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.     

Abiotic aging of water- soluble 3- hydroxybutyric acid oligomers as monitored by capillary zone electrophoresis

Oligomers of poly(3-hydroxybutyric acid) (P(3-HB)) were prepared by partial degradation of high molecular weight P(3-HB) dissolved in 1,2-dichloroethane/water mixture in the presence of p-toluene sulfonic acid (p-TSA). The water-soluble fraction of the resulting oligomers was extracted from the mixture with neutral sodium phosphate buffer. Capillary zone electrophoresis showed that the aqueous extracts were composed of two series of oligomers. The first one was composed of one to seven P(3-HB) oligomers (O(3-HB)). In contrast, the second series was composed of four oligomers characterized by the presence of a terminal C=C bond [O’(3-HB)]. Both series of oligomers behaved differently insofar as their fate in aqueous medium was concerned. The 0(3-HB) compounds were stable over a period of 2 months. On the other hand, the population of the O’(3-HB) oligomers varied, the proportion of oligomers increasing with aging time.     

Earthworms and the Degradation of Lactic Acid–Based Stereocopolymers

Two lactic acid–based stereocopolymers, namely 50/50 and 96/4 L/D poly(l-lactic-co-d-lactic acids) and corresponding oligomers, were allowed to age under different conditions in order to investigate their toxicity and that of some potential degradation by-products, namely lactic acid and sodium and calcium lactates, to earthworms. Degradation characteristics in various worm-free and worm-containing media were also investigated under various conditions including direct feeding using impregnated paper or coated tree-leaves, model composting, and vermi composting. Data were compared with abiotic degradation in sterile neutral phosphate buffer. Last but not least, a novel method aimed at assessing the bioassimilation of degradable polymers and oligomers was utilized, which is based on the monitoring of weight changes of a population of starved worms when the worms are given the polymeric or oligomeric compounds as potential nutrients. The work shows that high molar mass poly(lactic acids) can be ingested by earthworms provided they are disintegrated first. However, they cannot be bioassimilated before hydrolytic degradation generates oligomers. The involvement of microorganisms in the bioassimilation is discussed.     

Crystal and Thermal Properties of PLLA/PDLA Blends Synthesized by Direct Melt Polycondensation

We herein report the effects of the component ratio and method of blending on the synthesis of stereocomplex poly(lactic acid) (SC-PLA) based on poly(l-lactic acid) (PLLA) and poly(d-lactic acid) (PDLA) prepolymers. PLLA and PDLA were prepared by direct melt polycondensation of lactic acid (DMP). Combined with the dual catalyst system, PLA prepolymers with M_w more than 20,000 were prepared by DMP. PLLA was mixed by powder blending or melt blended with PDLA. It is revealed that melt-point and spherulite growth rate of SC-PLA is strongly dependent on the perfection of SC structure. The melt point of PLA can be increased by nearly 50 °C because of the particular strong intermolecular interaction between PLLA and PDLA chains. Solid-state polycondensation (SSP) is an efficient method to increase the molecular weight of SC-PLA, but it can have a negative effect on the regularity of linear chains of SC-PLA. Thermogravimetry analyzer (TGA) results show that SC structure cannot cause the delay reaction on the thermal degradation of PLA.     

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases

Five extracellular PHB depolymerases of bacteria isolated from various sources were purified to electrophoretic homogeneity and compared with known extracellular PHB depolymerase fromAlcaligenes faecalis T1. The molecular mass of these enzymes were all around 40–50 kDa. Nonionic detergent, diisopropylfluorophosphate and dithiothreitol inhibited the PHB depolymerase activity of all these enzymes. Trypsin abolished PHB depolymerase activity, but not theD-3-hydroxybutyric acid dimer hydrolase activity of all the enzymes. These results showed that the basic properties of these PHB depolymerases resemble those of theA. faecalis T1 enzyme. Analysis ofN-terminal amino acid sequence of the purified enzymes revealed that these enzymes includingA. faecalis T1 enzyme fall into three groups.     

