High-load terephthalic acid degradation and diverse bioproduct formation by novel Rhodococcus strains.

Pharmacotherapy is the therapeutic mainstay in epilepsy; however, in about 30% of patients, epileptic seizures are drug-resistant. A ketogenic diet (KD) is an alternative therapeutic option. The mechanisms underlying the anti-seizure effect of a KD are not fully understood. Epileptic seizures lead to an increased energy demand of neurons. An improvement in energy provisions may have a protective effect. C8 and C10 fatty acids have been previously shown to activate mitochondrial function in vitro. This could involve sirtuins (SIRTs) as regulatory elements of energy metabolism. The aim of the present study was to investigate whether ß-hydroxybutyrate (ßHB), C8 fatty acids, C10 fatty acids, or a combination of C8 and C10 (250/250 µM) fatty acids, which all increase under a KD, could up-regulate SIRT1, -3, -4, and -5 in HT22 hippocampal murine neurons in vitro. Cells were incubated for 1 week in the presence of these metabolites. The sirtuins were measured at the enzyme (fluorometrically), protein (Western blot), and gene expression (PCR) levels. In hippocampal cells, the C8, C10, and C8 and C10 incubations led to increases in the sirtuin levels, which were not inferior to a ßHB incubation as the 'gold standard'. This may indicate that both C8 and C10 fatty acids are important for the antiepileptic effect of a KD. A KD may be replaced by nutritional supplements of C8 and C10 fatty acids, which could facilitate the diet.

Plastics, such as polyethylene terephthalate (PET) from water bottles, are polluting our oceans, cities, and soils. While a number of Pseudomonas species have been described that degrade aliphatic polyesters, such as polyethylene (PE) and polyurethane (PUR), few from this genus that degrade the semiaromatic polymer PET have been reported. In this study, plastic-degrading bacteria were isolated from petroleum-polluted soils and screened for lipase activity that has been associated with PET degradation. Strains and consortia of bacteria were grown in a liquid carbon-free basal medium (LCFBM) with PET as the sole carbon source. We monitored several key physical and chemical properties, including bacterial growth and modification of the plastic surface, using scanning electron microscopy (SEM) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. We detected by-products of hydrolysis of PET using 1H-nuclear magnetic resonance (1H NMR) analysis, consistent with the ATR-FTIR data. The full consortium of five strains containing Pseudomonas and Bacillus species grew synergistically in the presence of PET and the cleavage product bis(2-hydroxyethyl) terephthalic acid (BHET) as sole sources of carbon. Secreted enzymes extracted from the full consortium were capable of fully converting BHET to the metabolically usable monomers terephthalic acid (TPA) and ethylene glycol. Draft genomes provided evidence for mixed enzymatic capabilities between the strains for metabolic degradation of TPA and ethylene glycol, the building blocks of PET polymers, indicating cooperation and ability to cross-feed in a limited nutrient environment with PET as the sole carbon source. The use of bacterial consortia for the biodegradation of PET may provide a partial solution to widespread planetary plastic accumulation.IMPORTANCE While several research groups are utilizing purified enzymes to break down postconsumer PET to the monomers TPA and ethylene glycol to produce new PET products, here, we present a group of five soil bacteria in culture that are able to partially degrade this polymer. To date, mixed Pseudomonas spp. and Bacillus spp. biodegradation of PET has not been described, and this work highlights the possibility of using bacterial consortia to biodegrade or potentially to biorecycle PET plastic waste.

Enzymatic degradation of polyethylene terephthalate (PET) has dramatically advanced through protein engineering of PETase, accelerating the biocatalytic depolymerization process. However, the subsequent microbial valorization of PET-derived intermediates, such as bis (2-hydroxyethyl) terephthalate (BHET) and terephthalic acid (TPA), remains limited because of restricted availability and suboptimal activity of specific biocatalytic enzymes. In this study, eight microbial species were isolated from the enriched cultures of marine plastic waste, using 1% PET, BHET and TPA as the sole carbon source. Stenotrophomonas was the only species detected in all the cultures. Strain WED208 was isolated from a BHET-enriched culture and selected for its potential role in plastic degradation. Phylogenetic analysis based on the 16S rRNA gene revealed 99.4% similarity to Stenotrophomonas riyadhensis LMG 33162T; however, it exhibited distinct physiological and genomic features. Strain WED208 degraded approximately 30% of BHET into mono (2-hydroxyethyl) terephthalate (MHET) over 30 days but did not catalyze further conversion to TPA. Comparative analysis identified a putative BHETase (WED208_02958) containing conserved catalytic residues (Ser90, Asp217, and His245). Structural modeling and protein-ligand docking analysis confirmed a key interaction between Ser90 and the ester bond of BHET, supporting the microbial hydrolytic function. Although strain WED208 could degrade neither PET nor TPA, its genome harbored two putative PETase genes and key enzymes potentially involved in TPA degradation, including diol dehydrogenase (tphB), MFS transporter (pcaK), and LysR-type transcriptional regulator (lysR). These findings suggest that WED208 is a promising microbial resource for enzyme engineering and has potential use in microbial consortia to enhance PET biodegradation and upcycling.

