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.     

Processing and characterization of a new biodegradable composite made of a PHB/V matrix and regenerated cellulosic fibers

In this study, a biodegradable composite consisting of a degradable continuous cellulosic fiber and a degradable polymer matrix—poly(3-hydroxybutyrate)-co-poly(3-hydroxyvalerate (PHB/V with 19% HV)—was developed. The composite was processed by impregnating the cellulosic fibers on-line withPHB/V powder in a fluidization chamber. The impregnated roving was then filament wound on a plate and hot-pressed. The resulting unidirectional composite plates were mechanically tested and optically characterized by SEM. The fiber content was 9.9 ±0.9 vol% by volumetric determination. The fiber content predicted by the rule of mixture for unidirectional composites was 13.8 ±1.4 vol%. Optical characterization showed that the fiber distribution was homogeneous and a satisfactory wetting of the fibers by the matrix was achieved. Using a blower to remove excess matrix powder during processing increased the fiber content to 26.5 ±3.3 vol % (volumetric) or 30.0 ±0.4 vol% (rule of mixture). The tensile strength of the composite parallel to the fiber direction was 128 ±12 MPa (10 vol% fiber) up to 278 ±48 MPa (26.5 vol% fiber), compared to 20 MPa for the PHB/V matrix. The Young’s modulus was 5.8 ±0.5 GPa (10 vol% fiber) and reached 11.4 ±0.14 GPa (26.5 vol% fiber), versus 1 GPa for the matrix.     

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.     

PHB/V in Extrusion Coating of Paper and Paperboard: Part I: Study of Functional Properties

Extrusion-coating experiments were carried out in the pilot line at Tampere Univesity of Technology (Institute of Paper Converting). Typical paper and paperboard substrates were coated with commercially produced 3-hydroxybutyrate/3-hydroxyvalerate. The resulting physical properties of extrusion-coated composite structures were studied. Adhesion between PHB/V and a fiber-based substrate was rather poor, regardless of typically used pretreatments (corona and flame). On the other hand, adhesion was sufficient (mode of failure was fiber tear as the materials were separated) when the substrate was primed with an acrylic-based primer. The surface energy and polarity of PHB/V were much higher than the respective ones of LDPE. Curling of PHB/V was reduced by the addition of wax or tall oil rosin into the base polymer.     

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.     

Degradation of poly(3-hydroxybutyrate), PHB,by bacteria and purification of a novel PHB depolymerase fromComamonas sp.

Bacteria capable of growing on poly(3-hydroxybutyrate), PHB, as the sole source of carbon and energy were isolated from various soils, lake water, activated sludge, and air. Although all bacteria utilized a wide variety of monomeric substrates for growth, most of the strains were restricted to degrade PHB and copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate, P(3HB-co-3HV). Five strains were also able to decompose a homopolymer of 3-hydroxyvalerate, PHV. Poly(3-hydroxyoctanoate), PHO, was not degraded by any of the isolates. One strain, which was identified asComamonas sp., was selected, and the extracellular depolymerase of this strain was purified from the medium by ammonium sulfate precipitation and by chromatography on DEAE-Sephacel and Butyl-Sepharose 4B. The purified PHB depolymerase was not a glycoprotein. The relative molecular masses of the native enzyme and of the subunits were 45,000 or 44,000, respectively. The purified enzyme hydrolyzed PHB, P(3HB-co-3HV), and—at a very low rate—also PHV. Polyhydroxyalkanoates, PHA, with six or more carbon atoms per monomer or characteristic substrates for lipases were not hydrolyzed. In contrast to the PHB depolymerases ofPseudomonas lemoignei andAlcaligenes faecalis T₁, which are sensitive toward phenylmethylsulfonyl fluoride (PMSF) and which hydrolyze PHB mainly to the dimeric and trimeric esters of 3-hydroxybutyrate, the depolymerase ofComamonas sp. was insensitive toward PMSF and hydrolyzed PHB to monomeric 3-hydroxybutyrate indicating a different mechanism of PHB hydrolysis. Furthermore, the pH optimum of the reaction catalyzed by the depolymerase ofComamonas sp. was in the alkaline range at 9.4.     

Biodegradability of Thermally Aged PHB,PHB-V,and PCL in Soil Compostage

The biodegradability of poly--hydroxybutyrate (PHB), poly--hydroxybutyrate-co-valerate (PHB-V) and poly--caprolactone (PCL) were examined following thermal aging in an oven for 192, 425 and 600 h. Different temperatures, 100, 120 and 140°C for PHB and PHB-V and 30, 40 and 50^oC for PCL were used to assess the influence of this parameter on biodegradation. The biodegradability tests were done in soil compostage at pH 11.0 and involved measuring the residual mass of polymer. Thermal analysis of the polymers was done using a differential scanning calorimeter (DSC). The melting temperature and crystallinity were also determined. Thermal ageing increased the biodegradability only for PHB at 120 and 140^oC, and there was no correlation between crystallinity and the biodegradation of the polymers.     