Characterization of a poly(3-hydroxybutyrate) depolymerase fromaureobacterium saperdae: Active site and kinetics of hydrolysis studies

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase was purified fromAureobacterium saperdae cultural medium by using hydrophobic interaction chromatography. The isolated enzyme was composed of a single polypeptide chain with a molecular mass of 42.7 kDa as determined by SDS-PAGE and by native gel filtration on TSK-HW-55S. The enzyme was not a glycoprotein. Its optimum activity occurred at pH 8.0 and it showed a broad pH stability, ranging from pH 3 to pH 11.N-Bromosuccinamide and 2-hydroxy-5-nitrobenzyl bromide completely inactivated the enzyme, suggesting the involvement of tryptophan residues at the active site of the protein. The enzyme was very sensitive to diisopropyl fluorophosphate and diazo-dl-norleucine methyl ester, showing the importance of serine and carboxyl groups. The modification of cysteine residues byp-hydroxy mercuricbenzoate did not cause a loss of activity, whereas dithiothreitol rapidly inactivated the enzyme, revealing the presence of disulfide bonds.A saperdae depolymerase acted on the surface layer of PHB films and the degradation proceeded by surface erosion releasing monomers and dimers of 3-hydroxybutric acid. The degradation of PHB films byA. saperdae depolymerase was partially inhibited in the presence of excess amounts of enzyme. This phenomenon, already observed by Mukaiet al. with poly(hydroxyalkanoates) depolymerases fromAlcaligenes faecalis, Pseudomonas pickettii, andComamonas testosteroni, was analyzed according to the kinetic model proposed by these authors. The experimental data evidenced a general agreement with the kinetic model, although higher initial degradation rates were found withA. saperdae depolymerase.     

Application of recombinant gene technology for production of polyhydroxyalkanoic acids: Biosynthesis of poly(4-hydroxybutyric acid) homopolyester

Screening of a large number of bacteria revealed several strains, which utilize 1,4-butanediol and/or 4-hydroxybutyric acid (4HB) as a carbon source for growth and for synthesis of polyhydroxyalkanoic acids (PHA) containing 4HB as one constituent among others (mostly 3-hydroxybutyric acid). However, none of the wild-type strains investigated in this study was able to produce a homopolyester consisting solely of 4HB. Only several poly(3-hydroxybutyric acid)-leaky mutants ofAlcaligenes eutrophus strain JMP222 synthesized poly(4HB) homopolyester, which amounted to approximately 10% (w/w) of the cellular dry matter. If the PHA synthase structural gene ofA. eutrophus strain H16 was expressed in these mutants, the amount of poly(4HB) was increased to approximately 30% (w/w). The occurrence of poly(4HB) was demonstrated by gas chromatographic as well as¹H and¹³C nuclear magnetic resonance spectroscopic analysis.Paper presented at the Bio/Environmentally Degradable Polymer Society—Second National Meeting, August 19–21, 1993, Chicago, Illinois.     

Abiotic Hydrolysis of Some Poly-D-glucaramides and Subsequent Microbial Utilization/Degradation

Carbohydrate acid amides, diamides and polyamides have been proposed to be utilized as nitrogen plant fertilizers or fertilizer components, and experiments with Brassica rapa demonstrated a positive biological response when these compounds were used as the only source of fixed nitrogen for plant growth. The present study was carried out with the aim of elucidating the mechanism of degradation of these polymers in both soil/compost and in liquid media and the role of microorganisms in this process. The results obtained suggest that a major route of degradation of polyglucaramides in the environment is their abiotic hydrolysis/release of the diacid and diamine building block units of these polymers, which are then utilized for growth by microorganisms. In cell-free crude extracts from enrichment cultures obtained with different poly-D-glucaramides, no enzyme activities catalyzing the release of diamines from these compounds were detected.     

Effects of Electron-Beam Irradiation and Ultraviolet Light (365 nm) on Polylactic Acid Plastic Films

Strips of Ca-I [polylactic acid (PLA) monolayer plastic films from Cargill Dow Polymers LLC, Minnetonka, MN] cut in the machine and nonmachine directions were irradiated with an electron beam using a CIRCLE III Linear Accelerator (MeV Industries S.A., Jouy-en-Josas, Cedex, France). The effects of 33-kGy irradiation on the physical properties of the Ca-I strips were studied. In addition, the effects of ultraviolet (UV) light (365-nm) illumination on the degradation of three PLA plastic films, Ch-I (PLA monolayer plastic films from Chronopol, Golden, CO), GII (PLA trilayer plastic films from Cargill Dow Polymers LLC), MN), and Ca-I (PLA monolayer plastic films from Cargill Dow Polymers LLC) were analyzed by a modified ASTM D5208-91 method. Results showed that irradiation had decreased the weight-average molecular weight (M _w), stress at break, percentage of elongation, and strain energy of Ca-I by 35.5, 26.7, 32.3, and 44.8%, respectively (P < 0.01). The shelf life of the irradiated Ca-I strips at 5°C and <20 ± 5% RH was about 6 months. The degradation rate of Ch-I, GII, and Ca-I with no UV light treatment at 55°C and 10% RH was 2512, 5618, and 3785 M _w/week, respectively. Under the UV light illumination (365 nm), the degradation rate of Ch-I, GII, and Ca-I, was 2982, 8722, and 7467 M _w/week, respectively. Hence, the degradation rate of GII and Ca-I was increased 55 and 97% by UV light (P < 0.008), respectively. This trend was not observed in Ch-I because its starting M _w (78,000 g/mol) was close to the tensile strength lost range (50,000 to 75,000 g/mol) of PLA films. To our knowledge, this is the first study to demonstrate that UV light does further enhance the degradation of PLA films.     

