Metagenome-derived SusD-homologs affiliated with Bacteroidota bind to synthetic polymers.

Starch utilization system (Sus)D-homologs are well known for their carbohydrate-binding capabilities and are part of the sus operon in microorganisms affiliated with the phylum Bacteroidota. Until now, SusD-like proteins have been characterized regarding their affinity toward natural polymers. In this study, three metagenomic SusD homologs (designated SusD1, SusD38489, and SusD70111) were identified and tested with respect to binding to natural and non-natural polymers. SusD1 and SusD38489 are cellulose-binding modules, while SusD70111 preferentially binds chitin. Employing translational fusion proteins with superfolder GFP (sfGFP), pull-down assays, and surface plasmon resonance (SPR) has provided evidence for binding to polyethylene terephthalate (PET) and other synthetic polymers. Structural analysis suggested that a Trp triad might be involved in protein adsorption. Mutation of these residues to Ala resulted in an impaired adsorption to microcrystalline cellulose (MC), but not so to PET and other synthetic polymers. We believe that the characterized SusDs, alongside the methods and considerations presented in this work, will aid further research regarding bioremediation of plastics. IMPORTANCE: SusD1 and SusD38489 can be considered for further applications regarding their putative adsorption toward fossil-fuel based polymers. This is the first time that SusD homologs from the polysaccharide utilization loci (PUL), largely described for the phylum Bacteroidota, are characterized as synthetic polymer-binding proteins.

Molecular engineering of PETase for efficient PET biodegradation.

The widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems. Compared with traditional thermomechanical or chemical PET cycling, the biodegradation of PET may offer a more feasible solution. Though the PETase from Ideonalla sakaiensis (IsPETase) displays interesting PET degrading performance under mild conditions; the relatively low thermal stability of IsPETase limits its practical application. In this study, enzyme-catalysed PET degradation was investigated with the promising IsPETase mutant HotPETase (HP). On this basis, a carbohydrate-binding module from Bacillus anthracis (BaCBM) was fused to the C-terminus of HP to construct the PETase mutant (HLCB) for increased PET degradation. Furthermore, to effectively improve PET accessibility and PET-degrading activity, the truncated outer membrane hybrid protein (FadL) was used to expose PETase and BaCBM on the surface of E. coli (BL21with) to develop regenerable whole-cell biocatalysts (D-HLCB). Results showed that, among the tested small-molecular weight ester compounds (p-nitrophenyl phosphate (pNPP), p-Nitrophenyl acetate (pNPA), 4-Nitrophenyl butyrate (pNPB)), PETase displayed the highest hydrolysing activity against pNPP. HP displayed the highest catalytic activity (1.94 µM(p-NP)/min) at 50 °C and increased longevity at 40 °C. The fused BaCBM could clearly improve the catalytic performance of PETase by increasing the optimal reaction temperature and improving the thermostability. When HLCB was used for PET degradation, the yield of monomeric products (255.7 µM) was ∼25.5 % greater than that obtained after 50 h of HP-catalysed PET degradation. Moreover, the highest yield of monomeric products from the D-HLCB-mediated system reached 1.03 mM. The whole-cell catalyst D-HLCB displayed good reusability and stability and could maintain more than 54.6 % of its initial activity for nine cycles. Finally, molecular docking simulations were utilized to investigate the binding mechanism and the reaction mechanism of HLCB, which may provide theoretical evidence to further increase the PET-degrading activities of PETases through rational design. The proposed strategy and developed variants show potential for achieving complete biodegradation of PET under mild conditions.

Efficient biodegradation of Polyethylene terephthalate (PET) plastic by Gordonia sp. CN2K isolated from plastic contaminated environment.

