Enzymatic degradation of poly([R,S] β-hydroxybutyrate)

The synthetic analogue of a bacterially produced polyester, poly(-hydroxybutyrate) (PHB) was synthesized from racemic -butyrolactone using anin situ trimethyl aluminum-water catalyst. The polymer was fractionated into samples differing in molecular weight and isotactic diad content. The latter was closely related to degree of crystallinity. The biodegradation of these fractions were examined by monitoring mass loss over time in the presence of anAlcaligenes faecalis T1 extracellular bacterial poly(-hydroxybutyrate) depolymerase. The fraction with high isotactic diad tacticity content showed little or no degradation over a 50 hour incubation period, whereas the fraction of intermediate isotactic diad content degraded in a continuous steady fashion at a rate that was less than that for bacterial PHB. The low isotactic diad fraction underwent a rapid initial degradation, followed by no further mass loss. The presence of stereoblocks in the polymer structure of the various fractions was an influence on the degree of susceptibility towards degradation and is related to sample crystallinity.     

Degradation of poly(β-hydroxyalkanoates) and polyolefin blends in a municipal wastewater treatment facility

Six types of plastics and plastic blends, the latter composed at least partially of biodegradable material, were exposed to aerobically treated wastewater (activated sludge) to ascertain their biodegradability. In one study, duplicate samples of 6% starch in polypropylene, 12% starch in linear low-density polyethylene, 30% polycaprolactone in linear low-density polyethylene, and poly(-hydroxybutyrate-co-hydroxyvalerate) (PHB/V), a microbially produced polyester, were exposed to activated sludge for 5 months, and changes in mass, molecular weight average, and tensile properties were measured. None of the blended material showed any sign of degradation. PHB/V, however, showed a considerable loss of mass and a significant loss of tensile strength. In a second study, PHB/V degraded rapidly, but another type of microbial polymer which forms a thermoplastic elastomer, poly(-hydroxyoctanoate), did not degrade. These results illustrate the potential for disposal and degradation of PHB/V in municipal wastewater.     

A method for the isolation of microorganisms producing extracellular long-side-chain poly (β-hydroxyalkanoate) depolymerase

A simple method was developed for the preparation of an autoclavable, long-side-chain poly (-hydroxyalkanoate) (LSC-PHA) colloidal suspension, which was used as a substrate for enzymatic degradation and to prepare agar overlay plates for the isolation of microorganisms producing extracellular LSC-PHA depolymerase. Six cultures producing extracellular LSC-PHA depolymerase were isolated from a composted hydrocarbon-contaminated soil. All were pseudomonads or related bacteria. All (with the possible exception ofXanthomonas maltophilia) could produce LSC PHA. Except forX. maltophilia none could hydrolyze poly (-hydroxybutyrate). Screening of sevenPseudomonas strains known to accumulate LSC PHA showed that all were negative for extracellular LSC-PHA depolymerase production. It was concluded that extracellular LSC-PHA depolymerase producers are found mostly in the genusPseudomonas but that they are relatively uncommon.     

Distribution of Poly(β-propiolactone) Aerobic Degrading Microorganisms in Different Environments

The distribution of degading microorganisms of high molecular weight poly(-propiolactone) (PPL), whose individual structural units are similar to those of poly(-hydroxybutyrate) (PHB) and poly(€-caprolactone) (PCL), was examined. Despite the fact that PPL is a chemosynthetic polymer, many kinds of PPL-degrading microorganisms were found to be distributed as resident populations widely in natural environments. A total of 77 strains of PPL-degrading microorganisms was isolated. From standard physiological and biochemical tests, at least 41 strains were referred to as Bacillus species. Microbial degradation of fibrous PPL proceeded rapidly in some enrichment cultures but was not as complete as that of PHB. Most of the isolated PPL-degrading microorganisms were determined to be PCL degraders and/or PHB degraders. Therefore, it can be assumed that mostly PPL is recognized by the microorganisms as PHB or another natural substrate of the same type as which PCL is regarded. Microbial degradation of PPL was confirmed by some Bacillus strains from type culture collections. The similarity of microbial degradation between PPL and PCL was found to be very close.     

The Effect of UV-B Irradiation on the Biodegradability of Poly-β-Hydroxybutyrate (PHB) and Poly-ɛ-Caprolactone (PCL)

The biodegradability of poly--hydroxybutyrate and poly--caprolactone in soil compostage before and after irradiation of the polymers for 192, 425, and 600 h in a Weather-Ometer was examined. The biodegradability tests were done in soil compostage at pH 7.0, 9.0, and 11.0 to assess the influence of this parameter on degradation. The rate of degradation was directly proportional to the soil alkalinity. Poly--hydroxybutyrate showed the greatest weight loss and aging in a Weather-Ometer did not significantly increase the biodegradation, except when the polymer was aged for 425 h and buried in soil compostage of pH 11.0.     