Effects of Temperature and Relative Humidity on Polylactic Acid Plastic Degradation

Three high molecular weight (120,000 to 200,000 g mol^–1) polylactic acid (PLA) plastic films from Chronopol (Ch-I) and Cargill Dow Polymers (GII and Ca-I) were analyzed for their degradation under various temperature and relative humidity (RH) conditions. Two sets of plastic films, each containing 11 samples, were randomly hung in a temperature/humidity-controlled chamber by means of plastic-coated paper clips. The tested conditions were 28, 40, and 55°C at 50 and 100% RH, respectively, and 55°C at 10% RH. The three tested PLA films started to lose their tensile properties when their weight-average molecular weight (M _w) was in the range of 50,000 to 75,000 g mol^–1. The average degradation rate of Ch-I, GII, and Ca-I was 28,931, 27,361, and 63,025 M _w/week, respectively. Hence, GII had a faster degradation rate than Ch-I and Ca-I under all tested conditions. The degradation rate of PLA plastics was enhanced by the increase in temperature and relative humidity. This trend was observed in all three PLA plastics (Ca-I, GII, and Ch-I). Of the three tested films, Ch-I was the first to lose its mechanical properties, whereas Ca-I demonstrated the slowest loss, with mechanical properties under all tested conditions.     

Degradation of Polylactic Acid (PLA) Plastic in Costa Rican Soil and Iowa State University Compost Rows

In this study the degradation of polylactic acid (PLA) plastic films in Costa Rican soil and in a leaf composting environment was investigated. Three types of PLA films were used: Ch-I, (PLA monolayer plastic films from Chronopol, Golden, CO), GII (PLA trilayer plastic films from Cargill Dow Polymers LLC, Minnetonka, MN), and Ca-I (PLA monolayer plastic films from Cargill Dow Polymers LLC). The average soil temperature and moisture content in Costa Rica were 27°C and 80%, respectively. The average degradation rate of PLA plastic films in the soil of the banana field was 7675 M _w/week. Two compost rows were set up at the Iowa State University (ISU) (Ames) compost site. Temperature and relative humidity of the compost rows were kept at 55 ± 5°C and 50 ± 10% RH, respectively. The degradation rates of GII and Ca-I in the compost rows were 113,290 and 71,283 M _w/week, respectively. Therefore, it was estimated that in Costa Rican soil and in compost rows, PLA would be visibly degraded in 6 months and in 3 weeks, respectively.     

Gene Cloning and Characterization of a Poly(<Emphasis Type="SmallCaps">l</Emphasis>-Lactic Acid) Depolymerase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Strain DS04-T

A gene encoding a poly(l-lactic acid) (PLA) depolymerase from Pseudomonas sp. strain DS04-T was cloned and overexpressed in Escherichia coli. The recombinant PLA depolymerase with a molecular weight of 19.2 kDa was purified to homogeneity. The optimum pH and temperature of the PLA depolymerase are 8.5 and 60 °C, respectively. K⁺, Ca²⁺ and Ni²⁺ enhance the enzyme activity, while Na⁺, Zn²⁺, Mg²⁺, Cu²⁺, Fe²⁺, Mn²⁺ and Co²⁺ inhibit it. The inhibition of different chemicals on the PLA depolymerase activity were examined, in which EDTA was found to have a significantly inhibitory effect. The main degradation product of the depolymerase is identified as lactic acid monomer by mass spectrometric analysis. Physicochemical properties, substrate specificity and sequence analysis indicated that PME is a new type of PLA depolymerase.     

Degradation of Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Some Soil Aspergillus spp.

