Tire wear particle (TWP)-derived compounds may be of high concern to consumers when released in the root zone of edible plants. We exposed lettuce plants to the TWP-derived compounds diphenylguanidine (DPG), hexamethoxymethylmelamine (HMMM), benzothiazole (BTZ), N-phenyl-N'-(1,3-dimethylbutyl)-p-phenylenediamine (6PPD), and its quinone transformation product (6PPD-q) at concentrations of 1 mg L-1 in hydroponic solutions over 14 days to analyze if they are taken up and metabolized by the plants. Assuming that TWP may be a long-term source of TWP-derived compounds to plants, we further investigated the effect of leaching from TWP on the concentration of leachate compounds in lettuce leaves by adding constantly leaching TWP to the hydroponic solutions. Concentrations in leaves, roots, and nutrient solution were quantified by triple quadrupole mass spectrometry, and metabolites in the leaves were identified by Orbitrap high resolution mass spectrometry. This study demonstrates that TWP-derived compounds are readily taken up by lettuce with measured maximum leaf concentrations between ∼0.75 (6PPD) and 20 µg g-1 (HMMM). Although these compounds were metabolized in the plant, we identified several transformation products, most of which proved to be more stable in the lettuce leaves than the parent compounds. Furthermore, continuous leaching from TWP led to a resupply and replenishment of the metabolized compounds in the lettuce leaves. The stability of metabolized TWP-derived compounds with largely unknown toxicities is particularly concerning and is an important new aspect for the impact assessment of TWP in the environment.

Identifying Functional Groups that Determine Rates of Micropollutant Biotransformations Performed by Wastewater Microbial Communities.

The goal of this research was to identify functional groups that determine rates of micropollutant (MP) biotransformations performed by wastewater microbial communities. To meet this goal, we performed a series of incubation experiments seeded with four independent wastewater microbial communities and spiked them with a mixture of 40 structurally diverse MPs. We collected samples over time and used high-resolution mass spectrometry to estimate biotransformation rate constants for each MP in each experiment and to propose structures of 46 biotransformation products. We then developed random forest models to classify the biotransformation rate constants based on the presence of specific functional groups or observed biotransformations. We extracted classification importance metrics from each random forest model and compared them across wastewater microbial communities. Our analysis revealed 30 functional groups that we define as either biotransformation promoters, biotransformation inhibitors, structural features that can be biotransformed based on uncharacterized features of the wastewater microbial community, or structural features that are not rate-determining. Our experimental data and analysis provide novel insights into MP biotransformations that can be used to more accurately predict MP biotransformations or to inform the design of new chemical products that may be more readily biodegradable during wastewater treatment.

Towards more Sustainable Peptide- based Antibiotics: Stable in Human Blood, Enzymatically Hydrolyzed in Wastewater?

The emergence and spread of antibiotic resistance is a major societal challenge and new antibiotics are needed to successfully fight bacterial infections. Because the release of antibiotics into wastewater and downstream environments is expected to contribute to the problem of antibiotic resistance, it would be beneficial to consider the environmental fate of antibiotics in the development of novel antibiotics. In this article, we discuss the possibility of designing peptide-based antibiotics that are stable during treatment (e.g. in human blood), but rapidly inactivated through hydrolysis by peptidases after their secretion into wastewater. In the first part, we review studies on the biotransformation of peptide-based antibiotics during biological wastewater treatment and on the specificity of dissolved extracellular peptidases derived from wastewater. In the second part, we present first results of our endeavour to identify peptide bonds that are stable in human blood plasma and susceptible to hydrolysis by the industrially produced peptidase Subtilisin A.

Exploring the Specificity of Extracellular Wastewater Peptidases to Improve the Design of Sustainable Peptide-Based Antibiotics.