The degradation of gel-spun poly(β-hydroxybutyrate) “wool”

The apparent biodegradability and biocompatibility of the microbially produced polyester, poly(-hydroxybutyrate) (PHB), has been the focus of much research by a number of authors with regard to its potential for use in packaging and medical implantation devices. PHB has recently been produced by gel-spinning into a novel form, with one possible application being as a wound scaffolding device, designed to support and protect a wound against further damage while promoting healing by encouraging cellular growth on and within the device from the wound surface. This new nonwoven form combines a large volume with a low mass, has an appearance similar to that of cotton wool, and has been called wool because of this similarity. The hydrolytic degradation of this wool was investigated in an accelerated model of pH 10.6 and temperature 70°C. It was determined that the PHB wool gradually collapsed during degradation. The surface area-to-volume ratio was concluded to be a primary influencing factor. Degradation was characterized by a reduction in the glass transition temperatures and melting points and a fusion enthalpy peak of maximum crystallinity, (88%), which coincided with the point of matrix collapse.     

Distribution of poly(β-hydroxybutyrate) and poly(ε-caprolactone)aerobic degrading microorganisms in different environments

To assess the capacity of the natural environment for degrading plastics, the populations of poly(-hydroxybutyrate)(PHB)-and poly(-caprolactone)(PCL)-degrading aerobic microorganisms and their ratios to the total number of microorganisms in soil samples were estimated by the plate count method with agar medium containing emulsified PHB or PCL. The numbers of the degrading microorganisms were determined by counting colonies that formed clear zones on the plate. It was found that PHB- and PCL-degrading (depolymerizing) microorganisms are distributed over many kinds of material, including landfill leachate, compost, sewage sludge, forest soil, farm soil, paddy soil, weed field soil, roadside sand, and pond sediment. Of total colony counts, the percentages of PHB and PCL degrading microorganisms were 0.2–11.4 and 0.8–11.0%, respectively. The results suggest that many kinds of degrading microorganisms are present in each environment and that specific consortia differing in biodegradation capacity are constructed.     

Preparation conditions and swelling equilibria of biodegradable hydrogels prepared from microbial poly(γ-glutamic acid) and poly(ε-lysine)

Biodegradable hydrogels prepared by -irradiation from microbial poly(amino acid)s are reviewed. pH-sensitive hydrogels were prepared by means of -irradiation of poly(-glutamic acid) (PGA) produced byBacillus subtilis IFO3335 and poly(-lysine) (PL) produced byStreptomyces albulus in aqueous solutions. The preparation conditions, swelling equilibria, hydrolytic degradation, and enzymatic degradation of these hydrogels were studied. A hydrogel with a wide variety of swelling behaviors has been produced by -irradiation from a mixture solution of PGA and PL.Paper presented at the 4th International Workshop on Biodegradable Plastics and Polymers, October 11–14, 1995, Durham, New Hampshire, USA.     

Effects of Poly(Ethylene Glycol) on the Production of Poly(β-Hydroxybutyrate) by Azotobacter vinelandii UWD

Azotobacter vinelandii UWD, ATCC 53799, an engineered strain derived from Azotobacter vinelandii UW was used in the poly(ethylene glycol) (PEG)-modulated synthesis of poly(-hydroxybutyrate) (PHB). To the best of our knowledge, this is the first report on modulating the production of PHB by amending the fermentation broth with PEG using A. vinelandii UWD. It was determined that A. vinelandii UWD is prone to back-mutation to the parent strain; hence fermentation experiments require the use of the antibiotic rifampicin. Diethylene glycol (DEG) and PEGs with molecular weights of 400, 2000, and 3400 Da and pentaerythritol ethoxylate (PEE) were used in the modulated fermentation experiments in a concentration of 2% (w/v). The molecular weight of the resulting polymers was reduced by up to 78%. No impact on the productivity of the strain was observed. Spectroscopic evidence showed that PEG-modulated synthesis resulted in the covalent attachment of the ethylene glycol moiety only when a small molecule, DEG, was used. PEGs had the same effects on the polymer formation in terms of molecular weight reduction as DEG, but no spectroscopic evidence was found for the formation of a covalent linkage between PHB and higher molecular weight PEGs.     

