Biodegradation of polyethylene by the thermophilic bacterium Brevibacillus borstelensis

AIM: To select a polyethylene-degrading micro-organism and to study the factors affecting its biodegrading activity. METHODS AND RESULTS: A thermophilic bacterium Brevibaccillus borstelensis strain 707 (isolated from soil) utilized branched low-density polyethylene as the sole carbon source and degraded it. Incubation of polyethylene with B. borstelensis (30 days, 50 degrees C) reduced its gravimetric and molecular weights by 11 and 30% respectively. Brevibaccillus borstelensis also degraded polyethylene in the presence of mannitol. Biodegradation of u.v. photo-oxidized polyethylene increased with increasing irradiation time. Fourier Transform Infra-Red (FTIR) analysis of photo-oxidized polyethylene revealed a reduction in carbonyl groups after incubation with the bacteria. CONCLUSIONS: This study demonstrates that polyethylene--considered to be inert--can be biodegraded if the right microbial strain is isolated. Enrichment culture methods were effective for isolating a thermophilic bacterium capable of utilizing polyethylene as the sole carbon and energy source. Maximal biodegradation was obtained in combination with photo-oxidation, which showed that carbonyl residues formed by photo-oxidation play a role in biodegradation. Brevibaccillus borstelensis also degraded the CH2 backbone of nonirradiated polyethylene. SIGNIFICANCE AND IMPACT OF THE STUDY: Biodegradation of polyethylene by a single bacterial strain contributes to our understanding of the process and the factors affecting polyethylene biodegradation.     

Aspects of herbicide activity and persistence under low level polyethylene covers

In an experiment prepared in autumn, weed numbers were not affected by covering the soil with clear perforated polyethylene. Weed growth was enhanced, however, and the fresh weight at the time of polyethylene removal in spring was more than three times greater on the covered compared with the uncovered areas. In a similar experiment prepared in spring, covering with perforated polyethylene increased weed numbers by a factor of two and weed fresh weight by a factor of seven. The performance of a number of herbicides used in carrots, in terms of percentage reduction in weed number and weed fresh weight, was similar under covers and in the open. The most effective weed control was achieved with broad-spectrum herbicide treatments, particularly under the polyethylene, since surviving weed seedlings grew rapidly in the protected environment. When weed control was good, covering enhanced carrot yield. Measurement of the distribution of herbicide residues in the soil demonstrated that persistence was increased and movement in the soil decreased by covering with polyethylene.     

The role of the copper-binding enzyme – laccase – in the biodegradation of polyethylene by the actinomycete Rhodococcus ruber

Polyethylene is considered one of the most durable plastic polymers. Virtually, non-biodegradable polyethylene accumulates in the environment, thus posing an ecological threat to man and wildlife. We have previously isolated a strain of the actinomycete Rhodococcus ruber (designated C208; EC 1.10.3.2.) capable of utilizing and degrading polyethylene. Here, we report the role of the bacterial copper-binding enzyme, laccase, in the oxidation and degradation of polyethylene by this strain. Copper markedly affected the induction and activity of laccase, resulting in polyethylene degradation. mRNA quantification by RT-PCR, revealed a 13-fold increase in laccase mRNA levels, in copper-treated cultures compared with the untreated control. Addition of copper to C208 cultures containing polyethylene enhanced the biodegradation of polyethylene by 75%, as compared with the non-amended control. Furthermore, when an extracellular isoform of laccase collected from the media of copper-induced cells was incubated with polyethylene, reductions of 20% and 15% were obtained in the Average Molecular Weight (Mw) and Average Molecular Number (Mn) with the polymer, respectively. FTIR analysis of similar polyethylene films incubated with the extracellular laccase exhibited an increase in the carbonyl peak, indicating that enzymatic oxidation by laccase plays a major role in the biodegradation of polyethylene.     

Enzymic activity of polymer-protein complexes in water-miscible organic solvents]

The enzymic activity of noncovalent complexes of alpha-chymotrypsin with polyethylene glycol and a block-copolymer of polyethylene oxide and polypropylene oxide (proxanol) was studied in aqueous-organic media. It was shown that complex formation activated the enzyme in media with a high content of the organic solvent, whereas in systems containing more than 50% water the enzymic activity of complexes was the same as that of the native enzyme. The activation in polyethylene glycol-containing complexes was greater than in complexes with proxanol of the same molecular mass.     

