Polyester degradation by soil bacteria: identification of conserved BHETase enzymes in Streptomyces.

The rising use of plastic results in an appalling amount of waste which is scattered into the environment. One of these plastics is PET which is mainly used for bottles. We have identified and characterized an esterase from Streptomyces, annotated as LipA, which can efficiently degrade the PET-derived oligomer BHET. The Streptomyces coelicolor ScLipA enzyme exhibits varying sequence similarity to several BHETase/PETase enzymes, including IsPETase, TfCut2, LCC, PET40 and PET46. Of 96 Streptomyces strains, 18% were able to degrade BHET via one of three variants of LipA, named ScLipA, S2LipA and S92LipA. SclipA was deleted from S. coelicolor resulting in reduced BHET degradation. Overexpression of all LipA variants significantly enhanced BHET degradation. All variants were expressed in E. coli for purification and biochemical analysis. The optimum conditions were determined as pH 7 and 25 °C for all variants. The activity on BHET and amorphous PET film was investigated. S2LipA efficiently degraded BHET and caused roughening and indents on the surface of PET films, comparable to the activity of previously described TfCut2 under the same conditions. The abundance of the S2LipA variant in Streptomyces suggests an environmental advantage towards the degradation of more polar substrates including these polluting plastics.

Accuracy of arch expansion with two thermoplastic materials in Invisalign® patients: EX30® and SmartTrack®.

OBJECTIVE: The purpose of this retrospective study was to compare accuracy of arch expansion using two different thermoplastic materials in Invisalign aligners: EX30® (Polyethylene Terephthalate Glycol, or PETG) and SmartTrack® (polyurethane). METHODS: The study sample comprised 65 adult patients consecutively treated with Invisalign from two private practices: group 1 - treated with EX30® (358 teeth) and group 2 - treated with SmartTrack® (888 teeth). Six hundred and twenty-three measurements were assessed in three digital models throughout treatment: model 1 - initial, model 2 - predicted tooth position, and model 3 - achieved position. Sixteen reference points per arch were marked and, after best alignment, 2 points per tooth were copied from one digital model to another. Linear values of both arches were measured for canines, premolars, and first molars: on lingual gingival margins and cusp tips of every tooth. Comparisons were performed by Wilcoxon and Mann-Whitney test. RESULTS: Both termoplastic materials presented significant differences between predicted and achieved values for all measurements, except for the lower molar cusp tip in the SmartTrack® group. There is no statistical difference in the accuracy of transverse expansion between these two materials. Overall accuracy for EX30® aligners in maxilla and mandible were found to be 37 and 38%, respectively; and Smarttrack® presented an overall accuracy of 56.62% in the maxilla and 68.72% in the mandible. CONCLUSIONS: It is not possible to affirm one material expands better than the other. Further controlled clinical studies should be conducted comparing SmartTrack® and EX30® under similar conditions.

A new strategy for PET depolymerization: Application of bimetallic MOF-74 as a selective catalyst.

Large-volume production of poly(ethylene terephthalate) (PET), especially in the form of bottles and food packaging containers, causes problems with polymer waste management. Waste PET could be recycled thermally, mechanically or chemically and the last method allows to obtain individual monomers, but most often it is carried out in the presence of homogeneous catalysts, that are difficult to separate and reuse. In view of this, this work reports for the first time, application of bimetallic MOF-74 - as heterogeneous catalyst - for depolymerization of PET with high monomer (bishydroxyethyl terephthalate, BHET) recovery. The effect of type and amount of second metal in the MOF-74 (Mg/M) was systematically investigated. The results showed increased activity of MOF-74 (Mg/M) containing Co2+, Zn2+ and Mn2+ as a second metal, while the opposite correlation was observed for Cu2+ and Ni2+. It was found that the highest catalytic activity was demonstrated by the introduction of Mg-Mn into MOF-74 with ratio molar 1:1, which resulted in complete depolymerization of PET and 91.8% BHET yield within 4 h. Furthermore, the obtained catalyst showed good stability in 5 reaction cycles and allowed to achieve high-purity BHET, which was confirmed by HPLC analysis. The as-prepared MOF-74 (Mg/Mn) was easy to separate from the post-reaction mixture, clean and reuse in the next depolymerization reaction.