Calculation of carbon balances for evaluation of the biodegradability of polymers

Establishing carbon balances has been proven to be an applicable and powerful tool in testing biodegradability of polymers. In controlled degradation tests at a 4-L scale with the model polymer poly(-hydroxybutyrate) (PHB), it was shown that the degree of degradation could not be determined with satisfactory accuracy from CO₂ release alone. Instead, the course of degradation was characterized by means of establishing carbon balances for the degradation of PHB withAcidovorax facilis and a mixed culture derived from compost. Different analytical methods for determining the different carbon fractions were adapted to the particular test conditions and compared. Quantitative determination of biomass and residual polymer were the main problems in establishing carbon balances. Amounts of biomass derived from protein measurements depend strongly on assumptions of the protein content of the biomass. Selective oxidation of biomass with hypochlorite was used as alternative, but here problems arose from insoluble metabolic products. Determination of soluble components with the method of chemical oxygen demand (COD) also includes empirical assumptions but seems acceptable if the dissolved carbon fraction is in the range of some 10% total carbon. Results confirm both analytical assays and theoretical approaches, in ending up at values very close to 100%, within an acceptable standard deviation range under test conditions comparable to standard test practice.Paper presented at the Bio/Environmentally Degradable Polymer Society—Third National Meeting, June 6–8, 1994, Boston, Massachusetts.     

Synthesis and environmental degradation of polyesters based on poly (ε-caprolactone)

Poly (-caprolactone) (PCL), poly (-valerolactone) (PVL), poly (-caprolactone-co--valerolactone) [P(CL-co-VL)], and poly (-caprolactone-co-ethylene oxide-co--caprolactone) (PCL-PEO-PCL) were synthesized by ring-opening and diol-initiated polymerization of -caprolactone and -valerolactone. The degradation of the samples by chemical hydrolysis and in a soil burial test was evaluated. It was found that PCL, PVL, and P(CL-co-VL) degrade mainly enzymatically. The rate of degradation depends on their molecular weight, chemical structure, composition, and morphology. PCL-PEO-PCL block copolymers exhibit a repelling effect to the microorganisms in the soil, which depends on the molecular weight and relative amount of PEO block in the copolymer.     

Purification of Aspergillus fumigatus (Pdf1) Poly(β-hydroxybutyrate) (PHB) Depolymerase Using a New, Single-Step Substrate Affinity Chromatography Method: Characterization of the PHB Depolymerase Exhibiting Novel Self-Aggregation Behavior

The extracellular poly(-hydroxybutyrate) (PHB) depolymerase of Aspergillus fumigatus Pdf1 was purified by a new, simple, one-step affinity chromatography method using the substrate PHB. The purified enzyme was glycosylated, with the molecular mass of 40 KD, and exhibited a novel self-aggregation behavior by means of hydrophobic interaction that was resolved by Triton X-100 (TX-100) pretreatment of enzyme and also TX-100 incorporation in the native gel. The apparent K _m value of purified enzyme for PHB was 119 g/mL and 3-hydroxybutyrate was detected as the main endproduct of PHB hydrolysis. The depolymerase was insensitive to phenylmethyl sulfonyl fluoride (PMSF), sodium azide, ethylenediaminetetraacetic acid (EDTA), and para-chloromercuric benzoic acid (PCMB), but was inactivated by dithioerythritol (DTT) and showed specificity for short chain-length poly(-hydroxyalkanoates) (PHAs) such as PHB, poly(hydroxyvalerate) (PHV), and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Medium-chain-length PHA failed to get hydrolyzed. The enzyme, however, exhibited strong cross reactivity with the Comamonas sp. PHB depolymerase antibodies, but not with PHV depolymerase antibodies of Pseudomonas lemoignei. Southern hybridization and dot blot analysis of A. fumigatus Pdf1 genomic DNA with alkaline phosphatase labeled probes of P. lemoignei PHB and PHV depolymerase genes revealed no homology, although the enzyme hydrolyzed both PHB and PHV.     