Preparation of benzoyl dextran and its use in aqueous two-phase systems.

The graft modification of dextran with benzoyl groups has been studied. The factors that affect the degree of substitution of benzoyl dextran were investigated. Phase diagrams for aqueous two-phase systems composed of polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran have been determined. Phase separation was also obtained in aqueous solution of two benzoyl dextran polymers with different degrees of substitution. A four-phase system was obtained with a mixture of polyethylene glycol, dextran and two kinds of benzoyl dextrans. The partitioning of methylene blue and a Procion yellow HE-3G dextran derivative were studied in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems and in systems of two benzoyl dextrans differing in degree of substitution. The proteins bovine serum albumin and glucose-6-phosphate dehydrogenase were partitioned in polyethylene glycol/benzoyl dextran aqueous two-phase systems and the effect of the degree of substitution of benzoyl dextran was studied. Chlorella pyrenoidosa, thylakoid membrane vesicles, plasma membrane vesicles and chloroplasts were partitioned in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems, and in a polyethylene glycol/dextran/benzoyl dextran four-phase system.     

Cryoprotection of purified rat kidney transamidinase by polyethylene glycol.

Polyethylene glycol is a water-soluble polymer which is widely used in the pharmaceutical, cosmetic, and chemical industries. In this study, it is shown that polyethylene glycol is an effective cryoprotectant of rat kidney transamidinase purified from both the mitochondria and cytosol. Much of the activity is lost when the purified enzyme is frozen and thawed in sodium-potassium phosphate buffer in the absence of cryoprotectants. Polyethylene glycols with molecular weights of 4000 to 10,000 were effective cryoprotectants. However, polyethylene glycols with a molecular weight of 1000 or lower inhibited the purified enzyme. A concentration of only 0.01% polyethylene glycol 4000, 8000, or 10,000 was required for complete cryoprotection. In addition to polyethylene glycol, 0.5 mM ethylenediaminetetraacetic acid was required in the phosphate buffer for complete cryoprotection. The stabilization of purified transamidinase by polyethylene glycol will facilitate characterization experiments designed to compare the properties of the mitochondrial and cytosolic isozymes.     

Porcine pancreatic lipase partition in potassium phosphate-polyethylene glycol aqueous two-phase systems

This research study examined porcine pancreatic lipase partition in aqueous two-phase systems formed by polyethylene glycol-potassium phosphate at pH 6.0, 7.0 and 8.0, the effect of polymer molecular mass, and NaCl concentration. The enzyme was preferentially partitioned into the polyethylene glycol rich phase in systems with molecular mass 4000-8000, while with polyethylene glycol of 10,000 molecular mass it was concentrated in the phosphate rich phase. The enthalpic and entropic changes found due to the protein partition were negative for all the polyethylene glycol molecular mass systems assessed. Both thermodynamic functions were shown to be associated by an entropic-enthalpic compensation effect suggesting that the water structure ordered in the ethylene chain of polyethylene glycol plays a role in the protein partition. The addition of NaCl increased the lipase affinity to the top phase and this effect was most significant in the system polyethylene glycol 2000-NaCl 3%. This system yielded an enzyme recovery more than 90% with a purification factor of approximately 3.4.     

Colonization and Degradation of Thermally Oxidized High-Density Polyethylene by Aspergillus niger (ITCC No. 6052) Isolated from Plastic Waste Dumpsite

Plastic materials, particularly polyethylene, are the potential source of environmental pollution. In the present study, a fungal strain was isolated from plastic waste dumpsites capable of adhering to high-density polyethylene (HDPE) surface. The fungal strain was identified as Aspergillus niger (ITCC no. 6052). A visible increase in the growth of the fungi was observed on the surface of the polyethylene when cultured in minimal medium at 30°C and 120 rpm, for 1 month. Approximately 3.44% reduction (gravimetrically) in mass and 61% reduction in tensile strength of polyethylene was observed after 1 month of incubation with fungal isolate. Scanning electron microscope analysis showed hyphael penetration and cracks on the surface of polyethylene. A thick network of fungal hyphae forming a biofilm was also observed on the surface of the plastic pieces. The efficient biofilm formation on polyethylene surface by Aspergillus niger (ITCC no. 6052) is attributed to its high cell surface hydrophobicity. This study indicated that Aspergillus niger (ITCC no. 6052) has ability to degrade thermally oxidized polyethylene.     

