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Phylogenetic affiliation of soil bacteria that degrade aliphatic polyesters available commercially as biodegradable plastics.

Thirty-nine morphologically different soil bacteria capable of degrading poly(beta-hydroxyalkanoate), poly(epsilon-caprolactone), poly(hexamethylene carbonate), or poly(tetramethylene succinate) were isolated. Their phylogenetic positions were determined by 16S ribosomal DNA sequencing, and all of them fell into the classes Firmicutes and Proteobacteria. Determinations of substrate utilization revealed characteristic patterns of substrate specificities.

Bacteria↗

Bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology.

The discovery and chemical identification, in the 1920s, of the aliphatic polyester: poly(3-hydroxybutyrate), PHB, as a granular component in bacterial cells proceeded without any of the controversies which marked the recognition of macromolecules by Staudinger. Some thirty years after its discovery, PHB was recognized as the prototypical biodegradable thermoplastic to solve the waste disposal challenge. The development effort led by Imperial Chemical Industries Ltd., encouraged interdisciplinary research from genetic engineering and biotechnology to the study of enzymes involved in biosynthesis and biodegradation. From the simple PHB homopolyester discovered by Maurice Lemoigne in the mid-twenties, a family of over 100 different aliphatic polyesters of the same general structure has been discovered. Depending on bacterial species and substrates, these high molecular weight stereoregular polyesters have emerged as a new family of natural polymers ranking with nucleic acids, polyamides, polyisoprenoids, polyphenols, polyphosphates, and polysaccharides. In this historical review, the chemical, biochemical and microbial highlights are linked to personalities and locations involved with the events covering a discovery timespan of 75 years.

3-Hydroxybutyric Acid↗

Properties of a bacterium which degrades solid poly(tetramethylene succinate)-co-adipate, a biodegradable plastic.

Various microorganisms were screened for their ability to degrade poly(tetramethylene succinate)-co-(tetramethylene adipate) (PBSA). Strain BS-3, which was newly isolated from a soil sample, was selected as the best strain. From taxonomical studies, the strain was tentatively ascribed to belong to the genus Acidovorax, most probably to the species A. delafieldii. Strain BS-3 could degrade both solid and emulsified PBSA, and also emulsified poly(tetramethylene succinate). During the degradation, a lipase activity was observed in the culture broth. This lipase activity was induced more strongly by PBSA than by tributyrin or triolein which are typical substrates of lipase. These observations strongly suggest that this lipase was involved in the PBSA biodegradation in strain BS-3.

Adipates↗

Integrated production of biodegradable plastic, sugar and ethanol.

Poly 3-hydroxybutyric acid (PHB) and related copolymers can be advantageously produced when integrated into a sugarcane mill. In this favorable scenario, the energy necessary for the production process is provided by biomass. Carbon dioxide emissions to the environment are photosynthetically assimilated by the sugarcane crop and wastes are recycled to the cane fields. The polymer can be produced at low cost considering the availability of a low-price carbon source and energy.

Biodegradation, Environmental↗

A review of plastic waste biodegradation.

With more and more plastics being employed in human lives and increasing pressure being placed on capacities available for plastic waste disposal, the need for biodegradable plastics and biodegradation of plastic wastes has assumed increasing importance in the last few years. This review looks at the technological advancement made in the development of more easily biodegradable plastics and the biodegradation of conventional plastics by microorganisms. Additives, such as pro-oxidants and starch, are applied in synthetic materials to modify and make plastics biodegradable. Recent research has shown that thermoplastics derived from polyolefins, traditionally considered resistant to biodegradation in ambient environment, are biodegraded following photo-degradation and chemical degradation. Thermoset plastics, such as aliphatic polyester and polyester polyurethane, are easily attacked by microorganisms directly because of the potential hydrolytic cleavage of ester or urethane bonds in their structures. Some microorganisms have been isolated to utilize polyurethane as a sole source of carbon and nitrogen source. Aliphatic-aromatic copolyesters have active commercial applications because of their good mechanical properties and biodegradability. Reviewing published and ongoing studies on plastic biodegradation, this paper attempts to make conclusions on potentially viable methods to reduce impacts of plastic waste on the environment.