Since we rely entirely on plastics or their products in our daily lives, plastics are the invention of the hour. Polyester plastics, such as Polyethylene Terephthalate (PET), are among the most often used types of plastics. PET plastics have a high ratio of aromatic components, which makes them very resistant to microbial attack and highly persistent. As a result, massive amounts of plastic trash accumulate in the environment, where they eventually transform into microplastic (<5 mm). Rather than macroplastics, microplastics are starting to pose a serious hazard to the environment. It is imperative that these polymer microplastics be broken down. Through the use of enrichment culture, the PET microplastic-degrading bacterium was isolated from solid waste management yards. Bacterial strain was identified as Gordonia sp. CN2K by 16 S rDNA sequence analysis and biochemical characterization. It is able to use polyethylene terephthalate as its only energy and carbon source. In 45 days, 40.43 % of the PET microplastic was degraded. By using mass spectral analysis and HPLC to characterize the metabolites produced during PET breakdown, the degradation of PET is verified. The metabolites identified in the spent medium included dimer compound, bis (2-hydroxyethyl) terephthalate (BHET), mono (2-hydroxyethyl) terephthalate (MHET), and terephthalate. Furthermore, the PET sheet exposed to the culture showed considerable surface alterations in the scanning electron microscope images. This illustrates how new the current work is.

Engineering Escherichia coli to produce aromatic chemicals from ethylene glycol.

Microbial overproduction of aromatic chemicals has gained considerable industrial interest and various metabolic engineering approaches have been employed in recent years to address the associated challenges. So far, most studies have used sugars (mostly glucose) or glycerol as the primary carbon source. In this study, we used ethylene glycol (EG) as the main carbon substrate. EG could be obtained from the degradation of plastic and cellulosic wastes. As a proof of concept, Escherichia coli was engineered to transform EG into L-tyrosine, a valuable aromatic amino acid. Under the best fermentation condition, the strain produced 2 g/L L-tyrosine from 10 g/L EG, outperforming glucose (the most common sugar feedstock) in the same experimental conditions. To prove the concept that EG can be converted into different aromatic chemicals, E. coli was further engineered with a similar approach to synthesize other valuable aromatic chemicals, L-phenylalanine and p-coumaric acid. Finally, waste polyethylene terephthalate (PET) bottles were degraded using acid hydrolysis and the resulting monomer EG was transformed into L-tyrosine using the engineered E. coli, yielding a comparable titer to that obtained using commercial EG. The strains developed in this study should be valuable to the community for producing valuable aromatics from EG.

An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation.

Polyethylene terephthalate (PET) is a prevalent synthetic polymer that is known to contaminate marine and terrestrial environments. Currently, only a limited number of PET-active microorganisms and enzymes (PETases) are known. This is in part linked to the lack of highly sensitive function-based screening assays for PET-active enzymes. Here, we report on the construction of a fluorescent biosensor based on Comamonas thiooxidans strain S23. C. thiooxidans S23 transports and metabolizes TPA, one of the main breakdown products of PET, using a specific tripartite tricarboxylate transporter (TTT) and various mono- and dioxygenases encoded in its genome in a conserved operon ranging from tphC-tphA1. TphR, an IclR-type transcriptional regulator is found upstream of the tphC-tphA1 cluster where TPA induces transcription of tphC-tphA1 up to 88-fold in exponentially growing cells. In the present study, we show that the C. thiooxidans S23 wild-type strain, carrying the sfGFP gene fused to the tphC promoter, senses TPA at concentrations as low as 10 µM. Moreover, a deletion mutant lacking the catabolic genes involved in TPA degradation thphA2-A1 (ΔtphA2A3BA1) is up to 10,000-fold more sensitive and detects TPA concentrations in the nanomolar range. This is, to our knowledge, the most sensitive reporter strain for TPA and we demonstrate that it can be used for the detection of enzymatic PET breakdown products. IMPORTANCE Plastics and microplastics accumulate in all ecological niches. The construction of more sensitive biosensors allows to monitor and screen potential PET degradation in natural environments and industrial samples. These strains will also be a valuable tool for functional screenings of novel PETase candidates and variants or monitoring of PET recycling processes using biocatalysts. Thereby they help us to enrich the known biodiversity and efficiency of PET degrading organisms and enzymes and understand their contribution to environmental plastic degradation.

Heterogeneous Nucleation of Supersaturated Water Vapor onto Sub-10 nm Nanoplastic Particles.