Proposed mechanism for bacterial metabolism of polyacrylate

A possible aerobic degradative pathway for polyacrylate was examined with trimer (1,3,5-pentane tricarboxylic acid; PTCA)-utilizing bacteria. A few metabolic products from PTCA accumulated in culture filtrates and reaction mixtures of washed cells. Fraction A was detected as a main metabolite by high-performance liquid chromatography. A small amount of fraction B was concomitant with fraction A. Another fraction, C, was also detected. These compounds were suggested by liquid chromatography-mass spectrometry analyses to be 1,3,5-(1- or 2-pentene)tricarboxylic acid (fraction A or B) and 1,3,5-(2-oxopentane)tricarboxylic acid (fraction C). Fraction A was quickly further metabolized by washed cells, but fraction B was only gradually degraded. From these results, the metabolic pathway for polyacrylate is suggested to be quite similar to-oxidation for saturated fatty acids. The degradation of PTCA by washed cells was slower than that by growing cells and was inhibited by 5 mM NaN₃. This suggests that the metabolism is linked to a respiratory chain or energy-producing system of bacteria which can aerobically assimilate PTCA.Paper presented at the Bio/Environmentally Degradable Polymer Society—Second National Meeting, August 19–21, 1993, Chicago, Illinois.     

Synthesis and biodegradation of poly(γ-butyrolactone-co-l-lactide)

Biodegradable polyesters were synthesized by ring-opening copolymerization of -butyrolactone (BL) and its derivatives withl-lactide (LLA). Although tetraphenyl tin was the main catalyst used, other organometallic catalysts were used as well.¹H and¹³C NMR spectra showed that poly(BL-co-LLA)s were statistical and that their number-average molecular weights were as high as 7×10⁴. The maximum BL content obtained from copolymerization BL/LLA was around 17%. TheT _m andT _g values of the copolymers showed a gradual depression with an increase in BL content. NoT _m was obtained for the copolymers containing more than 13 mol% BL. The biodegradability of the copolyesters was evaluated by enzymatic hydrolysis and nonenzymatic hydrolysis tests. The enzymatic hydrolysis was carried out at 37°C for 24 h using lipases fromRhizopus arrhizus andR. delemar. Hydrolyses by both lipases showed that an increase in BL content of the copolymer resulted in enhanced biodegradability. Nonenzymatic accelerated hydrolysis of copolymers at 70°C was found to increase proportionally to their exposure time. The hydrolysis rate of these copolymers was considerably faster than that of PLLA. The higher hydrolyzability was recorded for the BL-rich copolymers. The copolymerization of -methyl--butyrolactone (MBL) or -ethyl--butyrolactone (EBL) with LLA resulted in relatively LA-rich copolymers.     

Hydrolytic and enzymatic degradation of poly(γ-glutamic acid) hydrogels and their application in slow-release systems for proteins

The hydrolytic and enzymatic degradation of newly developed hydrogels, produced by cross-linking purified poly(-glutamic acid) (PGA) with dihaloalkane compounds, was studied and is reported in this paper. Analysis of hydrolysis of the hydrogel as a function of pH indicated that the hydrolysis occurred slowly at neutral pH, but fast in both acidic and alkaline solutions, while the polymer could be hydrolyzed rapidly only in acidic solutions. The ester bonds were more sensitive to hydrolysis than peptide bonds. The biodegradability of the hydrogel and polymer was further confirmed when enzymatic degradation was studied by three enzymes (cathepsin B, pronase E, and trypsin), which were able to cleave both ester and peptide bonds gradually. A slow-release system for porcine somatotropin (pST) formed by using the hydrogel as matrix to entrap the hormone was evaluatedin vitro andin vivo. Results demonstrated that the hydrogel was able to release the hormone for a period of 20–30 days and indicated its potential application in slow-release systems for bioactive materials, especially macromolecules, such as peptides and proteins.     

Radiation Effects on Blends of Poly(ε-Caprolactone) and Diatomites

Poly(-caprolactone) (PCL) was blended with diatomaceous earth (diatomite) and irradiated with -rays to introduce cross-linking between PCL molecules or both components. The unwashed diatomite containing a little of a volatile component showed high efficiency of introduction of cross-linking, whereas that with no volatile component showed low efficiency of introduction of cross-linking. Elongational viscosity, melt viscosity, and modulus of PCL/diatomite blend irradiated at various doses were significantly improved. Enzymatic degradation of the PCL/diatomite blend became faster than that of the PCL, though that of the blend irradiated became slower.     

Properties of a Poly(3-hydroxybutyrate) Depolymerase from Penicillium funiculosum

A poly(3-hydroxybutyrate) (PHB) depolymerase was purified from a fungus, Penicillium funiculosum (IFO6345), with phenyl-Toyopearl and its properties were compared with those of other PHB depolymerases. The molecular mass of the purified enzyme was estimated at about 33 kDa by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The pH optimum and pI were 6.5 and 6.5, respectively. The purified protein showed affinity to Con A-Sepharose, indicating that it is a glycoprotein. Diisopropylfluorophosphate and dithiothreitol inhibited the depolymerase activity completely. The N-terminal amino acid sequence of the purified enzyme was TALPAFNVNPNSVSVSGLSSGGYMAAQL, which contained a lipase box sequence. This purified enzyme is one of the extracellular PHB depolymerase which belong to serine esterase. The purified enzyme showed relatively strong hydrolytic activity against 3-hydroxybutyrate oligomers compared with its PHB-degrading activity. PHB-binding experiments showed that P. funiculosum depolymerase has the weakest affinity for PHB of all the depolymerases examined.     