<p indent="0mm">With the increasing amount of plastic waste and the decreasing fossil energy, catalytic pyrolysis has become a promising technology for the production of fuels and chemicals from plastic waste. Polyethylene terephthalate (PET), a widely used plastic derived from non-renewable petroleum resources, is ubiquitous in daily life. However, the vast majority of PET is discarded within a year of use and is notoriously resistant to natural degradation, leading to significant environmental accumulation and pollution. This widespread waste and the inability to effectively recycle PET contribute to severe environmental pollution and resource depletion. In light of global trends toward sustainable development, circular economy principles, and pollution prevention, the development of efficient, sustainable, and environmentally friendly methods for processing waste PET has become an urgent priority. Among the potential solutions, catalytic pyrolysis has gained increasing attention as an effective strategy for recovering valuable chemical feedstocks from waste plastics. In this paper, we propose a novel strategy for catalytic pyrolysis of PET in a hydrogen (H₂) atmosphere to produce high-value-added chemical products, such as terephthalic acid (TPA), benzoic acid, and olefins. To this end, a NiMo catalyst is designed and investigated for its performance in PET catalytic pyrolysis. The paper aims to explore the structure-activity relationship between the NiMo catalyst and PET, assess the influence of various reaction conditions on the catalytic pyrolysis process, and elucidate the reaction mechanism for the formation of TPA. In particular, the high value conversion of waste poly(ethylene terephthalate) (PET) to aromatic products rich in terephthalic acid (TPA) is successfully achieved by pyrolysis using NiMo bimetallic catalysts in H₂. The catalytic performance of NiMo/Al₂O₃ and NiMo/ZSM-5 for pyrolysis of PET in a H₂ atmosphere at different temperatures (350–550℃) was investigated. In addition, NiMo catalysts with different Ni contents (Mo: 25wt%, Ni: 1wt%–10wt%) were prepared for the pyrolysis of PET in a H₂ atmosphere to evaluate the effect of Ni on the catalytic reaction. The experimental results show that the performance of NiMo catalyst is far superior to that of Ni or Mo catalyst. When NiMo/Al₂O₃ (Ni-5wt%) is used at 450℃, the highest aromatic compound yield of 62.6wt% is obtained and the TPA content reached 65.1%. Meanwhile, for the target product TPA, the catalytic performance of NiMo/ZSM-5 is slightly lower than NiMo/Al₂O₃. The excellent catalytic performance of the NiMo catalyst is due to a combination of effects. The characterization results show that active centers of the NiMo catalyst are dominated by MoO₂ and Ni. The promotion effect of Ni on the catalytic reaction is not only due to its own excellent hydrogenation performance, but also limits the agglomeration growth of MoO₂ particles, promotes the dispersion of MoO₂, lowers the activation temperature of the catalyst to increase the active sites, improves the thermal stability of the catalyst, and enriches the acidic sites of the catalyst. In addition, the mechanism of PET catalytic pyrolysis in a H₂ atmosphere was elucidated by <italic>in situ</italic>-GC. The main pathway for the conversion of PET to TPA is the transfer of H_β and the cleavage of alkoxy groups to generate carboxylic acids and vinyl esters, followed by the hydrocracking of vinyl esters into carboxyl groups and alkenes. This work provides valuable insights into the potential for improving the sustainability and efficiency of waste PET recycling, addressing both environmental concerns and resource management challenges.