Nanoplastic pollution by atmospheric transport processes is a recently discovered environmental problem on a global scale that is attributed to the dispersion of aerosolized nanoplastics. However, knowledge about the basic physicochemical properties of aerosol nanoplastic particles is scarce. Here, we present experiments on the heterogeneous nucleation of supersaturated water vapor onto sub-10 nm polyethylene terephthalate (PET) seeds. We determined onset saturation ratios for the activation of PET seeds in comparison to the well-documented reference system of silver particles, resulting in lower onset saturation ratios of the PET seeds compared to silver seeds. By using different PET bulk materials for the generation of nanoparticles, we report a strong material dependence of the onset saturation ratios, pointing to a strong effect of additives from commodity plastics in heterogeneous nucleation. Moreover, our results show a strong dependence on nucleation temperature that might be of immediate atmospheric relevance. Our work can be considered as an initial step in airborne nanoplastic detection by condensation techniques, and we anticipate our study to serve as a basis for further research that will eventually allow assessing the impact of nanoplastic dispersion on atmospheric processes.

Impregnation of polyethylene terephthalate (PET) grafts with BMP-2 loaded functional nanoparticles for reconstruction of anterior cruciate ligament.

Current artificial ligaments based on polyethylene terephthalate (PET) are associated with some disadvantages due to their hydrophobicity and low biocompatibility. In this study, we aimed to modify the surface of PET using polyethylene glycol (PEG)-terminated polystyrene (PS)-linoleic nanoparticles (PLinaS-g-PEG-NPs). We accomplished that BMP-2 in two different concentrations encapsulated in nanoparticles with an efficiency of 99.71 ± 1.5 and 99.95 ± 2.8%. While the dynamic contact angle of plain PET surface reduced from 116° to 115° after a measurement periods of 10 s, that of PLinaS-g-PEG-NPs modified PET from 80° to 17.5° within 0.35 s. According to in vitro BMP2 release study, BMP-2 was released 13.12 ± 1.76% and 45.47 ± 1.78% from 0.05 and 0.1BMP2-PLinaS-g-PEG-NPs modified PET at the end of 20 days, respectively. Findings from this study revealed that BMP2-PLinaS-g-PEG-NPs has a great potential to improve the artificial PET ligaments, and could be effectively applied for ACL reconstruction.

Antarctic Polyester Hydrolases Degrade Aliphatic and Aromatic Polyesters at Moderate Temperatures.

Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics in the packaging industry, and consequently has become one of the main components of plastic waste found in the environment. However, several microorganisms have been described to encode enzymes that catalyze the depolymerization of PET. While most known PET hydrolases are thermophilic and require reaction temperatures between 60°C and 70°C for an efficient hydrolysis of PET, a partial hydrolysis of amorphous PET at lower temperatures by the polyester hydrolase IsPETase from the mesophilic bacterium Ideonella sakaiensis has also been reported. We show that polyester hydrolases from the Antarctic bacteria Moraxella sp. strain TA144 (Mors1) and Oleispira antarctica RB-8 (OaCut) were able to hydrolyze the aliphatic polyester polycaprolactone as well as the aromatic polyester PET at a reaction temperature of 25°C. Mors1 caused a weight loss of amorphous PET films and thus constitutes a PET-degrading psychrophilic enzyme. Comparative modeling of Mors1 showed that the amino acid composition of its active site resembled both thermophilic and mesophilic PET hydrolases. Lastly, bioinformatic analysis of Antarctic metagenomic samples demonstrated that members of the Moraxellaceae family carry candidate genes coding for further potential psychrophilic PET hydrolases. IMPORTANCE A myriad of consumer products contains polyethylene terephthalate (PET), a plastic that has accumulated as waste in the environment due to its long-term stability and poor waste management. One promising solution is the enzymatic biodegradation of PET, with most known enzymes only catalyzing this process at high temperatures. Here, we bioinformatically identified and biochemically characterized an enzyme from an Antarctic organism that degrades PET at 25°C with similar efficiency to the few PET-degrading enzymes active at moderate temperatures. Reasoning that Antarctica harbors other PET-degrading enzymes, we analyzed available data from Antarctic metagenomic samples and successfully identified other potential enzymes. Our findings contribute to increasing the repertoire of known PET-degrading enzymes that are currently being considered as biocatalysts for the biological recycling of plastic waste.

Towards synthetic PETtrophy: Engineering Pseudomonas putida for concurrent polyethylene terephthalate (PET) monomer metabolism and PET hydrolase expression.

BACKGROUND: Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts. RESULTS: P. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive. CONCLUSION: Our results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.

Atomic force microscopy characterization of polyethylene terephthalate grafting with poly(styrene sulfonate).