Correlating the Spatial Distribution of Atmospheric Ammonia with δ15N Values at an Ammonia Release Site

A field ammonia (NH₃) release experiment and open top chambers containing moorland monoliths continuously fumigated with NH₃ or sprayed with NH₄Cl were used to assess the potential for using ¹⁵N values in determining the area of influence around a point NH₃ emission source. ¹⁵N values are being increasingly used as environmental tracers and we tested the hypothesis that the ¹⁵N signal from an NH₃ emission source is observable in nearby vegetation. Using modified monitoring devices, atmospheric NH₃ concentrations were found to decrease with distance from source, with ¹⁵N values also reflecting this trend, producing a signal shift with changing concentration. Open top chamber studies of ¹⁵N values of Calluna vulgaris (L.) Hull indicated a correlation with deposition treatments in current year shoots. Analysis of Calluna shoots from the NH₃ release showed a similar trend of ¹⁵N enrichment. Significant linear correlations between ¹⁵N and percent N in plant material were found, both in the controlled conditions of the open top chambers and at the NH₃ release site, illustrating the possible use of this technique in N deposition biomonitoring.     

Aerobic thermophilic bacteria enhance biogas production

The enhancing effect of aerobic thermophilic (AT) bacteria on the production of biogas from anaerobically digested sewage sludge (methanogenic sludge) was investigated. Sewage sludge (5%, w/w) was incubated at 65°C with shaking for a few months to prepare the AT seed sludge. AT sludge was prepared by incubation of the AT seed sludge (5%, v/v) and sewage sludge (5%, w/w) at 65°C with shaking. The addition of this AT sludge (1.2% ± 0.5% of total volatile solids) to methanogenic sludge enhanced the production of biogas. The optimum volume of the addition and the pretreatment temperature of the AT sludge for optimum biogas production were 5% (v/v) and 65°C. Batch-fed anaerobic digestion was covered with the addition of various AT sludges. The AT sludge prepared with the AT seed sludge improved the biogas production by 2.2 times relative to that from the sewage sludge addition. The addition of sludge without AT seed sludge weakly enhanced biogas production. An aerobic thermophilic bacterium (strain AT1) was isolated from the AT seed sludge. Strain AT1 grew well in a synthetic medium. The production of biogas from the anaerobic digestion of sewage sludge was improved by the addition of 5% (v/v) AT1 bacterial culture compared with that from the sewage sludge addition. The addition of AT1 culture reduced the volatile solids by 21%, which was higher than the 12.6% achieved with the sewage sludge addition. The AT1 bacterial culture enhanced the biogas production more than the AT seed sludge. The phylogenetic analysis of the 16S rRNA gene revealed that strain AT1 is closely related to Geobacillus thermodenitrificans (100% sequence similarity). The improvement in the production of biogas with the AT sludge could be caused by thermophilic bacterial activity in the AT sludge.     

Detection of a Toxic Product Released by a Polyurethane-Containing Film Using a Composting Test Method Based on a Mineral Bed

The degradation of a film containing a 4,4diphenyl methane diisocyanate (MDI) poly(€-caprolactone)-based polyurethane was followed in a test system based on a mineral solid bed designed to facilitate analysis of break-down products released under composting conditions. The use of a mineral solid bed can help extraction and analytical procedures which could be hindered by the heterogeneous nature of compost. The fermentation conditions are typical of the composting environment and generate a powerfully degradative environment. The film fully disintegrated within 30 days of treatment. Analysis on the mineral bed extracts showed that: (i) about 40% of the initial polyurethane was still present in the bed extracts; (ii) this residue was strongly degraded in the poly(€-caprolactone) part, while the urethane part was almost completely recovered (from 80 to 95%, according to the measurement method); (iii) 4,4 diamino diphenyl methane (MDA), a very dangerous product of MDI, was released during biodegradation. The results indicate that a mineral bed can be employed to study degradation and metabolites formation in solid phase fermentation and that the MDI-based polyurethanes are not susceptible of a full degradation during composting and maintain the potential of a slow release of MDA into the environment after soil application.