The conversion of the petrochemical polymer polyethylene terephthalate (PET) to a biodegradable plastic polyhydroxyal-kanoate (PHA) is described here. PET was pyrolised at 450 degrees C resulting in the production of a solid, liquid, and gaseous fraction. The liquid and gaseous fractions were burnt for energy recovery, whereas the solid fraction terephthalic acid (TA) was used as the feedstock for bacterial production of PHA. Strains previously reported to grow on TA were unable to accumulate PHA. We therefore isolated bacteria from soil exposed to PET granules at a PET bottle processing plant From the 32 strains isolated, three strains capable of accumulation of medium chain length PHA (mclPHA) from TA as a sole source of carbon and energy were selected for further study. These isolates were identified using 16S rDNA techniques as P. putida (GO16), P. putida (GO19), and P. frederiksbergensis (GO23). P. putida GO16 and GO19 accumulate PHA composed predominantly of a 3-hydroxydecanoic acid monomer while P. frederiksbergensis GO23 accumulates 3-hydroxydecanoic acid as the predominant monomer with increased amounts of 3-hydroxydodecanoic acid and 3-hydroxydodecenoic acid compared to the other two strains. PHA was detected in all three strains when nitrogen depleted below detectable levels in the growth medium. Strains GO16 and GO19 accumulate PHA at a maximal rate of approximately 8.4 mg PHA/l/h for 12 h before the rate of PHA accumulation decreased dramatically. Strain GO23 accumulates PHA at a lower maximal rate of 4.4 mg PHA/l/h but there was no slow down in the rate of PHA accumulation over time. Each of the PHA polymers is a thermoplastic with the onset of thermal degradation occurring around 308 degrees C with the complete degradation occurring by 370 degrees C. The molecular weight ranged from 74 to 123 kDa. X-ray diffraction indicated crystallinity of the order of 18-31%. Thermal analysis shows a low glass transition (-53 degrees C) with a broad melting endotherm between 0 and 45 degrees C.

The throwaway culture related to the single-use materials such as polyethylene terephthalate (PET) has created a major environmental concern. Recycling of PET waste into biodegradable plastic polyhydroxyalkanoate (PHA) creates an opportunity to improve resource efficiency and contribute to a circular economy. We sequenced the genome of Pseudomonas umsongensis GO16 previously shown to convert PET-derived terephthalic acid (TA) into PHA and performed an in-depth genome analysis. GO16 can degrade a range of aromatic substrates in addition to TA, due to the presence of a catabolic plasmid pENK22. The genetic complement required for the degradation of TA via protocatechuate was identified and its functionality was confirmed by transferring the tph operon into Pseudomonas putida KT2440, which is unable to utilize TA naturally. We also identified the genes involved in ethylene glycol (EG) metabolism, the second PET monomer, and validated the capacity of GO16 to use EG as a sole source of carbon and energy. Moreover, GO16 possesses genes for the synthesis of both medium and short chain length PHA and we have demonstrated the capacity of the strain to convert mixed TA and EG into PHA. The metabolic versatility of GO16 highlights the potential of this organism for biotransformations using PET waste as a feedstock.

Terephthalic acid (TPA), a major monomer of polyethylene terephthalate (PET), represents a significant challenge in plastic waste management due to its persistence in the environment. In this study, we report a newly developed bacterial consortium capable of using TPA as the sole carbon source in a defined mineral medium. The consortium achieved stationary phase within five days and metabolized approximately 85% of the available TPA. Metabolite analysis by high-performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS/MS) revealed the activation of the benzoate degradation pathway during TPA catabolism. Additionally, the consortium secreted commercially relevant metabolites such as cis,cis-muconic acid and catechol into the culture medium. Genetic profiling using a reverse transcription quantitative polymerase chain reaction (RT-qPCR) and 16S rRNA sequencing identified Paraburkholderia fungorum as the dominant species, suggesting it plays a key role in TPA degradation. The ability of this microbial community to efficiently convert TPA into high-value by-products offers a promising and potentially economically sustainable approach to addressing plastic pollution.