Systematic screening of 45 soil fungi for degradation polyhydroxyalkanoic acids (PHAs) has led to the selection of 6 potent Aspergillus isolates belonging to A. flavus, A. oryzae, A. parasiticus, and A. racemosus. Degradation of PHAs as determined by tube assay method revealed that these Aspergillus spp. were more efficient in degrading poly(3-hydroxybutyrate) [P(3HB)] compared to copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (P3HB-co-16% 3HV). Moreover, the extent of degradation in mineral base medium was much better than those in complex organic medium. For all the Aspergillus spp. tested, maximum degradation was recorded at a temperature of 37°C with significant inhibition of growth. The optimum pH range for degradation was 6.5–7.0 with degradation being maximum at pH 6.8. The extent of polymer degradation increased with increase in substrate concentration, the optimum concentration for most of the cultures being 0.4% and 0.2% (w/v) for P(3HB) and P(3HB-co-16%3HV) respectively. Supplementation of the degradation medium with additional carbon sources exerted significant inhibitory effect on both P(3HB) and P(3HB-co-16%3HV) degradation.     

Degradation of Poly(acrylic acid)s with Phenolic Side-Chains by Manganese Peroxidase from White Rot Fungi

Poly(acrylic acid)s (PAAs) with various functional groups, such as phenolic hydroxyl, amino, and aldehyde groups, in the side-chains were treated with manganese peroxidase (MnP) prepared from the culture of lignin-degrading white rot fungi. While no change in the M_w of PAA without a functional group was observed after a 24-h MnP treatment, the M_w␣of␣PAA␣with p-aminophenol as side-chains decreased from 90,000 to 59,000, and that with␣o-aminophenol from 70,000 to 26,000. MnP treatment also decreased the M_w of PAA with a p-aminoaniline or aldehyde group. Furthermore, the MnP treatment generated a significant depolymerization of the cross-linked PAA with p-aminophenol from an insoluble polymer to water soluble products. These results suggest that functional groups generating radicals can act as elemental devices and induce degradation of the PAA main chain.     

Biodegradation of acrylic acid polymers and oligomers by mixed microbial communities in activated sludge

The biodegradability (mineralization to carbon dioxide) of acrylic acid oligomers and polymers was studied in activated sludge obtained from continuous-flow activated sludge (CAS) systems exposed to mixtures of low molecular weight (Mw < 8000) poly(acrylic acid)s and other watesoluble polymers [poly(ethylene glycol)s] in influent wastewater. Dilute preparations of activated sludge from the CAS units were tested for their ability to mineralize acrylic acid monomer and dimer, as well as a series of model acrylic acid oligomers and polymers (Mw 500, 700, 1000, 2000, and 4500), as sole carbon and energy sources. Complete mineralization of acrylic acid monomer and dimer was observed in low-biomass sludge preparations previously exposed to the polymer mixture, based on carbon dioxide production and residual dissolved organic carbon analyses. Extensive (though incomplete) degradation was also observed for the low molecular weight acrylic acid oligomers (Mw 500 and 700), but degradation dropped off sharply for the 1000, 2000, and 4500 Mw polymers. Radiochemical (¹⁴C) data also confirmed the low degradation potential of the 1000, 2000, and 4500 Mw materials. Degradation of two commercial poly(ethylene glycol)s at 1000 and 3400 Mw was complete and comparable to that of the acrylic acid monomer and dimer. Our results indicate that mixed populations of activated sludge microorganisms can extensively metabolize acrylic acid oligomers of seven units or less. Complete mineralization, however, could be confirmed only for the monomer and dimer material, and carbon mass balance data suggested that the true molecular weight cutoff for complete biodegradation was significantly less than the 500–700 Mw range tested.     

Fungal Degradation of the Bioplastic PHB (Poly-3-hydroxy- butyric acid)

PHB (poly-3-hydroxybutyric acid) is a thermoplastic polyester synthesized by Ralstonia eutropha and other bacteria as a form of intracellular carbon and energy storage and accumulated as inclusions in the cytoplasm of these bacteria. The degradation of PHB by fungi from samples collected from various environments was studied. PHB depolymerization was tested in vials containing a PHB-containing medium which were inoculated with isolates from the samples. The degradation activity was detected by the formation of a clear zone below and around the fungal colony. In total, 105 fungi were isolated from 15 natural habitats and 8 lichens, among which 41 strains showed PHB degradation. Most of these were deuteromycetes (fungi imperfecti) resembling species of Penicillium and Aspergillus and were isolated mostly from soils, compost, hay, and lichens. Soil-containing environments were the habitats from which the largest number of fungal PHB degraders were found. Other organisms involved in PHB degradation were observed. A total number of 31 bacterial strains out of 67 isolates showed clear zones on assay medium. Protozoa, possible PHB degraders, were also found in several samples such as pond, soil, hay, horse dung, and lichen. Lichen, a fungi and algae symbiosis, was an unexpected sample from which fungal and bacterial PHB degraders were isolated.     