Bacteria↗

Biodegradation of degradable plastic polyethylene by phanerochaete and streptomyces species.

The ability of lignin-degrading microorganisms to attack degradable plastics was investigated in pure shake flask culture studies. The degradable plastic used in this study was produced commercially by using the Archer-Daniels-Midland POLYCLEAN masterbatch and contained pro-oxidant and 6% starch. The known lignin-degrading bacteria Streptomyces viridosporus T7A, S. badius 252, and S. setonii 75Vi2 and fungus Phanerochaete chrysosporium were used. Pro-oxidant activity was accelerated by placing a sheet of plastic into a drying oven at 70 degrees C under atmospheric pressure and air for 0, 4, 8, 12, 16, or 20 days. The effect of 2-, 4-, and 8-week longwave UV irradiation at 365 nm on plastic biodegradability was also investigated. For shake flask cultures, plastics were chemically disinfected and incubated-shaken at 125 rpm at 37 degrees C in 0.6% yeast extract medium (pH 7.1) for Streptomyces spp. and at 30 degrees C for the fungus in 3% malt extract medium (pH 4.5) for 4 weeks along with an uninoculated control for each treatment. Weight loss data were inconclusive because of cell mass accumulation. For almost every 70 degrees C heat-treated film, the Streptomyces spp. demonstrated a further reduction in percent elongation and polyethylene molecular weight average when compared with the corresponding uninoculated control. Significant (P < 0.05) reductions were demonstrated for the 4- and 8-day heat-treated films by all three bacteria. Heat-treated films incubated with P. chrysosporium consistently demonstrated higher percent elongation and molecular weight average than the corresponding uninoculated controls, but were lower than the corresponding zero controls (heat-treated films without 4-week incubation). The 2- and 4-week UV-treated films showed the greatest biodegradation by all three bacteria. Virtually no degradation by the fungus was observed. To our knowledge, this is the first report demonstrating bacterial degradation of these oxidized polyethylenes in pure culture.

Journal Article↗

Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae.

We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C4-C6 and C3-C8 chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.

Adipates↗

Biodegradation of plastics.

Widespread studies on the biodegradation of plastics have been carried out in order to overcome the environmental problems associated with synthetic plastic waste. Recent work has included studies of the distribution of synthetic polymer-degrading microorganisms in the environment, the isolation of new microorganisms for biodegradation, the discovery of new degradation enzymes, and the cloning of genes for synthetic polymer-degrading enzymes.

Bacteria↗

Plasticization of biodegradable polymers for use in controlled release.

The design of a plasticized, biodegradable polymeric material, suitable for application as a controlled-release system, was attempted. A poly(DL-lactic acid) oligomer was plasticized with 1,2-propylene glycol and glycerol. The latter plasticizer showed poor compatibility whereas 1,2-propylene glycol was compatible with the polymer up to high concentrations. The mixtures prepared displayed considerable depression of processing temperature and enhanced delivery of salicylic acid, in the early stages of release. It seemed, therefore, feasible to produce systems which allow easy and safe processing and can be injected into a body cavity, without the need for surgical retrieval after completion of the release. Furthermore, the differential rate of drug delivery might be of profound interest for cases where elevated drug doses are necessary in the beginning of treatment.

Biodegradation, Environmental↗

Bioplastic biodegradability shapes microbial communities in a coastal brackish environment.