Immobilization of nitrifying bacteria by polyethylene glycol prepolymer

The effects of immobilizing materials on the activity of nitrifying bacteria were investigated by using 11 kinds of prepolymers of polyethylene glycol. Relative respiratory activity of immobilized nitrifying bacteria with polyethylene glycol metacrylate prepolymer was higher than that of polyethylene glycol acrylate prepolymer, and there was a tendency for relative respiratory activity to be higher with a prepolymer of greater molecular weight. With the polyethylene glycol prepolymer, there was a drastic improvement over the conventional method of immobilization by acrylamide in the relative respiratory activity of the pellet. Inorganic synthetic wastewater was treated under a high loading rate of 1.14 kg-N/m³·d. Influent NH₄-N could be removed to 2 mg/l or less and the nitrogen removal was 90%.     

Colonization,biofilm formation and biodegradation of polyethylene by a strain of<Emphasis Type="Italic"> Rhodococcus ruber</Emphasis>

A two-step enrichment procedure led to the isolation of a strain of Rhodococcus ruber (C208) that utilized polyethylene films as sole carbon source. In liquid culture, C208 formed a biofilm on the polyethylene surface and degraded up to 8% (gravimetrically) of the polyolefin within 30 days of incubation. The bacterial adhesion to hydrocarbon assay and the salt aggregation test both showed that the cell-surface hydrophobicity of C208 was higher than that of three other isolates which were obtained from the same consortium but were less efficient than C208 in the degradation of polyethylene. Mineral oil, but not nonionic surfactants, enhanced the colonization of polyethylene and increased biodegradation by about 50%. Fluorescein diacetate (FDA) hydrolysis and protein content analysis were used to test the viability and biomass density of the C208 biofilm on the polyethylene, respectively. Both FDA activity and protein content of the biofilm in a medium containing mineral oil peaked 48–72 h after inoculation and then decreased sharply. This finding apparently reflected rapid utilization of the mineral oil adhering to the polyethylene. The remaining biofilm population continued to proliferate moderately and presumably played a major role in biodegradation of the polyethylene. Fourier transform infrared spectra of UV-photooxidized polyethylene incubated with C208 indicated that biodegradation was initiated by utilization of the carbonyl residues formed in the photooxidized polyethylene     

Evaluation of water stress control with polyethylene glycols by analysis of guttation

The water relations of pepper plants (Capsicum frutescens L.) under conditions conducive to guttation were studied to evaluate the control of plant water stress with polyethylene glycols. The addition of polyethylene glycol 6000 to the nutrient solution resulted in water relations similar to those expected in soil at the same water potentials. Specifically, xylem pressure potential in the root and leaf became more negative during a 24-hour treatment period, while osmotic potential of the root xylem sap remained constant. The decrease in pressure potential was closely correlated with the decrease in osmotic potential of the nutrient solution. In contrast, the addition of polyethylene glycol 400 to the nutrient medium resulted in a reduction of osmotic potential in the root xylem sap; this osmotic adjustment in the xylem was large enough to establish an osmotic gradient for entry of water and cause guttation at a nutrient solution osmotic potential of −4.8 bars. Pressure potential in the root and leaf xylem became negative only at nutrient solution osmotic potentials lower than −4.8 bars. About half of the xylem osmotic adjustment in the presence of polyethylene glycol 400 was caused by increased accumulation of K⁺, Na⁺, Ca²⁺, and Mg²⁺ in the root xylem. These studies indicate that larger polyethylene glycol molecules such as polyethylene glycol 6000 are more useful for simulating soil water stress than smaller molecules such as polyethylene glycol 400.     

Reagents for the Preparation of Chromophorically Labeled Polyethylene Glycol-Protein Conjugates

We have developed a new class of reagents (2) for the covalent attachment of polyethylene glycol to proteins. These reagents (2) are the monomethoxypolyethylene glycol esters of 4-fluoro-3-nitrobenzoic acid. The reaction of 2 with lysine ε-amino groups produces a chromophore which can be used to quantitate the polyethylene glycol to protein molar ratio. Bovine (Zn, Cu) superoxide dismutase was used as a model protein for conjugation with 2. When monomethoxypolyethylene glycol of average molecular weight 2105 was used, a conjugate was obtained with a polyethylene glycol to protein molar ratio of 8.88 retaining 100% of native enzymatic activity; monomethoxypolyethylene glycol of average molecular weight 5210 yielded a conjugate with a polyethylene glycol to protein molar ratio of 9.96 retaining 73% of native enzymatic activity.     