Construction, Characterization and Biological Activity of Chiral and Thermally Stable Nanostructured Poly(Ester-Imide)s as Tyrosine-Containing Pseudo-Poly(Amino Acid)s

In this paper we studied the synthesis of biodegradable optically active poly(ester-imide)s containing different amino acid residues in the main chain. These pseudo-poly(amino acid)s were synthesized by polycondensation of N,N′-(pyromellitoyl)-bis-l-tyrosine dimethyl ester as a diphenolic monomer and two chiral trimellitic anhydride-derived diacid monomers containing s-valine and l-methionine. The direct polycondensation reaction of these diacids with aromatic diol was carried out in a system of tosyl chloride (TsCl), pyridine (Py) and N,N′-dimethylformamide (DMF) as a condensing agent. The structures and morphology of these polymers were studied by FT-IR, ¹H-NMR, powder X-ray diffraction, field emission scanning electron microscopy (FE-SEM), specific rotation, elemental and thermogravimetric analysis (TGA) techniques. TGA profiles indicate that the resulting PEIs have a good thermal stability. Morphology probes showed these polymers were noncrystalline and nanostructured polymers. The monomers and prepared polymers were buried under the soil to study the sensitivity of the monomers and the obtained polymers to microbial degradation. The high microbial population and prominent dehydrogenase activity in the soil containing polymers showed that the synthesized polymers are biologically active and microbiologically biodegradable. Wheat seedling growth in the soil buried with synthetic polymers not only confirmed non-toxicity of polymers but also showed possibility of phyto-remediation in polymer-contaminated soils.     

The Effect of Nanoclays on the Properties of PLLA-modified Polymers Part 1: Mechanical and Thermal Properties

Organically modified montmorillonite clays were incorporated at a 5% loading level into film grade of poly-L-lactic acid (PLLA) using a variety of masterbatches based on either semi-crystalline or amorphous poly-(lactic acid), as well as biodegradable aromatic aliphatic polyester. The PLLA masterbatches and compounded formulations were prepared using a twin screw compounding extruder, while the films were prepared using a single screw cast film extruder. The thermal and mechanical properties of the films were examined in order to determine the effect of the clay and different carriers on the polymer–clay interactions. In the optimal case, when a PLLA-based masterbatch was used, the tensile modulus increased by 30%, elongation increased by 40%, and the cold crystallization temperature decreased by 15 °C, compared to neat PLLA. The properties improvement of PLLA films containing nano clays demonstrated the possibility to extend the range of biodegradable film applications, especially in the field of packaging.     

Synthesis and Characterization of Lactic Acid Oligomers: Evaluation of Performance as Poly(Lactic Acid) Plasticizers

The use of fully bio-based and biodegradable materials for massive applications, such as food packaging, is an emerging tendency in polymer research. But the formulations proposed in this way should preserve or even increase the functional properties of conventional polymers, such as transparency, homogeneity, mechanical properties and low migration of their components to foodstuff. This is not always trivial, in particular when brittle biopolymers, such as poly(lactic acid) (PLA), are considered. In this work the formulation of innovative materials based on PLA modified with highly compatible plasticizers, i.e. oligomers of lactic acid (OLAs) is proposed. Three different synthesis conditions for OLAs were tested and the resulting additives were further blended with commercial PLA obtaining transparent and ductile materials, able for films manufacturing. These materials were tested in their structural, thermal and tensile properties and the best formulation among the three materials was selected. OLA with molar mass (M_n) around 1,000 Da is proposed as an innovative and fully compatible and biodegradable plasticizer for PLA, able to replace conventional plasticizers (phthalates, adipates or citrates) currently used for films manufacturing in food packaging applications.     

Degradation of Post-consumer PLA: Hydrolysis of Polymeric Matrix and Oligomers Stabilization in Aqueous Phase

Degradation of post-consumer PLA to lactic acid was analysed in order to assess the economic feasibility of the PLA chemical recycling process. Hydrolysis of PLA, in batch reactor, was analysed in the temperature range of 443–473 K, under autogenous pressure and a constant PLA to water ratio (equal to approximately 0.11 by weight), without the use of a catalyst. The experimental results suggest that the complete degradation of PLA can be obtained using relatively low reaction-times with the production of a mixture containing the monomer and traces of the dimer of lactic acid. The overall process was modelled using a two-step process: bulk degradation of PLA (in the solid or molten phase) with the solubilisation of low molecular weight oligomers, and their subsequent hydrolysis in water (stabilization). The model describes the trend of oligomer concentrations in the aqueous phase and PLA conversion as a function of time with both high accuracy and agreement with experimental results.