We aimed to investigate epidermal lipid profiles and their association with skin microbiome compositions in children with atopic dermatitis (AD). Specimens were obtained by skin tape stripping from 27 children with AD and 18 healthy subjects matched for age and sex. Proteins and lipids of stratum corneum samples from nonlesional and lesional skin of AD patients and normal subjects were quantified by liquid chromatography tandem mass spectrometry. Skin microbiome profiles were analyzed using bacterial 16S rRNA sequencing. Ceramides with nonhydroxy fatty acids (FAs) and C18 sphingosine as their sphingoid base (C18-NS-CERs) N-acylated with C16, C18 and C22 FAs, sphingomyelin (SM) N-acylated with C18 FAs, and lysophosphatidylcholine (LPC) with C16 FAs were increased in AD lesional skin compared to those in AD nonlesional skin and that of control subjects (all P < 0.01). SMs N-acylated with C16 FAs were increased in AD lesional skin compared to control subjects (P < 0.05). The ratio of NS-CERs with long-chain fatty acids (LCFAs) to short-chain fatty acids (SCFAs) (C24-32:C14-22), the ratio of LPC with LCFAs to SCFAs (C24-30:C16-22) as well as the ratio of total esterified omega-hydroxy ceramides to total NS-CERs were negatively correlated with transepidermal water loss (rho coefficients = -0.738, -0.528, and -0.489, respectively; all P < 0.001). The proportions of Firmicutes and Staphylococcus were positively correlated to SCFAs including NS ceramides (C14-22), SMs (C17-18), and LPCs (C16), while the proportions of Actinobacteria, Proteobacteria, Bacteroidetes, Corynebacterium, Enhydrobacteria, and Micrococcus were negatively correlated to these SCFAs. Our results suggest that pediatric AD skin shows aberrant lipid profiles, and these alterations are associated with skin microbial dysbiosis and cutaneous barrier dysfunction.

Metabolic engineering of Pseudomonas putida KT2440 for upcycling of terephthalic acid into levulinic acid.

Enzymatic degradation of PET using a novel thermophilic PETase

The global interest in fatty acids is steadily rising due to their wealth of industrial potential ranging from cosmetics to biofuels. Unfortunately, certain fatty acids, such as monounsaturated lauric acid with a carbon atom chain length of twelve (C12 fatty acids), cannot be produced cost and energy-efficiently using conventional methods. Biosynthesis using microorganisms can overcome this drawback. However, rewiring a microbe’s metabolome for increased production remains challenging. To overcome this, sophisticated genome-wide metabolic network models have become available. These models predict the effect of genetic perturbations on the metabolism, thereby serving as a guide for metabolic pathways optimization. In this work, we used constraint-based modeling in combination with the algorithm Optknock to identify gene deletions in Escherichia coli that improve C12 fatty acid production. Nine gene targets were identified that, when deleted, were predicted to increase C12 fatty acid titers. Targets play a role in anaplerotic reactions, amino acid synthesis, carbon metabolism, and cofactor-balancing. Subsequently, we constructed the corresponding (combinatorial) deletion mutants to validate the in silico predictions in vivo. Our highest producer (ΔmaeB Δndk ΔpykA) reaches a titer of 6.7 mg/L, corresponding to a 7.5-fold increase in C12 fatty acid production. This study demonstrates that model-guided metabolic engineering is a useful tool to improve C12 fatty acid production.Key points•Escherichia coli as a promising biofactory for unsaturated C12 fatty acids.•Optknock to identify non-obvious gene deletions for increased C12 fatty acids.•7.5-fold higher C12 fatty acid production achieved by deleting maeB, ndk, and pykA.

Transesterification reactions between poly(ethylene terephthalate) (PET), hydroquinone diacetate (HQDA), and terephthalic acid (TA), were conducted via the melt polymerization route with the objective of analyzing the copolyesterification kinetics of a phase separated system. At first homopolymerization of HQDA and TA were conducted at 50 mol % composition of each monomer. Then the polymerization kinetics of four compositions [PET 30/70 (HQDA + TA), PET 40/60 (HQDA + TA), PET 50/50 (HQDA + TA), PET 60/40 (HQDA + TA) with 30 : 35 : 35, 40 : 30 : 30, 50 : 25 : 25, and 60 : 20 : 20 mol % PET, HQDA, and TA] were investigated. The following assumptions were made to make kinetic analysis tractable. HQDA and TA combine to form acetic acid and higher oligomers. The oligomer subsequently adds on to the PET chain to give a copolymer of PET/HQDA/TA, the product of interest. The reaction between PET, HQDA, and TA proceeds in a heter-ogeneous two-phase system consisting of PET-rich and PET-poor regions. The reaction sequence is HQDA and TA react to form a dimer and subsequently the dimer is added onto the PET chain. This reaction sequence is assumed to be valid for the PET-rich and PET-poor phases. Both these reactions were assumed to be second order with respect to the reactants. Reactions wherein the dimer reacts with HQDA or TA to form acetic acid exist, but the probabilities of these processes are small with respect to the main reaction postulated above, thus maintaining the overall mass balance. Moles of acetic acid found experimentally were computed using a standard procedure. The rate constant k under the conditions of phase separation was determined. The rate constant in the presence of PET was higher than that observed in the HQDA and TA reaction. An Arrhenius plot revealed that the catalyst plays a marginal role. Microscopic analysis revealed that the HQDA and TA polymer were nonmelting while copolyesters PET 30/70 (HQDA + TA) to PET 60/40 (HQDA + TA) melted and were liquid crystalline. © 1996 John Wiley & Sons, Inc.