Acceleration a yeast-based biodegradation process of polyethylene terephthalate microplastics by Tween 20: Efficiency, by-product analysis, and metabolic pathway Prediction.

Polyethylene terephthalate is a widely produced plastic polymer that exhibits considerable biodegradation resistance, making its derived microplastics ubiquitous environmental pollutants. In this study, a new yeast strain (Vanrija sp. SlgEBL5) was isolated and found to have lipase and esterase-positive capabilities for degrading polyethylene terephthalate microplastics. This isolate changed the microplastic surface charge from -19.3 to +31.0 mV and reduced more than 150 µm of its size in addition to reducing the intensity of the terephthalate, methylene, and ester bond functional groups of the polymer in 30 days. Tween 20 as a chemical auxiliary treatment combined with biodegradation increased the microplastic degradation rate from 10 to 16.6% and the thermal degradation rate from 85 to 89%. Releasing less potentially hazardous by-products like 1,2 diethyl-benzene despite the higher abundance of long-chain n-alkanes, including octadecane and tetracosane was also the result of the bio + chemical treatment. Altogether, the findings showed that Vanrija sp. SlgEBL5 has the potential as a biological treating agent for polyethylene terephthalate microplastics, and the simultaneous bio + chemical treatment enhanced the biodegradation rate and efficiency.

A waste-to-wealth conversion of plastic bottles into effective carbon-based adsorbents for removal of tetracycline antibiotic from water.

Currently, plastic waste and antibiotic wastewater are two of the most critical environmental problems, calling for urgent measures to take. A waste-to-wealth strategy for the conversion of polyethylene terephthalate (PET) plastic bottles into value-added materials such as carbon composite is highly recommended to clean wastewater contaminated by antibiotics. Inspired by this idea, we develop a novel PET-AC-ZFO composite by incorporating PET plastic-derived KOH-activated carbon (AC) with ZnFe2O4 (ZFO) particles for adsorptive removal of tetracycline (TTC). PET-derived carbon (PET-C), KOH-activated PET-derived carbon (PET-AC), and PET-AC-ZFO were characterized using physicochemical analyses. Central composite design (CCD) was used to obtain a quadratic model by TTC concentration (K), adsorbent dosage (L), and pH (M). PET-AC-ZFO possessed micropores (d ≈ 2 nm) and exceptionally high surface area of 1110 m2 g-1. Nearly 90% TTC could be removed by PET-AC-ZFO composite. Bangham kinetic and Langmuir isotherm were two most fitted models. Theoretical maximum TTC adsorption capacity was 45.1 mg g-1. This study suggested the role of hydrogen bonds, pore-filling interactions, and π-π interactions as the main interactions of the adsorption process. Thus, a strategy for conversion of PET bottles into PET-AC-ZFO can contribute to both plastic recycling and antibiotic wastewater mitigation.

Probing Enzymatic PET Degradation: Molecular Dynamics Analysis of Cutinase Adsorption and Stability.

Understanding the mechanisms influencing poly(ethylene terephthalate) (PET) biodegradation is crucial for developing innovative strategies to accelerate the breakdown of this persistent plastic. In this study, we employed all-atom molecular dynamics simulation to investigate the adsorption process of the LCC-ICCG cutinase enzyme onto the PET surface. Our results revealed that hydrophobic, π-π, and H bond interactions, specifically involving aliphatic, aromatic, and polar uncharged amino acids, were the primary driving forces for the adsorption of the cutinase enzyme onto PET. Additionally, we observed a negligible change in the enzyme's tertiary structure during the interaction with PET (RMSD = 1.35 Å), while its secondary structures remained remarkably stable. Quantitative analysis further demonstrated that there is about a 24% decrease in the number of enzyme-water hydrogen bonds upon adsorption onto the PET surface. The significance of this study lies in unraveling the molecular intricacies of the adsorption process, providing valuable insights into the initial steps of enzymatic PET degradation.