Evaluation of viscosities of aqueous two-phase systems containing protein

The dynamic viscosities of aqueous polyethylene glycol, aqueous bovine serum albumin, and polyethylene glycol-bovine serum albumin-water solutions were measured at temperatures of 15, 20, 25, 30 and 35 degree C. To estimate the viscosity values of polyethylene glycol-bovine serum albumin-water solutions, a one parameter Grunberg-like model which was satisfactorily used earlier by the present author for polyethylene glycol-dextran-water solutions was employed. The disposable parameter a for our temperature range was estimated as 3.71. The relative errors varying from 0.29 to 18.98 in absolute value indicates that the Grunberg-like model works perfectly for polymer-protein solutions as well.     

Release of membrane constituents following polyethylene glycol treatment of HEp-2 cells.

HEp-2 cell monolayers were treated with 40% polyethylene glycol for 5 min which resulted in fusion during the subsequent incubation period. A loss of cell membrane components was detected in the polyethylene glycol-treated as well as phosphate buffer/saline-treated control cells, however the polyethylene glycol-treated cells released nearly twice the amount of [14C]acetate-labeled material and [3H]glycerol-labeled lipids into culture fluids than the control cells. It was further detected that the polyethylene glycol-treated cells released only approximately half the amount of protein, glycoprotein, and glycolipid as the control cells. These results suggest that polyethylene glycol exerts a differential mode of action against cell surface components and causes the treated cells to release membrane components rich in lipids but relatively low in protein and carbohydrate-containing components.     

Ethylene Oxide Resistance of Nondesiccated and Desiccated Spores of Bacillus subtilis var. niger Hermetically Sealed in Various Polymeric Films

The resistance to destruction of spores of Bacillus subtilis var. niger hermetically sealed in various polymeric films and exposed to ethylene oxide with and without relative humidity was determined. The effect of desiccation was also determined. The order of increased resistance to sterilization with regard to type of polymeric film was found to be: polyethylene equal to polyvinyl chloride, less than nylon, less than cellophane/polyethylene laminate, less than phenoxy, less than mylar/polyethylene laminate. Desiccated spores sealed in various polymeric films were much more resistant to ethylene oxide sterilization than nondesiccated spores. Relative humidity was an important factor in ethylene oxide sterilization with spores not sealed in polymeric films. However, with spores hermetically sealed in polyethylene, added relative humidity was an insignificant factor in the sterilization process.     

Wear-induced loss of mass in reversed total shoulder arthroplasty with conventional and inverted bearing materials

The notching phenomenon is one of the major concerns with reversed total shoulder arthroplasty. Repetitive contact between the humeral implant and the scapula (mechanical notching) produces progressive abrasion of the implant if the moving part is made of polyethylene. Its debris may then lead to active osteolysis (biological notching). Inversion of bearing materials, i.e. Glenosphere made of polyethylene and humeral Inlay made of metal, aims at the reduction of this phenomenon. However, the question arises if the tribological behavior would then be different. On an experimental setup, the gravimetric wear of both material configurations was measured after loading and moving over 500,000 cycles. The abrasion of the polyethylene Inlay due to mechanical notching was calculated by means of 3D CAD models with different notching stages. The loss of mass due to gravimetric wear was compared to the loss of mass caused by mechanical notching. After 500,000 cycles the measured amount of wear of the polyethylene components was between 8 and 10 mg for both tribological pairings. The calculated loss of mass of the polyethylene Inlay caused by mechanical notching ranged from 73 to 3881 mg. The results of this study indicate that the gravimetric polyethylene wear in the estimated life-time is very low and not significantly different between both material configurations. However, the polyethylene abrasion due to mechanical notching in the configuration with polyethylene Inlay is by far more important than any gravimetric wear. These results support the continued use of inverted bearings in reversed total shoulder arthroplasty.     

