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.     

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.     

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.     

Characterization and enzymatic degradation of a cross-linked bacterial polyester

The bacterial polyester, poly(-hydroxybutyrate-co--hydroxyvalerate) (PHB/V), was cross-linked with 1, 5, 7, 10, 20, and 30 wt% benzoyl peroxide by thermal decomposition reactions. Solvent extractions were carried out to determine the cross-linked fractions of the films. The sol/gel data were used to estimate cross-link densities. Films of PHB/V cross-linked with 10% benzoyl peroxide were placed in contact with purified depolymerase A secreted byP. lemoignei. These samples exhibited weight loss rates which were half that of un-cross-linked PHB/V, but the network was degraded completely by the enzyme. The results of this study suggest that anendo-type enzymatic degradation may occur, in addition to theexo-type activity, which is normally presumed to occur with theP. lemoignei depolymerase system.     

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.     

Assessing the biodegradation potential of polymers in screening- and long-term test systems

This paper presents a test scheme for assessing the biodegradation potential of polymers, starting with aquatic screening systems (aerobic and anaerobic) and continuing to long-term systems. At the end of the scheme the material has to prove its behavior under the relevant disposal conditions. Aerobic screening was performed mainly under aquatic conditions, but also in soil, using BOD-respirometry. Carbon balances were performed to obtain a better evaluation of the biodegradation potential. Under anaerobic conditions, biodegradation in an aquatic medium was followed by measuring CH₄ and CO₂ production. Polymers not fully degraded in the screening systems were tested in aquarium systems for at least 1 year. Biodegradation was followed by monitoring the DOC released in the water, mass loss, and microbial growth on the samples and in the water as well as via FTIR spectroscopy and SEM pictures. Results are presented for the polymers PHB, PHBV, PCL, Mater-Bi AI05H and ZF03U, and Bioceta. By combining the data from the screening with the aquarium system, a good picture of the degradation behavior of the polymers is obtained.Paper presented at the Bio/Environmentally Degradable Polymer Society—Third National Meeting, June 6–8, 1994, Boston, Massachusetts.     

Kinetics of Aerobic Polymer Degradation in Soil by Means of the ASTM D 5988-96 Standard Method

The standard test method ASTM D 5988-96 for determining the degree and rate of aerobic biodegradation of plastic materials in contact with soil was applied to poly(3-hydroxybutyrate) and poly(-caprolactone). The method proved to be reliable and supplied reproducible measurements of CO₂ production, provided potassium (instead of barium) hydroxide was used as a trapping solution. The trends of CO₂ evolution, as a function of time, observed for the degradation of polymer powders in soil are similar to those predicted by simple first order kinetics in solution. The experimental data are described by a Michaelis–Menten type model, which accounts for the heterogeneity of the polymer-soil system. The kinetic equation deduced predicts the degradation rate to the proportional to the exposed polymer surface area.     

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.     

Physical properties and biodegradability of blends containing poly(ε-caprolactone) and tropical starches

In order to assess feasibility of tropical starches (sago and cassava starches) as biodegradable plastic materials, blending with poly(-caprolactone) (PCL), a biodegradable polymer, was carried out. It was confirmed that the physical properties (tensile strength and elongation) of PCL/sago and PCL/cassava blends were similar to those of PCL/corn blend, suggesting that sago and cassava starches can also be blended with PCL for production of biodegradable plastic. However, the properties of all PCL/starch blends were still low compared with those of polyethylene. Enzymatic degradability evaluation showed that lipase degradation of PCL and-amylase degradation of starch increased as the starch content in the blend increased. Burial test of the blends for 1, 3, and 5 months was carried out and the rate of degradation of the PCL/sago blend was confirmed to be slower than those of PCL/corn and PCL/cassava blends. Observation of the film blends structure by scanning electron microscope revealed that the starch was dispersed in a PCL continuous phase. Furthermore, changes in the film surface before and after enyzme treatments were observed.     

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.     

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.     

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.     