Polyethylene terephthalate (PET)-degrading bacteria in the pelagic deep-sea sediments of the Pacific Ocean.

Polyethylene terephthalate (PET) plastic pollution is widely found in deep-sea sediments. Despite being an international environmental issue, it remains unclear whether PET can be degraded through bioremediation in the deep sea. Pelagic sediments obtained from 19 sites across a wide geographic range in the Pacific Ocean were used to screen for bacteria with PET degrading potential. Bacterial consortia that could grow on PET as the sole carbon and energy source were found in 10 of the 19 sites. These bacterial consortia showed PET removal rate of 1.8%-16.2% within two months, which was further confirmed by the decrease of carbonyl and aliphatic hydrocarbon groups using attenuated total reflectance-Fourier-transform infrared analysis (ATR-FTIR). Analysis of microbial diversity revealed that Alcanivorax and Pseudomonas were predominant in all 10 PET degrading consortia. Meanwhile, Thalassospira, Nitratireductor, Nocardioides, Muricauda, and Owenweeksia were also found to possess PET degradation potential. Metabolomic analysis showed that Alcanivorax sp. A02-7 and Pseudomonas sp. A09-2 could turn PET into mono-(2-hydroxyethyl) terephthalate (MHET) even in situ stimulation (40 MPa, 10 °C) conditions. These findings widen the currently knowledge of deep-sea PET biodegrading process with bacteria isolates and degrading mechanisms, and indicating that the marine environment is a source of biotechnologically promising bacterial isolates and enzymes.

High versatility of polyethylene terephthalate (PET) waste for the development of batteries, biosensing and gas sensing devices.

Continuously growing adoption of electronic devices in energy storage, human health and environmental monitoring systems increases demand for cost-effective, lightweight, comfortable, and highly efficient functional structures. In this regard, the recycling and reuse of polyethylene terephthalate (PET) waste in the aforementioned fields due to its excellent mechanical properties and chemical resistance is an effective solution to reduce plastic waste. Herein, we review recent advances in synthesis procedures and research studies on the integration of PET into energy storage (Li-ion batteries) and the detection of gaseous and biological species. The operating principles of such systems are described and the role of recycled PET for various types of architectures is discussed. Modifying the composition, crystallinity, surface porosity, and polar surface functional groups of PET are important factors for tuning its features as the active or substrate material in biological and gas sensors. The findings indicate that conceptually new pathways to the study are opened up for the effective application of recycled PET in the design of Li-ion batteries, as well as biochemical and catalytic detection systems. The current challenges in these fields are also presented with perspectives on the opportunities that may enable a circular economy in PET use.

Design and construction of chemical-biological module clusters for degradation and assimilation of poly(ethylene terephthalate) waste.

The rising accumulation of poly(ethylene terephthalate) (PET) waste presents an urgent ecological challenge, necessitating an efficient and economical treatment technology. Here, we developed chemical-biological module clusters that perform chemical pretreatment, enzymatic degradation, and microbial assimilation for the large-scale treatment of PET waste. This module cluster included (i) a chemical pretreatment that involves incorporating polycaprolactone (PCL) at a weight ratio of 2% (PET:PCL = 98:2) into PET via mechanical blending, which effectively reduces the crystallinity and enhances degradation; (ii) enzymatic degradation using Thermobifida fusca cutinase variant (4Mz), that achieves complete degradation of pretreated PET at 300 g/L PET, with an enzymatic loading of 1 mg protein per gram of PET; and (iii) microbial assimilation, where Rhodococcus jostii RHA1 metabolizes the degradation products, assimilating each monomer at a rate above 90%. A comparative life cycle assessment demonstrated that the carbon emissions from our module clusters (0.25 kg CO2-eq/kg PET) are lower than those from other established approaches. This study pioneers a closed-loop system that seamlessly incorporates pretreatment, degradation, and assimilation processes, thus mitigating the environmental impacts of PET waste and propelling the development of a circular PET economy.

