Toxicity screening of biodegradable polymers. I. Selection and evaluation of cell culture test methods

A screening test method for potential toxicity of biodegradable plastics on humans and the environment was selected and evaluated with samples of cellulose acetate, Bionolle, polyhydroxybutyrate-co-valerate (Biopol), and polycaprolactone (Tone polymer). Among the standardin vitro tests using animal cell cultures for the evaluation of biomedical materials, the test by direct contact and the test with extract were examined. Qualitative and quantitative determinations of the cell viability and morphology indicate that the test with extracts can be easily performed, providing reproducible and comparable results for all materials. Using the cell culture test with the extract of sterile samples, an estimation of the toxicity of a new polymeric material can be obtained within a few weeks.     

Assessment of the Viability of NIH3T3 Fibroblast Cells Cultured in Polymer Matrices with rhGH

Developing a biomaterial that functions as a scaffold (osteoconductor), combined with a growth factor (osteoinductor), is of great interest for clinical application in oral and maxillofacial surgery. Growth hormone (GH) is a good candidate, as it is a major regulator of postnatal bone growth and remodeling. Pure PLGA and 70/30 PLGA/PCL matrices were prepared by the solvent evaporation method, combined or not with GH, and tested for toxicity and cell viability using an MTT assay with NIH3T3 mouse cells (ATCC). Cell toxicity was assessed at 24, 48, 72 h, and 7 days of biomaterial exposure to culture medium. All polymers had high cell viability rates. However, from 48 h onwards, the groups with GH-polymer combinations had better results than the polymer groups without association with GH when compared to the control group. At 7 days of culture, only the pure PLGA matrix showed a significant difference from the control group. These results may suggest a preference of cells for the presence of GH in the biomaterial in culture medium, especially in the PLGA matrix. GH appeared to contribute to the increase in cell viability observed at some assessment time points, especially when combined with PLGA as compared to pure PLGA.     

Thermal and mechanical properties of poly(lactic Acid) and poly(ethylene/butylene Succinate) blends

In this study, blends of poly (lactic acid) (PLA) with poly(ethylene/butylene succinate) (Bionolle) have been investigated for their thermal and mechanical properties as a function of the concentration of Bionolle. Differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), and tensile tests were used to characterize the blends. From the results of the DMA and DSC, it was found that this blend system was not miscible within the compositions studied. DSC results showed that adding Bionolle aids in crystallization of PLA. It was observed that increasing the Bionolle concentration led to a slight increase in the strain-at-break of the blends but a decrease in the Young’s modulus and ultimate tensile strength. Biaxially oriented films showed an increase in tensile strength, modulus, and strain-at-break.     

Improving the two-step remediation process for CCA-treated wood: Part II. Evaluating bacterial nutrient sources

Remediation processes for recovery and reuse of chromated-copper-arsenate- (CCA) treated wood are not gaining wide acceptance because they are more expensive than landfill disposal. One reason is the high cost of the nutrient medium used to culture the metal-tolerant bacterium, Bacillus licheniformis, which removes 70-100% of the copper, chromium, and arsenic from CCA-treated southern yellow pine (CCA-SYP) in a two-step process involving oxalic acid extraction and bacterial culture. To reduce this cost, the nutrient concentration in the culture medium and the ratio of wood to nutrient medium were optimized. Maximum metal removal occurred when B. licheniformis was cultured in 1.0% nutrient medium and at a wood to nutrient medium ratio of 1:10. Also, malted barley, an abundant by-product of brewing, was evaluated as an alternative nutrient medium. Tests were done to determine absorption of metals by barley, and the results indicate that the barley acted as a biosorbent, removing heavy metals from the liquid culture after their release from CCA to SYP. For comparison, tests were also performed with no nutrient medium. Following bacterial remediation, 17% copper and 15% arsenic were removed from an aqueous slurry of CCA-SYP (no medium). When oxalic acid extraction preceded the aqueous bacterial culture, 21% copper, 54% chromium, and 63% arsenic were removed. The two-step process (oxalic acid extraction and bacterial culture with nutrient medium) appears to be an effective, yet costly, way to remove metals.     