Polyethylene terephthalate (PET) is widely used to elaborate biomaterials and medical devices in particular for long-term implant applications but tuning their surface properties remains challenging. We investigate surface functionalization by grafting poly(sodium 4-styrene sulfonate, PNaSS) with the aim of enhancing protein adhesion and cellular activity. Elucidating the topography and molecular level organization of the modified surfaces is important for understanding and predicting biological activity. In this work, we explore several grafting methods including thermal grafting, thermal grafting in the presence of Mohr's salt, and UV activation. We characterize the different surfaces obtained using atomic force microscopy (AFM), contact angle (CA), and x-ray photoelectron spectroscopy (XPS). We observe an increase in the percentage of sulfur atoms (XPS) that correlates with changes in (CA), and we identify by AFM characteristic features, which we interpret as patches of polymers on the PET surfaces. This work demonstrates tuning of biomaterials surface by functionalization and illustrates the capability of AFM to provide insights into the spatial organization of the grafted polymer.

Liquid chromatography-mass spectrometry method for the determination of polyethylene terephthalate and polybutylene terephthalate cyclic oligomers in blood samples.

Food contact materials (FCM) polyethylene terephthalate (PET) and polybutylene terephthalate (PBT) used extensively in food packaging may contain cyclic oligomers which may migrate into food and thus cause toxic effects on human health. A simple, fast, and sensitive ultra-high-performance liquid chromatography method quadrupole time-of-flight mass spectrometer was developed for the analysis of 7 cyclic oligomers in post-mortem blood samples. The targeted analytes were separated on a Waters BEH C18 (150 × 2.1 mm, 1.7 µm) analytical column by gradient elution. Calibration curves were constructed based on standard solutions and blood samples and Student's t-test was applied to evaluate the matrix effect. The LODs ranged from 1.7 to 16.7 µg mL-1, while the method accuracy was assessed by recovery experiments and resulting within the range 84.2-114.6%. Such an analytical method for the determination of PET and PBT cyclic oligomers in biological samples is reported for the first time. The developed methodology allows the determination of these oligomers in blood providing a useful analytical tool to assess the exposure and thus the potential hazard and health risks associated with these non-intentionally added substances (NIAS) from PET and PBT FCM through food consumption. The method was validated and successfully applied to the analysis of 34 post-mortem whole blood samples. Polyethylene terephthalate trimer was detected in four of them, for the first time in literature.

Towards bio-upcycling of polyethylene terephthalate.

Over 359 million tons of plastics were produced worldwide in 2018, with significant growth expected in the near future, resulting in the global challenge of end-of-life management. The recent identification of enzymes that degrade plastics previously considered non-biodegradable opens up opportunities to steer the plastic recycling industry into the realm of biotechnology. Here, the sequential conversion of post-consumer polyethylene terephthalate (PET) into two types of bioplastics is presented: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based poly(amide urethane) (bio-PU). PET films are hydrolyzed by a thermostable polyester hydrolase yielding highly pure terephthalate and ethylene glycol. The obtained hydrolysate is used directly as a feedstock for a terephthalate-degrading Pseudomonas umsongensis GO16, also evolved to efficiently metabolize ethylene glycol, to produce PHA. The strain is further modified to secrete hydroxyalkanoyloxy-alkanoates (HAAs), which are used as monomers for the chemo-catalytic synthesis of bio-PU. In short, a novel value-chain for PET upcycling is shown that circumvents the costly purification of PET monomers, adding technological flexibility to the global challenge of end-of-life management of plastics.

Comparison of Detection Methods of Microplastics in Landfill Mineralized Refuse and Selection of Degradation Degree Indexes.

A landfill is an important sink of plastic waste and potential sources of microplastics (MPs) when mineralized refuse is reused. However, limitations are still present in quantifying MPs in mineralized refuse and assessing their degradation degree. In this study, laser direct infrared spectroscopy and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to identify MPs of mineralized refuse from a landfill. Although 25-113 items/g MPs were detected in particles subjected to flotation, 37.9-674 µg/g polyethylene terephthalate (PET) and 0.0716-1.01 µg/g polycarbonate (PC) were detected in the residual solids by LC-MS/MS, indicating a great amount of plastic polymers still presented in the residue. This suggests that the commonly used flotation-counting method will lead to significant underestimation of MP pollution in mineralized refuse, which might be due to the aging and aggregation process caused by the long-term landfill process. The ratio of "bisphenol A/PC" and "plasticizer/MPs" was found to be positively correlated and negatively correlated with the landfill age, respectively. Therefore, in addition to the spectral index such as the carbonyl index, new indexes based on the concentrations of polymers, free monomers, and plasticizers were proposed to characterize the degradation degree of MPs in a landfill.