Using marine mussels to assess the potential ecotoxicological effects of two different commercial microplastics.

Microplastics (MPs) in the aquatic environment pose a serious threat to biota, by being confounded with food. These effects occur in mussels which are filter-feeding organisms. Mussels from the genus Mytilus sp. were used to evaluate the ecotoxicological effects of two MPs, polypropylene (PP) and polyethylene terephthalate (PET), after 4 and 28-days. Measured individual endpoints were condition index and feeding rate; and sub-individual parameters, metabolism of phase I (CYP1A1, CYP1A2 and CYP3A4) and II (glutathione S-transferases - GSTs), and antioxidant defense (catalase - CAT). MPs decreased both condition index (CI) and feeding rate (FR). No alterations occurred in metabolic enzymes, suggesting that these MPs are not metabolized by these pathways. Furthermore, lack of alterations in GSTs and CAT activities suggests the absence of conjugation and oxidative stress. Overall, biochemical markers were not responsive, but non-enzymatic responses showed deleterious effects caused by these MPs, which may be of high ecological importance.

Management of ibuprofen in wastewater using electrospun nanofibers developed from PET and PS wastes.

Electrospinning is a promising technique for the beneficial use and recycling of plastic waste polymers using simple methodologies. In this study, plastic bottles and Styrofoam wastes have been used to develop polyethylene terephthalate (PET) and polystyrene (PS) nanofibers using electrospinning technique separately without any further purification. The effect of the concentration onto the nanofiber's morphology was studied. The fabricated nanofibers were characterized using Field Emission Scanning Electron Microscope (FE-SEM), Fourier Transformed Infrared Spectroscopy (ATR-FTIR), N2 adsorption/desorption analysis, and water contact angle (WCA). Furthermore, the prepared nanofibers were applied for the adsorption of ibuprofen (IBU) from wastewater. Some parameters that can influence the adsorption efficiency of nanofibers such as solution pH, wt.% of prepared nanofibers, drug initial concentration, and contact time were studied and optimized. The results show that the equilibrium adsorption capacity was achieved after only 10 min for 12 wt% PET nanofibers which is equivalent to 364.83 mg/g. For 12 wt% PS nanofibers, an equilibrium adsorption capacity of 328.42 mg/g was achieved in 30 min. The experimental data was fitted to five isotherm and four kinetics models to understand the complicated interaction between the nanofibers and the drug. Langmuir-Freundlich isotherm model showed the best fit for experimental data for both PET and PS nanofibers. The adsorption process was characterized by predominantly physical reaction rather than chemical adsorption for both materials. The reusability study revealed that the synthesized nanofibers maintain their ability to adsorb/desorb IBU for up to five cycles. The results obtained demonstrated that fabricated nanofibers from plastic wastes could perform promising adsorbents for the management of IBU in wastewater. However, further research is needed for the scaling-up the fabrication which is required for real-world applications.

Optimization and application of pretreatment method of microplastics detection in municipal solid waste landfills.

The landfill is one of the most important sources of microplastics (MPs). The pretreatment method is a precondition of microplastics study for the presence of complex substances in landfills. Therefore, it is essential to examine the impact of different pretreatment methods on the microplastics detection. A literature review and a comparison experiment on digestion solutions were performed to establish a comprehensive identification method for MPs in landfills. When exposed to of 30 % H2O2, minimal mass reduction of PE, PP and PET were 4.00 %, 3.00 % and 3.00 % respectively, and the least surface damage was observed in MPs, while exhibiting the most optimal peak value for infrared spectral characteristics. It is demonstrated that the effect of 30 % H2O2 dissolution was superior compared to 10 % KOH and 65 % HNO3. The method was subsequently utilized to investigate the distribution of MPs in a landfill. The dominant MPs were polyethylene (PE, 18.56-23.91 %), polyethylene terephthalate (PET, 8.80-18.66 %), polystyrene (PS, 10.31-18.09 %), and polypropylene (PP, 11.60-14.91 %). The comprehensive identification method of "NaCl density separation + 30 % H2O2 digestion + NaI density separation + sampling microscope + Mirco-FTIR" is suitable for the detection of MPs in landfills.