Water vapor permeability of biodegradable polymers

The water vapor transmission rates (WVTR) of several biodegradable polymers were evaluated to determine their suitability as water-resistant coatings and to understand WVTR better in terms of polymer structure. Values of WVTR at 25‡C ranged from 13 to 2900 g/m² /day and increased in the order PHBV PLA (cryst.) PLA (amorph.) PCL Bionolle BAK 1095 CAP CA. Values of WVTR were positively correlated with higher polymer solubility parameters, lower crystallinities, and higher free volumes. Although the WVTR of biodegradable polymers are much higher than those of good barrier materials such as low-density polyethylene, they are sufficient for short-term (hours to days) protection of polysaccharide-based materials against water. Product names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Photodegradation and Biodegradation Study of a Starch and Poly(Lactic Acid) Coextruded Material

To simulate the behavior of agricultural mulch coextruded poly(lactic acid)(PLA)/starch films, two stages were carried out. The first was an ultraviolet treatment (UV) at 315 nm, during which glass transition temperature T_g, weight, and molecular weight (MW) decreased and a separation between PLA and starch phase was observed. For the second stage, the mineralization of the carbon of the material was followed using the ASTM (D 5209–92 and 5338–92) and ISO/CEN (14852 and 14855) standard procedures. To measure the biodegradability of polymer material, the assessment of the carbon balance allowed determination of the distribution between the carbon rate used to the biomass synthesis or the respiration process (released CO₂), as well as the dissolved organic carbon into the culture medium and the carbon in the residual insoluble material. The influence of the nature of the medium and the standardized procedures on the final rate of biodegradation was investigated. Whatever the standardized method, the biodegradation percentage was significantly stronger in liquid medium (92.4–93.4) than on inert medium (80–83%). In the case of the compost process, only released CO₂ was measured and corresponded to 79.1–80.3%.     

Effect of microbial activity on the mobility of chromium in soils

The effect of microbial activity on the chemical state of chromium, in a contaminated soil located in the Rh?ne-Alpes region (France), has been investigated. This soil contained 4,700 mg kg(-1) Cr, with about 40% present in the soluble hexavalent form. Indigenous microbial activity was found to significantly reduce Cr(VI) to the less mobile form (III) when the soil was incubated at 30 degrees C in an aqueous medium containing glucose and nutrients. A Cr(VI)-reducing strain of Streptomyces thermocarboxydus was isolated from the contaminated soil. The strain was found to metabolize Cr(VI) in a similar manner as an exogenous inoculum of Pseudomonas fluorescens LB300, and to precipitate chromium as a Cr oxyhydroxide with a gammaCrOOH-like local structure. The Cr(VI)-reducing activity of S. thermocarboxydus was induced, or significantly accelerated, by the aggregation of bacterial cells or their adhesion to suspended solid particles, and was stimulated in pure culture by glycerol and chromate.     

Kinetics of microbial landfill methane oxidation in biofilters

A methane oxidizing biofilter system fitted to the passive venting system of a harbor sludge landfill in Germany was characterized with respect to the the methanotrophic population, methane oxidizing capacity, and reaction kinetics. Methanotrophic cell counts stabilized on a high level with 1.3 x 10(8) to 7.1 x 10(9) cells g dw(-1) about one year after first biofilter operation, and a maximum of 1.2 x 10(11) cells g dw(-1). Potential methane oxidizing activity varied between 5.3 and 10.7 microg h(-1) g dw(-1). Cell numbers correlated well with methane oxidation activities. Extrapolation of potential activities gave methane removal rates between 35 and 109 g CH4 h(-1) m(-3), calculated for 30 degrees C. Optimum temperature was 38 degrees C for freshly sampled biofilter material and 22 degrees C for a methanotrophic enrichment culture grown at 10 degrees C incubation temperature. Substrate kinetics revealed the presence of a low-affinity methane oxidizing community with a high Vmax of 1.78 micromol CH4 h(-1) g ww(-1) and a high K(M) of 15.1 microM. K(MO2) for methane oxidation was 58 microM. No substantial methane oxidizing activity was detected below 1.7-2.6 vol.-% O2 in the gaseous phase. Methane deprivation led to a decrease in methane oxidation activity within 5-9 weeks but could still be detected after 25 weeks of substrate deprivation and was fully restored within 3 weeks of continuous methane supply. Very high salt loads are leached from the novel biofilter material, expanded clay, yielding electric conductivity values of up to 15 mS cm(-1) in the leachate. Values > 6 mS cm(-1) were shown to depress methane consumption. Water retention characteristics of the material proved to be favourable for methane oxidizing systems with a gas permeable volume of 78% of bulk volume at field capacity water content. Correspondingly, no influence of water content on methane oxidation activity could be detected at water contents between 2.5 and 20 vol.-%.     

