Thermophilic microbial degradation of polyethylene succinate

This paper examined the biodegradability of a new aliphatic polyester, polyethylene succinate (PES), at a high incubation temperature of 50°C. The distribution and population of total colonies and of PES degrading micro organisms on polymer-emulsified agar plates were determined using the plate count and clear zone methods. The PES-decomposers were present in six of 10 soil samples and the total number ranged from 2.0×104 to 2.2×106 c.f.u./g of samples. Degrading microorganisms constituted between 20 and 80% of the total colonies on PES–agar plates. A single PES-degrading strain, TT96, was isolated and tested for its biodegrading capacity on PES powder and on other aliphatic polyesters: poly(beta-hydroxybutyrate) (PHB), polycaprolactone (PCL), poly(butylene succinate) (PBS), and poly(L-lactide) (PLA). Degraded films of PES and PBS were presented and compared using scanning electron microscopy. Strain TT96 was able to create clear zones on all the polymers used, except on PHB-agar plates. Liquid culture test after 2 weeks showed that TT96 completely degraded PCL powder but had very little activity on other samples. Scanning electron micrograph confirmed the microbial attack of TT96 on PES and PBS films. PES film surfaces were degraded more uniformly compared to PBS films which were decomposed only in some parts.     

Thermophilic microbial degradation of polyethylene succinate

This paper examined the biodegradability of a new aliphatic polyester, polyethylene succinate (PES), at a high incubation temperature of 50°C. The distribution and population of total colonies and of PES degrading micro organisms on polymer-emulsified agar plates were determined using the plate count and clear zone methods. The PES-decomposers were present in six of 10 soil samples and the total number ranged from 2.0×104 to 2.2×106 c.f.u./g of samples. Degrading microorganisms constituted between 20 and 80% of the total colonies on PES–agar plates. A single PES-degrading strain, TT96, was isolated and tested for its biodegrading capacity on PES powder and on other aliphatic polyesters: poly(beta-hydroxybutyrate) (PHB), polycaprolactone (PCL), poly(butylene succinate) (PBS), and poly(L-lactide) (PLA). Degraded films of PES and PBS were presented and compared using scanning electron microscopy. Strain TT96 was able to create clear zones on all the polymers used, except on PHB-agar plates. Liquid culture test after 2 weeks showed that TT96 completely degraded PCL powder but had very little activity on other samples. Scanning electron micrograph confirmed the microbial attack of TT96 on PES and PBS films. PES film surfaces were degraded more uniformly compared to PBS films which were decomposed only in some parts.     

Biotechnological production of (R)-3-hydroxybutyric acid monomer

The escalating problems regarding the treatment of plastic waste materials have led to development of biodegradable plastics. At present, a number of aliphatic polyesters; such as poly[(R)-3-hydroxybutyrate] (PHB), poly(l-lactide), polycaplolactone, poly(ethylene succinate) and poly(butylene succinate) have been developed. Among these aliphatic polyesters, PHB is one of the most attractive since it can undergo biodegradation at various environmental conditions and has properties similar to polypropylene. Although much effort has been made to produce PHB and its copolyesters from renewable resources or through microbial processes, their commercialization and widespread application are still not economically attractive compared to conventional non-biodegradable plastic. Moreover, wide application of PHB and its copolyesters as biodegradable plastic have not only been limited by the cost of production but also by their stinky smell during industrial processing. However, (R)-3-hydroxybutyric acid, a monomer of PHB has wide industrial and medical applications. (R)-3-hydroxybutyric acid can also serve as chiral precursor for synthesis of pure biodegradable PHB and its copolyesters. A number of options are available for production of (R)-3-hydroxybutyric acid. This review discusses each of these options to assess the alternatives that exist for production of pure biodegradable PHB and its copolyesters with good properties.     

Hydrolysis of polyesters by serine proteases

The substrate specificity of -chymotrypsin and other serine proteases, trypsin, elastase, proteinase K and subtilisin, towards hydrolysis of various polyesters was examined using poly(L-lactide) (PLA), poly(-hydroxybutyrate) (PHB), poly(ethylene succinate) (PES), poly(ethylene adipate) (PEA), poly(butylene succinate) (PBS), poly(butylene succinate-co-adipate) (PBS/A), poly[oligo(tetramethylene succinate)-co-(tetramethylane carbonate)] (PBS/C), and poly(-caprolactone) (PCL). -Chymotrypsin could degrade PLA and PEA with a lower activity on PBS/A. Proteinase K and subtilisin degraded almost all substrates other than PHB. Trypsin and elastase had similar substrate specificities to -chymotrypsin.     