Enzymatic textile recycling - best practices and outlook.

Recently, textiles and their end-of-life management have become the focus of public and political attention. In the European Union the revised waste framework directive defines textiles as municipal waste and stipulates their separate collection by 2025. In the context of these developments, this paper summarises briefly the current state-of-the-art in textile recycling. It is evident that recycling methods are not yet fully developed. This is especially the case with multi-material textiles, which are composed of two or more polymers that are incompatible for recycling. In the practical part of the communication, results are presented which show that enzymatic hydrolysis is a suitable process for recycling textiles made of cotton and polyester. After a complete removal of cotton, the remaining pure polyester fibres undergo a re-granulation and post-condensation step. The so obtained recycled polyester is fed back into the textile processing chain and finally towels are obtained. The main steering parameters of the enzymatic hydrolysis process are described. The study proves that solutions in accordance with the Circular Economy in the textile sector are available but an industrial implementation has not yet been realised.

Functional expression of polyethylene terephthalate-degrading enzyme (PETase) in green microalgae.

BACKGROUND: For decades, plastic has been a valuable global product due to its convenience and low price. For example, polyethylene terephthalate (PET) was one of the most popular materials for disposable bottles due to its beneficial properties, namely impact resistance, high clarity, and light weight. Increasing demand of plastic resulted in indiscriminate disposal by consumers, causing severe accumulation of plastic wastes. Because of this, scientists have made great efforts to find a way to biologically treat plastic wastes. As a result, a novel plastic degradation enzyme, PETase, which can hydrolyze PET, was discovered in Ideonella sakaiensis 201-F6 in 2016. RESULTS: A green algae, Chlamydomonas reinhardtii, which produces PETase, was developed for this study. Two representative strains (C. reinhardtii CC-124 and CC-503) were examined, and we found that CC-124 could express PETase well. To verify the catalytic activity of PETase produced by C. reinhardtii, cell lysate of the transformant and PET samples were co-incubated at 30 °C for up to 4 weeks. After incubation, terephthalic acid (TPA), i.e. the fully-degraded form of PET, was detected by high performance liquid chromatography analysis. Additionally, morphological changes, such as holes and dents on the surface of PET film, were observed using scanning electron microscopy. CONCLUSIONS: A PET hydrolyzing enzyme, PETase, was successfully expressed in C. reinhardtii, and its catalytic activity was demonstrated. To the best of our knowledge, this is the first case of PETase expression in green algae.

Oxidative stress-related effects induced by micronized polyethylene terephthalate microparticles in the Manila clam.

Microplastic (MP) contamination represents a serious threat for marine organisms. Several lab studies demonstrated adverse effects induced by exposure to different MP polymers toward diverse marine species. However, the information regarding toxicity of polyethylene terephthalate (PET) MPs is largely unknown. The present study was aimed at investigating the adverse effects induced by 7-day exposure to two concentrations (0.125 or 12.5 µg/ml) micronized, irregular shaped and variable size PET microparticles (PET-MPs) toward Manila clam (Ruditapes philippinarum). Histological analyses were performed to assess tissue damage on digestive glands, gonads, gut and gills, whereas oxidative stress-related effects, including the concentration of pro-oxidant molecules, activity of antioxidant (superoxide dismutase - SOD, catalase - CAT and glutathione peroxidase - GPx) and detoxifying (glutathione S-transferase - GST) enzymes, as well as levels of lipid peroxidation, were determined in gills and digestive gland. Our results showed that clams ingest and egest micronized PET-MPs, but no marked histological alterations to bivalve tissues occurred. Although PET-MPs did not produce oxidative stress in the digestive gland, these materials significantly altered oxidative status of gills, leading to lipid peroxidation. No apparent clear indication of a weakness of bivalve health status was obtained in this study.