Microorganisms are metabolically versatile and central to marine ecosystems, yet the potential of marine microbial communities to degrade different bioplastics and the effect of environmental factors are poorly understood. Employing multi-seasonal in situ and in vitro experiments, we assessed the biodegradation of six commonly used bio-based bioplastic materials at a coastal site in the brackish Baltic Sea and characterized the associated microbial communities using metagenomics and metatranscriptomics. Cellulose acetate (CA), polybutylene succinate (PBS), and polyhydroxybutyrate/valerate (PHB) degraded at varying rates across materials, seasons, and experimental settings, with up to 28% weight attrition after 97&#xa0;weeks in situ (CA) and 56% carbon loss as CO2 after 4 weeks in vitro (PBS). The three biodegraded plastics developed similar microbial communities that differed markedly from those on the other materials (cellulose acetate propionate, polyamide, and polyethylene) and in the water column. The main microbial populations on the biodegraded plastics included aerobic and facultative anaerobic heterotrophs with a broad capacity for carbohydrate metabolism. Populations with the potential for nitrogen fixation and denitrification were more prevalent on the biodegraded plastics, suggesting that bioplastic biodegradation is constrained by and coupled to the marine nitrogen cycle. Based on the metatranscriptomic signal of key genes involved in the initial hydrolysis of CA, PBS, and PHB, we identified diverse microbial populations that can potentially drive the biodegradation of these materials in the Baltic Sea, many of which encoded the potential to degrade multiple bioplastics. We propose the term 'bioplastisphere' to denote the distinctive microbial communities associated with biodegradable plastics.

Seawater↗

Anti-inflammatory prodrugs as plasticizers for biodegradable implant materials based on poly(3-hydroxybutyrate).

Salicylic and acetylsalicylic acid esters were tested as plasticizers for biodegradable poly(3-hydroxybutyrate) (PHB). The aim is the combination of plasticizing and anti-inflammatory properties in the fabrication of implant materials. Solution-cast films made of mixtures of PHB and 30% ester showed plasticization accompanied by a decrease of elastic modulus and an increase in elongation at break in comparison with pure PHB films. However, the number of usable plasticizers from the group of the salicylic acid and acetylsalicylic acid esters is limited. Short-chain derivatives are volatile while long-chain compounds tend to crystallize. In both cases PHB films embrittle within short time. Moreover, some derivatives show a fast release in an aqueous environment. As alternative nonsteroidal anti-inflammatory prodrugs arylpropionic acid esters were tested as plasticizers. The addition of ketoprofen ethyl ester led to PHB films with decreased brittleness. In summary, various esters of anti-inflammatory drugs show plasticizing effects on solution-cast PHB films comparable with those of commonly used citric acid esters.

Journal Article↗

Determining biodegradability of plastic materials under controlled and natural composting environments.

With the advent of recently promulgated Government regulations on plastics in Mauritius, a study was initiated to examine the biodegradability of two different types of plastic, namely Willow Ridge Plastics - PDQ-H additive (Plastic A) and Ecosafe Plastic - TDPA additive (Plastic B) under controlled and natural composting environments. The results obtained from the controlled composting environment showed that the cumulative carbon dioxide evolution for Plastic A was much higher than that for Plastic B. Plastic A therefore showed a higher level of biodegradation in terms of CO2 evolution than Plastic B. However, from the regression analysis, it was found that the level of CO2 varying with time fitted the sigmoid type curves with very high correlation coefficients (R2 values: 0.9928, 0.9921 and 0.9816, for reference material, inoculum and Plastic A, respectively). The corresponding F-values obtained from the ANOVA analysis together with significance levels of p<0.05 indicated that the three treatments analysed in the biodegradability experiment were significant. The other experiment was undertaken to observe any physical change of Plastics A and B as compared to a reference plastic, namely, compostable plastic bag (Mater-Bi product-Plastic C), when exposed to a natural composting environment. Thermophilic temperatures were obtained for about 3-5 days of composting and the moisture content was in the range of 60-80% throughout the degradation process. It was observed that after 55 days of composting, Plastic C degraded completely while Plastic A and Plastic B did not undergo any significant degradation. It can be concluded that naturally based plastic made of starch would degrade completely in a time frame of 60 days, whereas plastics with biodegradable additive would require a longer time.