Anaerobic Biodegradation of Poly-3-Hydroxybutyrate (PHB) by Sulfate Reducing Bacterium Desulfotomaculum sp.

Poly-3-hydroxybutyrate (PHB) film pieces were degraded by sulfate reducing Desulfotomaculum sp. incubated under anaerobic laboratory conditions. Degradation started with adherence of the microbial cells and followed by formation of black colonies on the film surface. Scanning electron microscopic (SEM) observations revealed the presence of bacteria and formation of small holes on the film. After 60 days of incubation at 30°C, 10 % weight loss in polymer and 13 % sulfate reduction in the medium was observed. According to gel permeation chromatography (GPC) analysis, the molecular weight of the PHB decreased after 30 days and did not decrease further at a more extended incubation period. Loss of weight of PHB does not seem to be correlated with molecular weight decrease.     

Substrate and binding specificities of bacterial polyhydroxybutyrate depolymerases

The substrate specificities of three extracellular polyhydroxybutyrate (PHB) depolymerases from Alcaligenes faecalis (PhaZ_Afa), Pseudomonas stutzeri (PhaZ_Pst), and Comamonas acidovorans (PhaZ_Cac), which are grouped into types A and B based on the position of a lipase box sequence in the catalytic domain, were examined for films of 12 different aliphatic polyesters. Each of these PHB depolymerases used was capable of hydrolyzing poly(3-hydroxybutyrate) (P(3HB)), poly(3-hydroxypropionate) (P(3HP)), poly(4-hydroxybutyrate) (P(4HB)), poly(ethylene succinate) (PESU), and poly(ethylene adipate) (PEA) but could not hydrolyze another seven polyesters. In addition, the binding characteristics of substrate binding domains from PhaZ_Afa, PhaZ_Cac, and PHB depolymerase from Comamonas testosteroni (PhaZ_Cte) were studied by using fusions with glutathione S-transferase (GST). All of fusion proteins adsorbed strongly on the surfaces of polyester granules of P(3HB), P(3HP), and poly(2-hydroxypropionate) (P(2HP)) which was not hydrolyzed by the PHB depolymerases used in this study, while they did not bind on Avicel and chitin granules. The adsorption kinetics of the fusion proteins to the surface of P(3HB) and P(2HP) granules were found to obey the Langmuir isotherm. The cross-area per molecule of fusion protein bound to P(3HB) granules was estimated to be 12±4 nm²/molecule. It has been suggested that the active sites in catalytic domains of PHB depolymerases have a similar conformational structure, and that several amino acids in substrate-binding domains of PHB depolymerases interact specifically with the surface of polyesters.     

Biodegradation kinetics of poly(3-hydroxybutyrate)-based biopolymer systems

The aim of this study was to evaluate and to compare the long-term kinetics curves of biodegradation of poly(3-hydroxybutyrate) (PHB), its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and a PHB/polylactic acid composite. The total weight loss and the change of average viscosity molecular weight were used as the parameters reflecting the biodegradation degree. The rate of biodegradation was analyzed in vitro in the presence of lipase and in vivo after film implantation in animal tissues. The morphology of the PHB film surface was studied by the atomic force microscopy technique. It was shown that PHB biodegradation involves both polymer hydrolysis and its enzymatic biodegradation. The results obtained in this study can be used for the development of various PHB-based medical devices.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Microbial degradation of aliphatic and aliphatic-aromatic co-polyesters

Biodegradable plastics (BPs) have attracted much attention since more than a decade because they can easily be degraded by microorganisms in the environment. The development of aliphatic-aromatic co-polyesters has combined excellent mechanical properties with biodegradability and an ideal replacement for the conventional nondegradable thermoplastics. The microorganisms degrading these polyesters are widely distributed in various environments. Although various aliphatic, aromatic, and aliphatic-aromatic co-polyester-degrading microorganisms and their enzymes have been studied and characterized, there are still many groups of microorganisms and enzymes with varying properties awaiting various applications. In this review, we have reported some new microorganisms and their enzymes which could degrade various aliphatic, aromatic, as well as aliphatic-aromatic co-polyesters like poly(butylene succinate) (PBS), poly(butylene succinate)-co-(butylene adipate) (PBSA), poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), poly(l-lactic acid) (PLA), poly(3-hydroxybutyrate) and poly(3-hydoxybutyrate-co-3-hydroxyvalterate) (PHB/PHBV), poly(ethylene terephthalate) (PET), poly(butylene terephthalate) (PBT), poly(butylene adipate-co-terephthalate (PBAT), poly(butylene succinate-co-terephthalate) (PBST), and poly(butylene succinate/terephthalate/isophthalate)-co-(lactate) (PBSTIL). The mechanism of degradation of aliphatic as well as aliphatic-aromatic co-polyesters has also been discussed. The degradation ability of microorganisms against various polyesters might be useful for the treatment and recycling of biodegradable wastes or bioremediation of the polyester-contaminated environments.     