Biodegradation of Poly(vinyl alcohol) and Bacterial Cellulose Composites by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The ability of fungal strains to attack a composite material obtained from poly(vinyl alcohol) (PVA) and bacterial cellulose (BC) is investigated. The fungal strain tested was Aspergillus niger. This fungal strain was able to change not only the polymer surface from smoother to rougher, but also to disrupt the polymer. The degradation results were confirmed by visual observations, scanning electron microscopy (SEM) analyses, X-ray diffraction analyses and FTIR spectra of the film samples. SEM micrographs confirmed the growth of fungi on the composite film surface. The degree of microbial degradation depends on culture medium and on composition of polymeric materials, especially on PVA content. The biodegradation process is accelerated by the presence of glucose in the culture medium as an easily available carbon source.     

Mechanism and Characterization of Polyamide 4 Degradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

We identified a biodegrading microorganism of polyamide (nylon) 4, a linear polymer of γ-aminobutyric acid (GABA). From activated sludge, the biodegrading bacteria strains of Pseudomonas sp. were isolated and identified by their taxonomic characteristics and nucleotide sequences of 16S rDNA. One strain, ND-11, was grown on a minimal medium containing polyamide 4 (PA4) as the sole carbon source. The strain produced GABA as a degradation intermediate, as identified by analyzing the NMR spectra of degraded products. The culture supernatant of strain ND-11 degraded the emulsified PA4 completely within one day. These results suggest that the ND-11 strain degraded PA4 using its extracellular enzymes to hydrolyze amide bonds.     

Evaluation of Cyto- and Genotoxicity of Poly(lactide-co-glycolide) Nanoparticles

This work reports on an analysis of the cyto- and genotoxicity of poly(lactide-co-glycolide) polymer nanoparticles, in an attempt to evaluate their mutagenic effects. Fibroblast (3T3) and human lymphocyte cell cultures were exposed to solutions containing three different concentrations of nanoparticles (5.4, 54 and 540 μg/mL, polymer mass/volume of solution). The nanoparticles were characterized in terms of their hydrodynamic diameters, zeta potentials and polydispersity indices. The morphology of the particles was determined by atomic force microscopy. The PLGA nanospheres presented a size of 95 nm, a zeta potential of −20 mV and a spherical morphology. Cellular viability assays using fibroblast cells showed no significant alterations compared with the negative control. A cytogenetic analysis of human lymphocyte cells showed no significant changes in the mitotic index in relation to the control, indicating that in the concentration range tested, the particles used in the experimental models did not present cyto- or genotoxicity. For the tests conducted in this work we can conclude that biodegradable and biocompatible PLGA nanospheres are not toxic in the cell cultures tested (fibroblast and lymphocyte cells) and in the range of concentrations employed. The results provide new information concerning the toxic effects of particles produced using PLGA.     

Application of large volume injection GC-MS to analysis of organic compounds in the extracts and leachates of municipal solid waste incineration fly ash