New antimicrobial peptides are emerging as promising alternatives to conventional antibiotics because of their specificity for target pathogens and their potential to be rapidly hydrolyzed (i.e., inactivated) by extracellular peptidases during biological wastewater treatment, thereby limiting the emergence and propagation of antibiotic resistance in the environment. However, little is known about the specificity of extracellular peptidases derived from wastewater microbial communities, which is a major impediment for the design of sustainable peptide-based antibiotics that can be hydrolyzed by wastewater peptidases. We used a set of natural peptides to explore the specificity of dissolved extracellular wastewater peptidases. We found that enzyme-catalyzed hydrolysis occurred at specific sites and that a subset of these hydrolyses was conserved across enzyme pools derived from three independent wastewater microbial communities. An analysis of the amino-acid residues flanking the hydrolyzed bonds revealed a set of residue motifs that were linked to enzyme-catalyzed hydrolysis and are therefore candidates for incorporation into new and sustainable peptide-based antibiotics.

Biotransformation of antibiotics: Exploring the activity of extracellular and intracellular enzymes derived from wastewater microbial communities.

Evaluating the activity of extracellular and intracellular enzymes derived from wastewater microbial communities is essential to improve our fundamental understanding of micropollutant removal during wastewater treatment. To study biotransformations with respect to enzyme biogeography, we developed a method to separate soluble extracellular, extracellular polymeric substance (EPS)-bound, and intracellular enzymes from wastewater microbial communities and assessed the protease and peptidase activity of the resulting enzyme pools. We also evaluated the biotransformation of six antibiotics (amoxicillin, ampicillin, clindamycin, daptomycin, linezolid, and vancomycin) in each enzyme pool because we expect that the kinetics, pathways, and biogeography of antibiotic biotransformations influence the selection of antibiotic resistance within wastewater microbial communities and in downstream environments. Our results demonstrated that biotransformation rate constants varied among the tested antibiotics, and that the observed rank order was consistent across three wastewater treatment plants. Importantly, many of the observed biotransformations eliminated the functional groups associated with antibiotic activity. Furthermore, we found that ß-lactam hydrolysis and daptomycin hydrolysis were catalyzed by enzymes extracted from the EPS, while none of the tested antibiotics were biotransformed by soluble extracellular enzymes. Finally, our results demonstrated that the number of enzyme-catalyzed antibiotic transformations was larger for intracellular than for extracellular enzymes. Together, this study provides novel insights on the kinetics, pathways, and biogeography of antibiotic biotransformations performed by wastewater microbial communities and can be used to inform pathway prediction or the development of biodegradable chemicals.

Photochemical Transformation of Poly(butylene adipate- co-terephthalate) and Its Effects on Enzymatic Hydrolyzability.

Biodegradable polyesters are being increasingly used to replace conventional, nondegradable polymers in agricultural applications such as plastic film for mulching. For many of these applications, poly(butylene adipate- co-terephthalate) (PBAT) is a promising biodegradable material. However, PBAT is also susceptible to photochemical transformations. To better understand how photochemistry affects the biodegradability of PBAT, we irradiated blown, nonstabilized, transparent PBAT films and studied their enzymatic hydrolysis, which is considered the rate-limiting step in polyester biodegradation. In parallel, we characterized the irradiated PBAT films by dynamic mechanical thermal analysis. The rate of enzymatic PBAT hydrolysis decreased when the density of light-induced cross-links within PBAT exceeded a certain threshold. Mass-spectrometric analysis of the enzymatic hydrolysis products of irradiated PBAT films provided evidence for radical-based cross-linking of two terephthalate units that resulted in the formation of benzophenone-like molecules. In a proof-of-principle experiment, we demonstrated that the addition of photostabilizers to PBAT films mitigated the negative effect of UV irradiation on the enzymatic hydrolyzability of PBAT. This work advances the understanding of light-induced changes on the enzyme-mediated hydrolysis of aliphatic-aromatic polyesters and will therefore have important implications for the development of biodegradable plastics.

Sustainable Polyester Elastomers from Lactones: Synthesis, Properties, and Enzymatic Hydrolyzability.