Degradation of polyethylene succinate (PES) by a new thermophilic <Emphasis Type="Italic">Microbispora</Emphasis> strain

Thermophilic actinomycetes were isolated from sediment of the Chingshuei hot spring in north Taiwan, and the strain HS 45-1 was selected from colonies which formed distinct clear zones on agar plate with emulsified polyethylene succinate (PES). The film of PES disappeared within 6 days in liquid cultures at 50°C. The strain HS 45-1 was also able to degrade poly (ε-carpolactone) (PCL) and poly (3-hydroxybutyrate) (PHB) films completely within 6 days in liquid cultures. Basing on the results of phynotypic characteristics, phylogenetic studies and DNA-DNA hybridization, strain HS 45-1 should be assigned to Micorbispora rosea subsp. taiwanensis.     

A Novel PHB Depolymerase from a Thermophilic Streptomyces Sp.

A novel PHB depolymerase from a thermophilic Streptomyces sp. MG was purified to homogeneity by hydrophobic interaction chromatography and gel filtration. The molecular mass of the purified enzyme was 43 kDa as determined by size exclusion chromatography and 41 kDa by SDS-PAGE. The optimum pH and temperature were 8.5 and 60 °C respectively. The enzyme was stable at 50 °C and from pH 6.5–8.5. The enzyme hydrolyzed not only bacterial polyesters, i.e. poly(3-hydroxybutyric acid and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), but also synthetic, aliphatic polyesters such as polypropiolactone, poly(ethylene adipate) and poly(ethylene succinate). Revisions requested 9 November 2005; Revisions received 12 December 2005     

Direct observation of poly(3-hydroxybutyrate) depolymerase adsorbed on polyester thin film by atomic force microscopy

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerases adsorbed on poly(L-lactide) (PLLA) thin film were directly observed by atomic force microscopy (AFM). A PLLA thin film of 100 nm thickness was prepared on a silicon wafer by spin-cast method. The PLLA thin film was treated at 220 degrees C and quenched to room temperature, resulting in the formation of a completely amorphous film with a smooth surface. Then, the PHB depolymerases from Pseudomonas stutzeri YM1006 and Ralstonia pickettii T1 were dispersed on the amorphous PLLA thin film. Direct AFM observation has revealed that the PHB depolymerases bind in an elliptic shape on the surface of the PLLA thin film and that a small ridge is created around each enzyme molecule. After removal of the enzymes with 40% ethanol aqueous solution, small hollows were found on the PLLA thin film. These results suggest that a PHB depolymerase interacts with polyester molecules during their adsorption to make a hollow on the substrate surface.     

Novel intracellular 3-hydroxybutyrate-oligomer hydrolase in Wautersia eutropha H16

Wautersia eutropha H16 (formerly Ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli. Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount is present in the poly(3-hydroxybutyrate) inclusion bodies of W. eutropha. PhaZc degraded various 3-hydroxybutyrate oligomers at a high specific activity and artificial amorphous poly(3-hydroxybutyrate) at a lower specific activity. Native PHB granules and semicrystalline PHB were not degraded by PhaZc. A PhaZ deletion mutation enhanced the deposition of PHB in the logarithmic phase in nutrient-rich medium. PhaZc differs from the hydrolases of W. eutropha previously reported and is a novel type of intracellular 3-hydroxybutyrate-oligomer hydrolase, and it participates in the mobilization of PHB along with other hydrolases.     