Development and characterization of a bacterial enzyme cascade reaction system for efficient and stable PET degradation.

The widespread use of polyethylene terephthalate (PET) in various industries has led to a surge in microplastics (MPs) pollution, posing a significant threat to ecosystems and human health. To address this, we have developed a bacterial enzyme cascade reaction system (BECRS) that focuses on the efficient degradation of PET. This system harnesses the Escherichia coli (E. coli) surface to display CsgA protein, which forms curli fibers, along with the carbohydrate-binding module 3 (CBM3) and PETases, to enhance the adsorption and degradation of PET. The study demonstrated that the BECRS achieved a notable PET film degradation rate of 3437 ± 148 µg/(d*cm²), with a degradation efficiency of 21.40% for crystalline PET MPs, and the degradation products were all converted to TPA. The stability of the system was evidenced by retaining over 80% of its original activity after multiple uses and during one month of storage. Molecular dynamics simulations confirmed that the presence of CsgA did not interfere with the enzymatic activity of PETases. This BECRS represents a significant step forward in the biodegradation of PET, particularly microplastics, offering a practical and sustainable solution for environmental pollution control.

Toxic effects of fragmented polyethylene terephthalate particles on the marine rotifer Brachionus koreanus: Based on ingestion and egestion assay, in vivo toxicity test, and multi-omics analysis.

Microplastics (MPs) are a major concern in marine ecosystem because MPs are persistent and ubiquitous in oceans and are easily consumed by marine biota. Although many studies have reported the toxicity of MPs to marine biota, the toxicity of environmentally relevant types of MPs is little understood. We investigated the toxic effects of fragmented polyethylene terephthalate (PET) MP, one of the most abundant MPs in the ocean, on the marine rotifer Brachionus koreanus at the individual and molecular level. No significant rotifer mortality was observed after exposure to PET MPs for 24 and 48 h. The ingestion and egestion assays showed that rotifers readily ingested PET MPs in the absence of food but not when food was supplied; thus, there were also no chronic effects of PET MPs. In contrast, intracellular reactive oxygen species levels and glutathione S-transferase activity in rotifers were significantly increased by PET MPs. Transcriptomic and metabolomic analyses revealed that genes and metabolites related to energy metabolism and immune processes were significantly affected by PET MPs in a concentration-dependent manner. Although acute toxicity of PET MPs was not observed, PET MPs are potentially toxic to the antioxidant system, immune system, and energy metabolism in rotifers.

Microplastics alter the microbiota-mediated phosphorus profiles at sediment-water interface: Distinct microbial effects between sediment and plastisphere.

Microplastics (MPs) are ubiquitous in freshwater sediments, raising concern about their potential impacts on ecosystem services. However, the specific impacts of microbiota mediated by MPs in sediment and plastisphere compartments on P availability remain elusive. This investigation conducted a series of microcosm experiments utilizing eutrophic lake sediment amended with fuel-based polyethylene terephthalate (PET), bio-based polylactic acid (PLA) MPs, and a natural cobblestone substrate to unravel their effects. The findings highlighted that MPs induced alterations in bacterial communities in both sediment and plastisphere, consequently modifying P availabilities at the sediment-water interface (SWI). In comparison to non-biodegradable PET, biodegradable PLA MPs presented higher proportions of specific bacteria and functional genes associated with P profiles, such as Firmicutes, Ignavibacteriota, and P mineralizing genes in the sediment and plastisphere. This, in turn, elevated the levels of soluble reactive P in the porewater by 54.19 % (0-1 cm), 55.81 % (1-3 cm), and 18.24 % (3-5 cm), respectively. Additionally, PLA obviously altered P immobilization capacity and bioavailability, increasing the organic P fraction. Whereas, inert cobblestone exhibited negligible influence on P biogeochemical processes during the incubation. Moreover, the biofilm communities and those in the surrounding sediment specifically contributed to the changes in P profiles at the SWI. The functional genes associated with P profiles in the sediment mainly concentrate on P mineralization and P uptake/transport. In the plastisphere, P activation genes are obviously affected under MP exposure. This study fills the knowledge gap concerning the repercussions of MPs on ecosystem services.