Chemically cross-linked elastomers are an important class of polymeric materials with excellent temperature and solvent resistance. However, nearly all elastomers are petroleum-derived and persist in the environment or in landfills long after they are discarded; this work strives to address these issues by demonstrating the synthesis of renewable, enzymatically hydrolyzable, and mechanically competitive polyester elastomers. The elastomers described were synthesized using a novel bis(ß-lactone) cross-linker and star-shaped, hydroxyl-terminated poly(γ-methyl-ε-caprolactone). Using model compounds, we determined that the bis(ß-lactone) cross-linker undergoes acyl bond cleavage to afford ß-hydroxyesters at the junctions. The mechanical properties of the cross-linked materials were tunable and competitive with a commodity rubber band. Furthermore, the elastomers demonstrated high thermal stability and a low glass transition (-50 °C), indicating a wide range of use temperatures. The polyester networks were also subjected to enzymatic hydrolysis experiments to investigate the potential for these materials to biodegrade in natural environments. We found that they readily hydrolyzed at neutral pH and environmentally relevant temperatures (2-40 °C); complete hydrolysis was achieved in all cases at temperature-dependent rates. The results presented in this work exemplify the development of high performance yet sustainable alternatives to conventional elastomers.

Polyol Structure Influences Enzymatic Hydrolysis of Bio-Based 2,5-Furandicarboxylic Acid (FDCA) Polyesters.

Polyesters of 2,5-furandicarboxylic acid (FDCA) have gained attention as they can be regarded as the bio-based alternatives to the petroleum-based polyesters of terephthalic acid. However, only little is known about the biodegradation and enzymatic hydrolysis of FDCA-based polyesters. This work aims to investigate the influence of different polyols on enzymatic hydrolysis of FDCA-based polyesters. A series of polyesters containing various polyols are synthesized and analyzed regarding susceptibility to enzymatic hydrolysis by cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1). FDCA-based polyesters' number average molecular weight (Mn ) ranged from 9360-35 800 g mol-1 according to gel permeation chromatography (GPC) analysis. Differential scanning calorimetry (DSC) analyses show decreasing glass transition temperature (Tg ) with increasing diol chain length. Crystallinity of all polyesters is below 1% except for polyesters containing 1,6-hexanediol, 1,8-octanediol, and 1,12-dodecanediol for which calculated crystallinities are 27, 37, and 30%, respectively. Thc_Cut1 hydrolyzes all tested polyesters with preference for polyesters containing 1,5-pentanediol and 1,9-nonanediol (57.7 ± 7.5 and 52.8 ± 4.0% released FDCA). Enzyme activity increases when the linear diol 1,3-propanediol is replaced by the branched analog 1,2-propanediol or ethoxy units are introduced into the polyester chain. The results will contribute to expand the knowledge of microbial biodegradation of FDCA-based polyesters.

Enzymatic Degradation of Aromatic and Aliphatic Polyesters by P. pastoris Expressed Cutinase 1 from Thermobifida cellulosilytica.

To study hydrolysis of aromatic and aliphatic polyesters cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1) was expressed in P. pastoris. No significant differences between the expression of native Thc_Cut1 and of two glycosylation site knock out mutants (Thc_Cut1_koAsn and Thc_Cut1_koST) concerning the total extracellular protein concentration and volumetric activity were observed. Hydrolysis of poly(ethylene terephthalate) (PET) was shown for all three enzymes based on quantification of released products by HPLC and similar concentrations of released terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were detected for all enzymes. Both tested aliphatic polyesters poly(butylene succinate) (PBS) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were hydrolyzed by Thc_Cut1 and Thc_Cut1_koST, although PBS was hydrolyzed to significantly higher extent than PHBV. These findings were also confirmed via quartz crystal microbalance (QCM) analysis; for PHBV only a small mass change was observed while the mass of PBS thin films decreased by 93% upon enzymatic hydrolysis with Thc_Cut1. Although both enzymes led to similar concentrations of released products upon hydrolysis of PET and PHBV, Thc_Cut1_koST was found to be significantly more active on PBS than the native Thc_Cut1. Hydrolysis of PBS films by Thc_Cut1 and Thc_Cut1_koST was followed by weight loss and scanning electron microscopy (SEM). Within 96 h of hydrolysis up to 92 and 41% of weight loss were detected with Thc_Cut1_koST and Thc_Cut1, respectively. Furthermore, SEM characterization of PBS films clearly showed that enzyme tretment resulted in morphological changes of the film surface.