Dynamic adsorption behavior of poly(3-hydroxybutyrate) depolymerase onto polyester surface investigated by QCM and AFM

Time-dependent adsorption behavior of poly(3-hydroxybutyrate) (PHB) depolymerase from Ralstonia pickettiiT1 on a polyester surface was studied by complementary techniques of quarts crystal microbalance (QCM) and atomic force microscopy (AFM). Amorphous poly(l-lactide) (PLLA) thin films were used as adsorption substrates. Effects of enzyme concentration on adsorption onto the PLLA surface were determined time-dependently by QCM. Adsorption of PHB depolymerase took place immediately after replacement of the buffer solutions with the enzyme solutions in the cell, followed by a gradual increase in the amount over 30 min. The amount of PHB depolymerase molecules adsorbed on the surface of amorphous PLLA thin films increased with an increase in the enzyme concentration. Time-dependent AFM observation of enzyme molecules was performed during the adsorption of PHB depolymerase. The phase response of the AFM signal revealed that the nature of the PLLA surface around the PHB depolymerase molecule was changed due to the adsorption function of the enzyme and that PHB depolymerase adsorbed onto the PLLA surface as a monolayer at a lower enzyme concentration. The number of PHB depolymerase molecules on the PLLA surface depended on the enzyme concentration and adsorption time. In addition, the height of the adsorbed enzyme was found to increase with time when the PLLA surface was crowded with the enzymes. In the case of higher enzyme concentrations, multilayered PHB depolymerases were observed on the PLLA thin film. These QCM and AFM results indicate that two-step adsorption of PHB depolymerase occurs on the amorphous PLLA thin film. First, adsorption of PHB depolymerase molecules takes place through the characteristic interaction between the binding domain of PHB depolymerase and the free surface of an amorphous PLLA thin film. As the adsorption proceeded, the surface region of the thin film was almost covered with the enzyme, which was accompanied by morphological changes. Second, the hydrophobic interactions among the enzymes in the adlayer and the solution become more dominant to stack as a second layer.     

A human antibody fragment with high affinity for biodegradable polymer film

Antibodies with high affinity for the surface of a solid material would be advantageous in biomaterial science as a protein device. A human antibody fragment that binds to poly(hydroxybutyrate) (PHB), a biodegradable polymer matter, was generated by a phage display system. Clone PH7-3d3 was isolated after several rounds of selection and prepared as a fragment of immunoglobulin variable regions (Fv). The quartz crystal microbalance technique showed that PH7-3d3 Fv completely inhibited PHB enzymatic degradation by competing with PHB depolymerase. Kinetic analysis based on surface plasmon resonance demonstrated that PH7-3d3 Fv bound to the PHB film with an equilibrium dissociation constant of 14 nM. The three-dimensional structure of PH7-3d3 Fv was resolved to 1.7 A, revealing that the complementarity determining regions (CDRs) in the Fv fragment form a relatively flat surface on which uncharged polar and aromatic amino acids are distributed in clusters. The structure of PH7-3d3 Fv was similar to that of PHB depolymerase in the orientation of aromatic residues in the binding sites. Alanine scanning mutagenesis demonstrated that these aromatic residues, especially tryptophan residues in CDRs, were critical in the interaction between PH7-3d3 Fv and PHB. Our results suggest the possible selection of an antibody fragment that binds a material surface in a manner similar to protein-ligand interaction.     

Degradation of polycaprolactone at 50 °C by a thermotolerant Aspergillus sp.

A thermotolerant Aspergillus sp. strain ST-01 degrading poly(-caprolactone) films was isolated. The polyester was degraded and assimilated giving 36 mg of cell from 100 mg sample and 10 mg yeast extract after 6 days at 50 °C. The degradation products were identified as succinic acid, butyric acid, valeric acid, and caproic acid. The isolate also degraded more than 90% film samples of polyhydroxybutyrate (PHB) and poly(tetramethylene succinate-co-tetramethylene adipate) at 50 °C.     

Comparison of cytocompatibility of zein film with other biomaterials and its degradability in vitro

Cytocompatibility of particle zein (Pzein) and film zein (Fzein) was evaluated and compared with polyhydroxybutyrate (PHB), its copolymer poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), polylactic acid (PLA), and collagen, using HL-7702 cells, in terms of cell attachment rate within 3 h, and cell viabilities at 3 and 6 days determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The zein degradation test was carried out using collagenase and trypsin, and the degradation product was added to the culture medium at different concentrations in order to examine the concentration-dependent cytotoxic effect. RESULT: The adhesion rate of the HL-7702 cells on both Pzein and Fzein was higher than that on collagen film. Cell viabilities were higher on both Pzein and Fzein than on films of PLA, PHB, PHBV, and collagen from fish skin. Zein can be degraded by both trypsin and collagenase, and the degradation product can enhance cell viability within a certain range of concentrations.     

Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli.