Aerobiosis↗

Novel biodegradable aromatic plastics from a bacterial source. Genetic and biochemical studies on a route of the phenylacetyl-coa catabolon.

Novel biodegradable bacterial plastics, made up of units of 3-hydroxy-n-phenylalkanoic acids, are accumulated intracellularly by Pseudomonas putida U due to the existence in this bacterium of (i) an acyl-CoA synthetase (encoded by the fadD gene) that activates the aryl-precursors; (ii) a beta-oxidation pathway that affords 3-OH-aryl-CoAs, and (iii) a polymerization-depolymerization system (encoded in the pha locus) integrated by two polymerases (PhaC1 and PhaC2) and a depolymerase (PhaZ). The complete assimilation of these compounds requires two additional routes that specifically catabolize the phenylacetyl-CoA or the benzoyl-CoA generated from these polyesters through beta-oxidation. Genetic studies have allowed the cloning, sequencing, and disruption of the genes included in the pha locus (phaC1, phaC2, and phaZ) as well as those related to the biosynthesis of precursors (fadD) or to the catabolism of their derivatives (acuA, fadA, and paa genes). Additional experiments showed that the blockade of either fadD or phaC1 hindered the synthesis and accumulation of plastic polymers. Disruption of phaC2 reduced the quantity of stored polymers by two-thirds. The blockade of phaZ hampered the mobilization of the polymer and decreased its production. Mutations in the paa genes, encoding the phenylacetic acid catabolic enzymes, did not affect the synthesis or catabolism of polymers containing either 3-hydroxyaliphatic acids or 3-hydroxy-n-phenylalkanoic acids with an odd number of carbon atoms as monomers, whereas the production of polyesters containing units of 3-hydroxy-n-phenylalkanoic acids with an even number of carbon atoms was greatly reduced in these bacteria. Yield-improving studies revealed that mutants defective in the glyoxylic acid cycle (isocitrate lyase(-)) or in the beta-oxidation pathway (fadA), stored a higher amount of plastic polymers (1.4- and 2-fold, respectively), suggesting that genetic manipulation of these pathways could be useful for isolating overproducer strains. The analysis of the organization and function of the pha locus and its relationship with the core of the phenylacetyl-CoA catabolon is reported and discussed.

Acetyl Coenzyme A↗

Methods for assessment of biodegradability of plastic films in soil.

Traditional and novel techniques were tested and compared for their usefulness in evaluating biodegrad-ability claims made for newly formulated "degradable" plastic film products. Photosensitized polyethylene (PE), starch-PE, extensively plasticized polyvinyl chloride (PVC), and polypropylene (PP) films were incorporated into aerobic soil. Biodegradation was measured for 3 months under generally favorable conditions. Carbon dioxide evolution, residual weight recovery, and loss of tensile strength measurements were supplemented, for some films, by gas chromatographic measurements of plasticizer loss and gel permeation chromatographic (GPC) measurement of polymer molecular size distribution. Six- and 12-week sunlight exposures of photosensitized PE films resulted in extensive photochemical damage that failed to promote subsequent mineralization in soil. An 8% starch-PE film and the plasticized PVC film evolved significant amounts of CO(2) in biodegradation tests and lost residual weight and tensile strength, but GPC measurements demonstrated that all these changes were confined to the additives and the PE and PVC polymers were not degraded. Carbon dioxide evolution was found to be a useful screening tool for plastic film biodegradation, but for films with additives, polymer biodegradation needs to be confirmed by GPC. Photochemical cross-linking of polymer strands reduces solubility and may interfere with GPC measurements of polymer degradation.

Journal Article↗

Spontaneous liberation of intracellular polyhydroxybutyrate granules in Escherichia coli.