Discovery and characterization of two novel polyethylene terephthalate hydrolases: One from a bacterium identified in human feces and one from the Streptomyces genus.

Polyethylene terephthalate (PET) is widely used for various industrial applications. However, owing to its extremely slow breakdown rate, PET accumulates as plastic trash, which negatively affects the environment and human health. Here, we report two novel PET hydrolases: PpPETase from Pseudomonas paralcaligenes MRCP1333, identified in human feces, and ScPETase from Streptomyces calvus DSM 41452. These two enzymes can decompose various PET materials, including semicrystalline PET powders (Cry-PET) and low-crystallinity PET films (gf-PET). By structure-guided engineering, two variants, PpPETaseY239R/F244G/Y250G and ScPETaseA212C/T249C/N195H/N243K were obtained that decompose Cry-PET 3.1- and 1.9-fold faster than their wild-type enzymes, respectively. The co-expression of ScPETase and mono-(2-hydroxyethyl) terephthalate hydrolase from Ideonella sakaiensis (IsMHETase) resulted in 1.4-fold more degradation than the single enzyme system. This engineered strain degraded Cry-PET and gf-PET by more than 40% and 6%, respectively, after 30 d. The concentrations of terephthalic acid (TPA) in the Cry-PET and gf-PET degradation products were 37.7% and 25.6%, respectively. The discovery of these two novel PET hydrolases provides opportunities to create more powerful biocatalysts for PET biodegradation.

Characterization of Thermotoga maritima Esterase Capable of Hydrolyzing Bis(2-hydroxyethyl) Terephthalate.

The gene-encoding carboxylesterase (TM1022) from the hyperthermophilic bacterium Thermotoga maritima (T. maritima) was cloned and expressed in Escherichia coli Top10 and BL21 (DE3). Recombinant TM1022 showed the best activity at pH 8.0 and 85 °C and retained 57% activity after 8 h cultivation at 90 °C. TM1022 exhibited good stability at pH 6.0-9.0, maintaining 53% activity after incubation at pH 10.0 and 37 °C for 6 h. The esterase TM1022 exhibited the optimum thermo-alkali stability and kcat/Km (598.57 ± 19.97 s-1mM-1) for pN-C4. TM1022 hydrolyzed poly(ethylene terephthalate) (PET) degradation intermediates, such as bis(2-hydroxyethyl) terephthalate (BHET) and mono(2-hydroxyethyl) terephthalate (MHET). The Km, kcat, and kcat/Km values for BHET were 0.82 ± 0.01 mM, 2.20 ± 0.02 s-1, and 2.67 ± 0.02 mM-1 s-1, respectively; those for MHET were 2.43 ± 0.07 mM, 0.04 ± 0.001 s-1, and 0.02 ± 0.001 mM-1 s-1, respectively. When purified TM1022 was added to the cutinase BhrPETase, hydrolysis of PET from drinking water bottle tops produced pure terephthalic acids (TPA) with 166% higher yield than those obtained after 72 h of incubation with BhrPETase alone as control. The above findings demonstrate that the esterase TM1022 from T. maritima has substantial potential for depolymerizing PET into monomers for reuse.

Microplastics and non-natural cellulosic particles in Spanish bottled drinking water.