Engineered Halomonas sp. Y3 enables highly efficient upcycling of poly(ethylene terephthalate) to polyhydroxyalkanoates.

The dipyruvylated glycolipid from Mycobacterium smegmatis (Saadat, S., and Ballou, C.E. (1983) J. Biol. Chem. 258, 1813-1818) has been shown to have the following structure in which FA1 is tetra- or hexadecanoic acid and FA2 is 2,4-dimethyl-2-eicosenoic acid. (formula; see text) The fast atom bombardment mass spectrum showed two major ions [M - H]- at m/z 1511 and 1539 (Mr 1512 and 1540) in a ratio of 1.4:1, suggesting that the glycolipid was a mixture of homologs that differed in fatty acid composition by 2 methylene groups. Analysis revealed C14, C16, and C22 fatty acids in ratios of 0.6:0.4:1.0, indicating that 60% of the molecules contained a C14 and C22 fatty acid whereas 40% contained a C16 and C22 fatty acid. The fragmentation pattern showed that a single glucose unit along with the smaller fatty acid could be lost to yield a tetrasaccharide with attached C22 fatty acid, and a second fragmentation yielded a trisaccharide containing 2 pyruvic acids but without attached fatty acid. The C14 and C16 fatty acids were identified as myristic and palmitic acid, whereas the C22 fatty acid was 2,4-dimethyl-2-eicosenoic acid. Precise localization of the fatty acids came from periodate oxidation and methylation analysis.

Transesterification reactions between poly(ethylene terephthalate) (PET), hydroquinone diacetate (HQDA), and terephthalic acid (TA), were conducted via the melt polymerization route with the objective of analyzing the copolyesterification kinetics of a phase separated system. At first homopolymerization of HQDA and TA were conducted at 50 mol % composition of each monomer. Then the polymerization kinetics of four compositions [PET 30/70 (HQDA + TA), PET 40/60 (HQDA + TA), PET 50/50 (HQDA + TA), PET 60/40 (HQDA + TA) with 30 : 35 : 35, 40 : 30 : 30, 50 : 25 : 25, and 60 : 20 : 20 mol % PET, HQDA, and TA] were investigated. The following assumptions were made to make kinetic analysis tractable. HQDA and TA combine to form acetic acid and higher oligomers. The oligomer subsequently adds on to the PET chain to give a copolymer of PET/HQDA/TA, the product of interest. The reaction between PET, HQDA, and TA proceeds in a heter-ogeneous two-phase system consisting of PET-rich and PET-poor regions. The reaction sequence is HQDA and TA react to form a dimer and subsequently the dimer is added onto the PET chain. This reaction sequence is assumed to be valid for the PET-rich and PET-poor phases. Both these reactions were assumed to be second order with respect to the reactants. Reactions wherein the dimer reacts with HQDA or TA to form acetic acid exist, but the probabilities of these processes are small with respect to the main reaction postulated above, thus maintaining the overall mass balance. Moles of acetic acid found experimentally were computed using a standard procedure. The rate constant k under the conditions of phase separation was determined. The rate constant in the presence of PET was higher than that observed in the HQDA and TA reaction. An Arrhenius plot revealed that the catalyst plays a marginal role. Microscopic analysis revealed that the HQDA and TA polymer were nonmelting while copolyesters PET 30/70 (HQDA + TA) to PET 60/40 (HQDA + TA) melted and were liquid crystalline. © 1996 John Wiley & Sons, Inc.