Despite receiving much attention as a biodegradable substitute for conventional non-biodegradable plastics, the commercial use of polyhydroxybutyrate (PHB) remains limited because of its high production cost. In order to reduce the recovery/purification cost, which forms over half of the total production cost, we have developed a new cultivation method which enables spontaneous liberation of PHB by modulation of the initial inoculum size and the medium composition in recombinant Escherichia coli harboring Alcaligenes eutrophus phbCAB genes. In flask cultivation using a low cell inoculum and 2x LB medium containing 21% glucose, autolysis of 80.2% as well as yields of 85.2 g/l of PHB and a PHB content of 99.0% (w/w) were obtained. The glucose conversion rate was 0.43. The strategies developed in this study can minimize complex and unfavorable efforts required for efficient recovery/purification processes, thereby enabling biodegradable plastic to be produced by the recombinant E. coli so as to compete with conventional non-biodegradable plastic.

Alcaligenes↗

Biodegradability of degradable plastic waste.

Plastic waste constitutes the third largest waste volume in Malaysian municipal solid waste (MSW), next to putrescible waste and paper. The plastic component in MSW from Kuala Lumpur averages 24% (by weight), whereas the national mean is about 15%. The 144 waste dumps in the country receive about 95% of the MSW, including plastic waste. The useful life of the landfills is fast diminishing as the plastic waste stays un-degraded for more than 50 years. In this study the compostability of polyethylene and pro-oxidant additive-based environmentally degradable plastics (EDP) was investigated. Linear low-density polyethylene (LLDPE) samples exposed hydrolytically or oxidatively at 60 degrees C showed that the abiotic degradation path was oxidative rather than hydrolytic. There was a weight loss of 8% and the plastic has been oxidized as shown by the additional carbonyl group exhibited in the Fourier transform infra red (FTIR) Spectrum. Oxidation rate seemed to be influenced by the amount of pro-oxidant additive, the chemical structure and morphology of the plastic samples, and the surface area. Composting studies during a 45-day experiment showed that the percentage elongation (reduction) was 20% for McD samples [high-density polyethylene, (HDPE) with 3% additive] and LL samples (LLDPE with 7% additive) and 18% reduction for totally degradable plastic (TDP) samples (HDPE with 3% additive). Lastly, microbial experiments using Pseudomonas aeroginosa on carbon-free media with degradable plastic samples as the sole carbon source, showed confirmatory results. A positive bacterial growth and a weight loss of 2.2% for degraded polyethylene samples were evident to show that the degradable plastic is biodegradable.

Air↗

In vitro results with special plastics for biodegradable endoureteral stents.

Internal ureteral stents are widely used in urologic practice for temporary urinary diversion, but all double-J catheters to date exhibit the same disadvantage; that is, they have to be removed endoscopically, necessitating further intervention. We tested different materials (designated G100X-15xLB and G100X-20xLB) to develop a biodegradable (biodissolvable) endoureteral stent that can be held in place without functional loss yet could be dissolved by changing the environment. The principle of the biochemical background is based on the physiological milieu of the urine. The plastics tested are stable in acidic and dissolve in alkaline conditions. In a first step, specimens of two polymers were placed in artificial urine of different pH over a period of 60 days and monitored for integrity (solution trial). In a second step, artificial urine was set in motion (744 mL/24 hours) an infusion pump (Volumed microVP 5000; Fresenius AG, Bad Homburg vdH, Germany) through an infusion set in which a 30-cm piece of the materials to test had been placed (ureter model). Below the inserted specimen, the lumen of the infusion tube was minimized to make obstruction by fragments more possible. In the solution trial, all specimens remained stable under physiologic conditions (pH 5.2) over a period of at least 30 days. The specimens dissolved completely when the pH was adjusted to an alkaline one (pH 7.9). In the ureter model, with pH values of 7.9, all specimens were decomposed after 20 hours, and no occlusion of the model occurred. Using acidic artificial urine, the specimens remained stable with a smooth consistent surface. The dissolution was not a standard chemical one; the materials broke into microscopically small pieces, with fragments of G100X-20xLB being smaller than those of G100X-15xLB. Our first in vitro results show that the tested materials are suitable for further development of biodissolvable endoureteral stents, dissolution of which can be steered by changing the urinary pH.

Biocompatible Materials↗