Organic solvent and water extracts of fly ash from a Milan (Italy) municipal solid waste incinerator (MSWI) were analyzed by large volume injection-gas chromatography-mass spectrometry (LVI-GC-MS) with programmable temperature vaporizer (PTV). Using injection volumes of 10-100 microl, typically over a hundred compounds were detected in organic solvent extracts and ca. 35% of them could be tentatively identified from their electron impact ionization mass spectra. A protocol for the determination of the maximum amount of a potential environmental pollutant available for leaching (availability test) was developed for four selected target compounds: pentachlorobenzene (PeCB), hexachlorobenzene (HxCB), o-terphenyl (o-TPH) and m-terphenyl (m-TPH). Key parameters, extraction time and liquid-to-solid ratio (L/S), were studied in more detail. Recoveries of PeCB, HxCB and o-TPH spiked into the fly ash samples at two concentration levels ranged from 38% to 53% for freshly spiked and from 14% to 40% for 40-day aged fly ash. Recoveries of m-TPH were 8% to 11% from freshly spiked and less than 3% from aged spiked fly ash. The native amounts in Milan MSWI fly ash, determined in an interlaboratory exercise using the developed protocol, were 31 ng/g PeCB, 34 ng/g HxCB, 72 ng/g o-TPH and 4.4 ng/g m-TPH. A separate methodology was developed for the determination of compounds extracted from fly ash by water (leaching test). Following 8-h sonication at L/S 20, the leached amounts of PeCB, HxCB and o-TPH were 1.1, 3.1 and 6.0 ng/g fly ash, respectively.     

Two-Step Cultivation Method for Microbial Disintegration of Tire Rubber Particles

A rubber-degrading strain of Nocardia was cultured on tread rubber particles from a truck tire by a two-step cultivation method. At the first step, the culture medium was either not agitated or stirred at very slow rate of 40 rpm for one or 2 weeks. At the second step, the culture medium was stirred at relatively higher stirring rate of 150 or 300 rpm for seven or six weeks. It was found that the rate of disintegration was greatly increased and the weight losses of the particles with diameters of about 2.3 mm were as high as 40% in the two-step method and only 20–30% in a one-step method in which the stirring rate was kept constant throughout the culture period. With this method, microbial colonization and disintegration were depressed particularly at the corners of the cubic particles and characteristic protuberant structures were observed at the corners after the removal of microbial cells by washing.     

In Vitro Cytotoxicity of Amylose-Based Bioplastic for Packaging Applications

Amylose containing polysaccharides are one of the most abundant and inexpensive naturally occurring biopolymers. Therefore, they are one of the most promising candidates to produce substitute plastics, especially in packaging applications. To determine the suitability for packaging applications, cytotoxicity of a modified amylose based bioplastic was investigated using NIH 3T3 Fibroblast cells from observation of cell morphology and MTS assay. Chemical durability of the amylose based bioplastic film was also studied by ion release and pH measurement after immersing the film into water. In vitro cytotoxicity (Cell morphology study and MTS assay) showed that the amylose based bioplastic film has in vitro biocompatibility and can be used for packaging applications. The ion release and pH measurement also supported the results.     

Large-scale production of poly(3-hydroxyvaleric acid) by fermentation ofChromobacterium violaceum,processing, and characterization of the homopolyester

A fed-batch process was developed, which allowed biotechnological production of the homopolyester poly(3-hydroxyvaleric acid) [poly(3HV)], in a mineral salts medium containing valeric acid as carbon source and complex nutrients as supplements byChromobacterium violaceum at a 10- and 300-L fermentation scale. This process yielded up to 40 g dry cell matter per L fermentation broth, and the cells contained up to 70% (w/w) poly(3HV). Poly(3HV), which was extracted from the cells with chloroform and was precipitated from this solvent with ethanol, was processed to test bars by injection molding or by press processing and to fibers by melt spinning. The unprocessed and processed poly(3HV) material was characterized with respect to the molecular weight and with respect to thermal, rheological, and mechanical properties. It was shown that it is possible to process biodegradable poly(3HV) thermoplastically and to obtain a polymer suitable for applications with low strength requirements.     

Ability of Fungal Strains to Degrade PVA Based Materials

The aim of this work was to select a fungal strain with degradative potential upon PVA based materials. The polymeric materials tested were PVA films which contain different percentages of PVA, starch and glycerol. These materials are of interest for food packaging applications, which presume to solve the problems concerning accumulation, disposal and degradation. Eleven strains were tested in solid culture for the ability to use PVA based composites as carbon and energy sources. The fungal strain selected was cultivated in liquid medium with different compositions. The scanning electron microscopy (SEM) investigations revealed the effects of microorganism growth upon polymeric films. Significant changes in polymer surface aspects were observed depending on the medium culture composition, the presence of supplementary carbon source facilitating microbial growth and degradation process.     