We studied recovery of poly(3-hydroxybutyric acid) (PHB) from Alcaligenes eutrophus and a recombinant Escherichia coli strain harboring the A. eutrophus poly(3-hydroxyalkanoic acid) biosynthesis genes. The amount of PHB degraded to a lower-molecular-weight compound in A. eutrophus during the recovery process was significant when sodium hypochlorite was used, but the amount degraded in the recombinant E. coli strain was negligible. However, there was no difference between the two microorganisms in the patterns of molecular weight change when PHB was recovered by using dispersions of a sodium hypochlorite solution and chloroform. To understand these findings, we examined purified PHB and lyophilized cells containing PHB by using a differential scanning calorimeter, a thermogravimetric analyzer, and nuclear magnetic resonance. The results of our analysis of lyophilized whole cells containing PHB with the differential scanning calorimeter suggested that the PHB granules in the recombinant E. coli strain were crystalline, while most of the PHB in A. eutrophus was in a mobile amorphous state. The stability of the native PHB in the recombinant E. coli strain during sodium hypochlorite treatment seemed to be due to its crystalline morphology. In addition, as determined by the thermogravimetric analyzer study, lyophilized cell powder of the recombinant E. coli strain containing PHB exhibited greater thermal stability than purified PHB obtained by chloroform extraction. The PHB preparations extracted from the two microorganisms had identical polymer properties.     

Nuclear Magnetic Resonance Studies of Poly(3-Hydroxybutyrate) and Polyphosphate Metabolism in Alcaligenes eutrophus

The metabolic pathways of poly(3-hydroxybutyrate) (PHB) and polyphosphate in the microorganism Alcaligenes eutrophus H16 were studied by ¹H, ¹³C, and ³¹P nuclear magnetic resonance (NMR) spectroscopy and by conventional analytical techniques. A. eutrophus cells accumulated two storage polymers of PHB and polyphosphate in the presence of carbon and phosphate sources under aerobic conditions after exhaustion of nitrogen sources. The solid-state cross-polarization/magic-angle spinning ¹³C NMR spectroscopy was used to study the biosynthetic pathways of PHB and other cellular biomass components from ¹³C-labeled acetate. The solid-state ¹³C NMR analysis of lyophilized intact cells grown on [1-¹³C]acetate indicated that the carbonyl carbon of acetate was selectively incorporated both into the carbonyl and methine carbons of PHB and into the carbonyl carbons of proteins. The ³¹P NMR analysis of A. eutrophus cells in suspension showed that the synthesis of intracellular polyphosphate was closely related to the synthesis of PHB. The roles of PHB and polyphosphate in the cells were studied under conditions of carbon, phosphorus, and nitrogen source starvation. Under both aerobic and anaerobic conditions PHB was degraded, whereas little polyphosphate was degraded. The rate of PHB degradation under anaerobic conditions was faster than that under aerobic conditions. Under anaerobic conditions, acetate and 3-hydroxybutyrate were produced as the major extracellular metabolites. The implications of this observation are discussed in connection with the regulation of PHB and polyphosphate metabolism in A. eutrophus.     

Microbial Degradation of an Aliphatic Polyester with a High Melting Point, Poly(Tetramethylene Succinate)

The biodegradability of poly(tetramethylene succinate) (PTMS), a synthetic aliphatic polyester with a high melting point, was evaluated. The ecological study showed that the distribution of PTMS-degrading microorganisms in soil environments was quite restricted compared with the distribution of microorganisms that degrade poly((epsilon)-caprolactone) (PCL), a polyester with a low melting point. However, in soil samples in which the formation of a clear zone was observed, PTMS-degrading microorganisms constituted 0.2 to 6.0% of the total number of microorganisms, which is very close to the percentage (0.8 to 8.0%) observed for PCL-degrading microorganisms. Five strains were isolated from colonies which formed distinct clear zones on agar plates with emulsified PTMS. In liquid cultures of the isolates with ground PTMS powder, strain HT-6, an actinomycete, showed the highest PTMS degrading activity. It assimilated about 60% of the ground PTMS powder after 8 days of cultivation. When a PTMS emulsion was used, a higher degradation rate was observed and more than 90% of the PTMS was assimilated in 6 days. PTMS degradation products were analyzed by gas chromatography, and it was found that 1,4-butanediol, 4-hydroxy n-butyrate, and succinic acid accumulated during cultivation. Degradation of PTMS film by the strain occurred in two steps: fragmentation and then the formation of hemispherical holes on the surface of the film. Strain HT-6 was also able to assimilate PCL and poly((beta)-hydroxybutyrate) (PHB). The crude enzyme showed a wide range of substrate specificity, being able to degrade low-molecular-weight PTMS, PCL, PHB, and even high-molecular-weight PTMS.