Contamination Levels in Recollected PET Bottles from Non-Food Applications and their Impact on the Safety of Recycled PET for Food Contact.

PET beverage bottles have been recycled and safely reprocessed into new food contact packaging applications for over two decades. During recollection of post-consumer PET beverage bottles, PET containers from non-food products are inevitably co-collected and thereby enter the PET recycling feed stream. To explore the impact of this mixing on the safety-in-use of recycled PET (rPET) bottles, we determined the concentrations of post-consumer substances in PET containers used for a range of non-food product applications taken from the market. Based on the chemical nature and amounts of these post-consumer substances, we evaluated their potential carry-over into beverages filled in rPET bottles starting from different fractions of non-food PET in the recollection systems and taking worst-case cleaning efficiencies of super-clean recycling processes into account. On the basis of the Threshold of Toxicological Concern (TTC) concept and Cramer classification tools, we present a risk assessment for potential exposure of the consumer to the identified contaminants as well as unidentified, potentially genotoxic substances in beverages. As a result, a fraction of 5% non-food PET in the recycling feed stream, which is very likely to occur in the usual recollection systems, does not pose any risk to the consumer. Our data show that fractions of up to 20%, which may sporadically be contained in certain, local recollection systems, would also not raise a safety concern.

New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes.

Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and ß-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria, Proteobacteria, and Bacteroidetes Within the Proteobacteria, the Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria, as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil.IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET hydrolases. Finally, we describe the biochemical traits of four novel PET hydrolases.

Determination of oligomers in virgin and recycled polyethylene terephthalate (PET) samples by UPLC-MS-QTOF.

An oligomer is a molecule that consists of a few monomer units. It can be formed during polymer manufacturing and also due to polymer degradation processes or even during use conditions. Since oligomers are not included in chemical databases, their identification is a complex process. In this work, the oligomers present in 20 different PET pellet samples have been determined. Two different sample treatment procedures, solvent extraction and total dissolution, were applied in order to select the most efficient one. The analyses were carried out by UPLC-MS-QTOF. The use of high resolution mass spectrometry allowed the structural elucidation of these compounds and their correct identification. The main oligomers identified were cyclic as well as lineal from the first, second, and third series. All of them were composed of terephthalic acid (TPA), diethylene glycol (DEG), and ethylene glycol (EG). Quantitative values were very different in both procedures. In total dissolution of PET samples, the concentration of oligomers was always, at least, 10 times higher than in solvent extraction; some of the compounds were only detected when total dissolution was used. Results showed that the oligomers with the highest concentration values were dimers and trimers, cyclic, as well as lineal, from the first and second series. The oligomer with the maximum concentration value was TPA2-EG-DEG that was found in all the samples in a concentration range from 2493 to 19,290 ng/g PET. No differences between virgin and recycled PET were found. Migration experiments were performed in two PET bottles, and results showed the transference of most of these oligomers to a fat food simulant (ethanol 95%). Graphical abstract Graphical abstract of the two procedures developd and optimized for identifying oligomers in PET pellets and in migration form PET bottles.

The Effect of Storage Time, Temperature and Type of Packaging on the Release of Phthalate Esters into PackedAcidic Liquids.

Acidic liquids such as verjuice, lemon juice and vinegar are frequently consumed in Iran. Different kinds of acidic liquids are packaged in polyethylene terephthalate (PET) and high-density polyethylene (HDPE) bottles. There is evidence indicating that phthalates can leach from PET and HDPE bottles into their contents. In this work the effect of storage time, temperature and bottle type on the migration of phthalates from packaging materials into acidic liquids is studied by analyzing the samples stored under different conditions, before storage and after 2, 4 and 6 months of storage. The determined mean phthalate concentrations in µg/L were: <0.04 to 0.501 in verjuice, <0.04 to 0.231 in lemon juice and <0.04 to 0.586 in vinegar. The highest concentrations of diethyl phthalate (DEP) and diethyl hexyl phthalate (DEHP) were found in PET and HDPE bottles, respectively. Results of analyses before and after storage indicate that under some storage conditions, the concentrations of DEP, DEHP and dibutyl phthalate (DBP) increased in acidic liquids. The possible migration of phthalic acid esters from plastic packaging materials into the contents was indicated by the results of the present study.