Investigation of hydrocyclones for the separation of shredded fridge plastics

The recycling of fridges produces a mixed plastic product of limited value. In order to maximise its value, the separation of the individual polymers that include high impact polystyrene (HIPS), acrylonitrile butadiene styrene (ABS), polyvinyl chloride (PVC) and polyethylene (PE) must take place. The effectiveness of a hydrocyclone system for the separation of these plastics, using both water and calcium chloride solutions, has been investigated. In addition a qualitative procedure for the determination of the HIPS content of a mixed ABS/HIPS product, by selective dissolution in R-limonene, has been developed. It was found that the effective density of separation depended on the particle size and aspect ratio. As the particle size and aspect ratio decreased, the separation became more efficient and the offset between separation density and hydrocyclone medium density decreased. This suggests that, for efficient density separation, closely sized, fine plastic fractions are required. Using these criteria, it was found that the removal of high density plastic such as PVC was readily achieved using a hydrocyclone. A partial separation of HIPS from ABS was found to be possible, taking advantage of a small density difference, using a hydrocyclone medium density of 1035kgm(-3).     

Biodegradable Composite Foams of PLA and PHBV Using Subcritical CO<Subscript>2</Subscript>

One key strategy for increasing the application potential for biodegradable plastics lies in improving the physical and mechanical characteristics, which can be attained by inducing a cellular morphology in the pure polymer with the aid of a blowing agent, as well as by blending two or more polymers with the desirable properties. This paper examines the effect that blending two biodegradable polymers has on the thermal properties and morphology of the resultant foams blown with carbon dioxide (CO₂). Polylactic acid (PLA), polyhydroxybutyrate-co-valerate (PHBV) and blends of both were foamed and characterized in terms of thermal characteristics, relative density, cell size, and foam morphology. The results indicate that although PLA and PHBV are immiscible, the presence of small quantities of PHBV (25 wt%) could lead to low density foams with finer, more uniform cells. Furthermore, the crystallinity of PHBV was found to be unaffected by the presence of PLA in the composite, which supports the immiscibility of PLA and PHBV.     

Effect of Poly(l-Lactide) and Poly(Butylene Succinate) on the Growth of Red Pepper and Tomato

Seeds of red pepper and tomato were sowed and cultivated in a soil blended with powdery poly(l-lactide) (PLLA), and poly(butylene succinate) (PBS). PBS depressed the growth of the two plants significantly even at a concentration as low as 5%, whereas PLLA up to 35% affected negligibly or even boosted the growth of the two plants. pH and number of microbial cells in the soil after 80 days of cultivation were almost the same independently whether the soil was blended with the two polymers or not. In contrast, the molecular weight of PBS decreased much faster than that of PLLA. Because succinic acid and 1,4-butane diol, from which PBS was synthesized, exhibited toxicity to both plant and animal cells to retard the germination rate of young radish seeds and to deform the morphology of HeLa cells significantly [1], the monomers and the oligomers produced from the PBS degradation should have a detrimental influence on the growth of the two plants.     

Biodegradation Behaviors of Poly(p-dioxanone) in Different Environment Media

This work was aimed at researching the aerobic biodegradation of poly(p-dioxanone) (PPDO), a novel kind of degradable polymer material, by simulating real-life conditions in a laboratory-scale test, specified by the standard methods based on two biodegradation environments, composting and aqueous media. To measure and describe the biodegradability of PPDO, not only had carbon dioxide produced by respiratory metabolism of microorganism been measured, which determines the ultimate aerobic biodegradability of chemical compounds, but also the detailed results of biodegradation were further characterized by monitoring physical, chemical and thermal properties changes of test materials at different incubation times in the two media, confirmed by using the appropriate analytical techniques. Scanning electron microscopy was used to observe the surface morphology, and the thermal performance of PPDO was characterized by differential scanning calorimetry. The changes of molecular weight were detected by intrinsic viscosity ([η]) and gel permeation chromatography, and the variations of the molecular structure were monitored by the nuclear magnetic resonance and FT-IR. The results show that PPDO has outstanding character of biodegradation and may be more adapted for biodegrading in liquid medium than in composting.