This investigation explored the presence of microplastics (MPs) and artificial cellulosic particles (ACPs) in commercial water marketed in single use 1.5 L poly(ethylene terephthalate) bottles. In this work we determined a mass concentration of 1.61 (1.10-2.88) µg/L and 1.04 (0.43-1.82) µg/L for MPs and ACPs respectively in five top-selling brands from the Spanish bottled water market. Most MPs consisted of white and transparent polyester and polyethylene particles, while most ACPs were cellulosic fibers likely originating from textiles. The median size of MPs and ACPs was 93 µm (interquartile range 76-130 µm) and 77 µm (interquartile range 60-96 µm), respectively. Particle mass size distributions were fitted to a logistic function, enabling comparisons with other studies. The estimated daily intake of MPs due to the consumption of bottled water falls within the 4-18 ng kg-1 day-1 range, meaning that exposure to plastics through bottled water probably represents a negligible risk to human health. However, it's worth noting that the concentration of plastic found was much higher than that recorded for tap water, which supports the argument in favour of municipal drinking water.

Improving plastic degrading enzymes via directed evolution.

Plastic degrading enzymes have immense potential for use in industrial applications. Protein engineering efforts over the last decade have resulted in considerable enhancement of many properties of these enzymes. Directed evolution, a protein engineering approach that mimics the natural process of evolution in a laboratory, has been particularly useful in overcoming some of the challenges of structure-based protein engineering. For example, directed evolution has been used to improve the catalytic activity and thermostability of polyethylene terephthalate (PET)-degrading enzymes, although its use for the improvement of other desirable properties, such as solvent tolerance, has been less studied. In this review, we aim to identify some of the knowledge gaps and current challenges, and highlight recent studies related to the directed evolution of plastic-degrading enzymes.

Combining analytical techniques to assess the translocation of diesel particles across an alveolar tissue barrier in vitro.

BACKGROUND: During inhalation, airborne particles such as particulate matter ≤ 2.5 µm (PM2.5), can deposit and accumulate on the alveolar epithelial tissue. In vivo studies have shown that fractions of PM2.5 can cross the alveolar epithelium to blood circulation, reaching secondary organs beyond the lungs. However, approaches to quantify the translocation of particles across the alveolar epithelium in vivo and in vitro are still not well established. In this study, methods to assess the translocation of standard diesel exhaust particles (DEPs) across permeable polyethylene terephthalate (PET) inserts at 0.4, 1, and 3 µm pore sizes were first optimized with transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-VIS), and lock-in thermography (LIT), which were then applied to study the translocation of DEPs across human alveolar epithelial type II (A549) cells. A549 cells that grew on the membrane (pore size: 3 µm) in inserts were exposed to DEPs at different concentrations from 0 to 80 µg.mL- 1 ( 0 to 44 µg.cm- 2) for 24 h. After exposure, the basal fraction was collected and then analyzed by combining qualitative (TEM) and quantitative (UV-VIS and LIT) techniques to assess the translocated fraction of the DEPs across the alveolar epithelium in vitro. RESULTS: We could detect the translocated fraction of DEPs across the PET membranes with 3 µm pore sizes and without cells by TEM analysis, and determine the percentage of translocation at approximatively 37% by UV-VIS (LOD: 1.92 µg.mL- 1) and 75% by LIT (LOD: 0.20 µg.cm- 2). In the presence of cells, the percentage of DEPs translocation across the alveolar tissue was determined around 1% at 20 and 40 µg.mL- 1 (11 and 22 µg.cm- 2), and no particles were detected at higher and lower concentrations. Interestingly, simultaneous exposure of A549 cells to DEPs and EDTA can increase the translocation of DEPs in the basal fraction. CONCLUSION: We propose a combination of analytical techniques to assess the translocation of DEPs across lung tissues. Our results reveal a low percentage of translocation of DEPs across alveolar epithelial tissue in vitro and they correspond to in vivo findings. The combination approach can be applied to any traffic-generated particles, thus enabling us to understand their involvement in public health.