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.     

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.     

Synthesis of copolyesters containing poly(ethylene terephthalate) and poly(ε-caprolactone) units and their susceptibility toPseudomonas sp. lipase

Copolyesters containing poly(ethylene terephthalate) (PET) and poly(-caprolactone) (PCL) were synthesized from PET and PCL homopolymers by transesterification reaction at 270°C in the presence of catalyst. The copolyesters were characterized by¹³C-NMR and differential scanning calorimetry (DSC). The degradation behavior of PCL byPseudomonas sp. lipase in buffer solution (pH 7) and tetrahydrofuran (THF) was investigated by gel permeation chromatography (GPC) and¹H-NMR. From these experiments, it was found thatPseudomonas sp. lipase acted endoenzymatically on PCL. Using this lipase, degradation tests for PET/PCL copolyesters whose PCL content was below 50% by weight were also performed in buffer solution (pH 7). However, evenPseudomonas sp. lipase with high degradation activity on PCL did not easily degrade the PCL unit in PET/PCL copolyesters.     

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.     

Degradation of L- and DL-lactic acid oligomers in the presence ofFusarium moniliforme andPseudomonas putida

Poly(-alkanoates) derived from lactic acid enantiomers are known to degrade easily hydrolytically in aqueous media. The ability of two microorganisms, a filamentous fungus,Fusarium moniliforme, and a bacterium,Pseudomonas putida, to assimilate the degradation by-products of poly(lactic acid) (PLA), namely, lactic acid, lactyllactic acid dimers, and higher oligomers, was investigated in liquid culture. To distinguish the influence of chirality on bioassimilation, two series of substrates were considered which derived from the racemic and the L-form of lactic acid, respectively. The fate of these compounds was monitored by HPLC. Under the selected conditions,DL- andL-lactic acids were totally used by the two microorganisms regardless of the enantiomeric composition. Both microorganisms degraded the LL-dimer rather rapidly. However,F. moniliforme acted more rapidly thanP. putida. It is likely that the DD-dimer also biodegraded but at a slower rate, especially in the case of the fungi. Higher racemic oligomers were slowly assimilated by the two microorganisms, whereas higher L-oligomers appeared biostable probably because of their crystallinity. A synergistic effect was observed when both microorganisms were present in the same culture medium containing racemic oligomers.Presented at the 4th International Workshop on Biodegradable Plastics and Polymers, October 11–14, 1995. Durham, New Hampshire.     

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.     

Biodegradation of Electrospun Poly-(ɛ-caprolactone) Non-woven Fabrics by Pure-Cultured Soil Filamentous Fungi

The biodegradation of electrospun nano-fibers of poly(-caprolactone) (PCL) was initially investigated with respect to the environmental application of PCL non-woven fabrics, using pure-cultured soil filamentous fungi, Aspergillus oryzae, Penicillium caseicolum, P. citrinum, Mucor sp., Rhizopus sp., Curvularia sp., and Cladosporium sp. Three kinds of non-woven PCL fabrics with different mean fiber diameters (330, 360, and 510 nm) were prepared by changing the viscosities of the pre-spun PCL solutions (150, 210, and 310 cPs, respectively). All of the pure-line soil filamentous fungi tested grew on the two fiber materials. Electron microscopy was used to observe the biodegradation processes revealing remarkable growth of two fungi, Rhizopus sp. and Mucor sp., along with the accompanying collapse of the nano-fiber matrices. In the biochemical oxygen demand (BOD) test, the biodegradation of the 330 nm PCL nano-fibers by Rhizopus sp. and Mucor sp. exceeded 20 and 30% carbon dioxide generation, respectively. The biodegradability of the PCL non-woven fabrics decreased with the mean fiber diameter and the 330 nm PCL nano-fiber that was made from 150 cPs solution (concentration, 7 wt%) exhibited the highest biodegradability. These results might offer some clues for the applications of the PCL non-woven fabrics having the controlled biodegradability in the environmental uses.     

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.