U.v.-induced blackening of rose petals

Blackening of petals is common in roses cv. Mercedes during the winter in unheated greenhouses clad with polyethylene films which are transparent to solar radiation of 300 nm wavelength and longer. Petal blackening was prevented when the rose plants were kept at 18°C or a higher temperature. Blackening was also prevented when the flower buds were covered with aluminum foil from dawn to dusk, or when flower buds were covered with polyethylene or PVC films, opaque to solar radiation in a u.v. range shorter than 350 nm even when they were exposed to outdoor conditions with minimum temperatures as low as 5°C. Blackening was not prevented when the flower buds were covered with aluminum foil only during the night, or when covered with polyethylene film transparent to u.v. radiation. Measurements of the spectral transmission of the various plastic materials showed that PVC film and the IR/VR-type of polyethylene were opaque to u.v. radiation. High levels of u.v. transmission were measured in the IR-type polyethylene and low levels of u.v. transmission were present under horticultural glass. The involvement of u.v.-B radiation in the phenomenon of rose petal blackening and its interaction with low temperature is discussed.     

Comparative molecular docking and molecular-dynamic simulation of wild-type- and mutant carboxylesterase with BTA-hydrolase for enhanced binding to plastic

According to the literature review, microbial degradation of polyethylene terephthalate by PETases has been detected effective and eco-friendly. However, the number of microorganisms capable of such feats is limited with some undesirable bioprospecting results. BTA-hydrolase has been already reported capable of degrading polyethylene terephthalate. Therefore, mutation by in silico site-directed mutagenesis means to introduce current isomer of PETase for polyethylene terephthalate degradative capability as a better approach to resolve this issue. This study aimed to use in silico site-directed mutagenesis to convert a carboxylesterase from Archaeoglobus fulgidus to BTA-hydrolase from Thermobifida fusca by replacing six amino acids in specific locations. This work was followed by molecular docking analysis with polyethylene terephthalate and polypropylene to compare their interactions. The best-docked enzyme-substrate complex was further subjected to molecular dynamics simulation to gauge the binding quality of the BTA-hydrolase, wild-type and mutant-carboxylesterase with only polyethylene terephthalate as a substrate. Results of molecular docking revealed lowest binding energy for the wild-type carboxylesterase-polypropylene complex (-7.5 kcal/mol). The root-mean-square deviation value was observed stable for BTA-hydrolase. Meanwhile, root-mean-square fluctuation was assessed with higher fluctuation for the mutated residue Lys178. Consequently, the Rg value for BTA-hydrolase-ligand complex (～1.68 nm) was the lowest compared to the mutant and wild-type carboxylesterase. The collective data conveyed that mutations imparted a minimal change in the ability of the mutant carboxylesterase to bind to polyethylene terephthalate.     

Bacterial Utilization of Ether Glycols

A soil bacterium capable of using oligo- and polyethylene glycols and ether alcohols as sole sources of carbon for aerobic growth was isolated. The effects of substituent groups added to the ether bonds on the acceptability of the compounds as substrates were studied. Mechanisms for the incorporation of two-carbon compounds were demonstrated by the observation that acetate, glyoxylate, ethylene glycol, and a number of the tricarboxylic acid cycle intermediates served as growth substrates in minimal media. The rate of oxidation of the short-chained ethylene glycols by adapted resting cells varied directly with increasing numbers of two-carbon units in the chains from one to four. The amount of oxygen consumed per carbon atom of oligo- and polyethylene glycols was 100% of theoretical, but only 67% of theoretical for ethylene glycol. Resting cells oxidized oligo- and polyethylene glycols with 2 to 600 two-carbon units in the chains. Longer chained polyethylene glycols (up to 6,000) were oxidized at a very slow rate by these cells. Dehydrogenation of triethylene glycol by adapted cells was observed, coupling the reaction with methylene blue reduction.     

Degradation of ethylene glycol and polyethylene glycols by methanogenic consortia.

Methanogenic enrichments capable of degrading polyethylene glycol and ethylene glycol were obtained from sewage sludge. Ethanol, acetate, methane, and (in the case of polyethylene glycols) ethylene glycol were detected as products. The sequence of product formation suggested that the ethylene oxide unit [HO-(CH2-CH2-O-)xH] was dismutated to acetate and ethanol; ethanol was subsequently oxidized to acetate by a syntrophic association that produced methane. The rates of degradation for ethylene, diethylene, and polyethylene glycol with molecular weights of 400, 1,000, and 20,000, respectively, were inversely related to the number of ethylene oxide monomers per molecule and ranged from 0.84 to 0.13 mM ethylene oxide units degraded per h. The enrichments were shown to best metabolize glycols close to the molecular weight of the substrate on which they were enriched. The anaerobic degradation of polyethylene glycol (molecular weight, 20,000) may be important in the light of the general resistance of polyethylene glycols to aerobic degradation.