Effects of higher-order structure of poly(3-hydroxybutyrate) on its biodegradation. II. Effects of crystal structure on microbial degradation

As one of a series of studies concerning the relationship between the higher-order structure and the biodegradability of a biodegradable plastic, the effects of the crystal structure of the plastic on microbial degradation were investigated. Bacterial poly(d-(–)-3-hydroxybutyrate) (PHB) films which had a wide range of crystallinity were prepared by the melt-quenching method. Results of the microbial degradation indicated that the development of crystallinity evidently depressed the microbial degradability. From scanning electron microscopy (SEM) observations, it is suggested that the microbial degradation proceeded in at least two manners. One was preferential degradation of the amorphous region leaving the crystalline lamellae intact, which was considered to be a homogeneous enzymatic degradation over the surface. The other was nonpreferential spherical degradation on the surface. The SEMs indicate that the spherical holes were the result of colonization by degrading bacteria. The holes varied in size and number with the change of crystal structure. Therefore, it is considered that the crystal structure of PHB also influenced the physiological behavior of the degrading bacteria on the PHB surface.     

Biodegradation of γ Irradiated Poly 3-hydroxybutyrate (PHB) Films Blended with Poly(Ethyleneglycol)

Poly(3-hydroxybutyrate) (PHB) was evaluated in blends with poly(ethyleneglycol) (PEG) of different weight average molecular weight (Mw = 300, 600, 1,000 and 6,000). Irradiation of the PHB/PEG films was carried out to different levels of irradiation doses (5 and 10 kGy) and the effects were investigated talking into consideration: thermal properties by differential scanning calorimetry (DSC), perforation resistance, water vapor transmission rate and biodegradation in simulated soil. The addition of plasticizer alters thermal stability and crystallinity of the blends. The improvement in perforation resistance due to irradiation was regarded to be a result of the crosslinking effect. Also, biodegradation assays resulted in mass retention improvements with increases in PEG molar masses, PEG concentration and irradiation dose. The irradiation process was shown to hamper the biodegradation mechanism.     

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.     

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.     

Microscopic Visualization of the Enzymatic Degradation of Poly(3HB-co-3HV) and Poly(3HV) Single Crystals by PHA Depolymerases From Pseudomonas lemoignei

As a complement to previous studies of the enzymatic degradation of folded chain lamellar single crystals of polyhydroxyalkanoates, single crystals of a number of polyhydroxyalkanoates were partially degraded with depolymerases from Pseudomonas lemoignei and examined by transmission electron microscopy. Single crystals of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate), bacterial poly(3-hydroxyvalerate), and synthetic poly(3-hydroxybutyrate) with 88% isotactic diads were degraded using purified extracellular PHA-depolymerases from P. lemoignei: PHB-depolymerase A, PHB-depolymerase B, and depolymerases from recombinant E. coli: PHB-depolymerase PhaZ4 (PHB-depolymerase E), PHB-depolymerase PhaZl (PHB-depolymerase C), and PHB-depolymerase PhaZ5 (PHB-depolymerase A). In contrast to previous results with single crystals of bacterial PHB, the predominant effect observed with all crystals was a significant narrowing of the lamellae. This suggests an edge attack mechanism which because of lateral disorder of the crystals leads to a narrowing of the crystalline lamellae as opposed to the splintering effect previously observed. The model suggested for the degradation of single crystals of bacterial PHB by PHB-depolymerases is refined to include the effects of lateral disorder caused by the introduction of valerate or repeat units of opposite stereochemistry into the single crystal.     

Biochemical and Genetic Characterization of an Extracellular Poly(3-Hydroxybutyrate) Depolymerase from Acidovorax Sp. Strain TP4

To determine the properties of enzymes from bacteria that degrade polypropiolactone (PPL), we isolated 13 PPL-degrading bacteria from pond water, river water, and soil. Nine of these strains were identified as Acidovorax sp., three as Variovorax paradoxus, and one as Sphingomonas paucimobilis. All the isolates also degraded poly(3-hydroxybutyrate) (PHB). A PPL-degrading enzyme was purified to electrophoretical homogeneity from one of these bacteria, designated Acidovorax sp. TP4. The purified enzyme also degraded PHB. The molecular weight of the enzyme was estimated as about 50,000. The enzyme activity was inhibited by diisopropylfluorophosphate, dithiothreitol, and Triton X-100. The structural gene of the depolymerase was cloned in Escherichia coli. The nucleotide sequence of the cloned DNA fragment contained an open reading frame (1476 bp) specifying a protein with a deduced molecular weight of 50,961 (491 amino acids). The deduced overall sequence was very similar to that of a PHB depolymerase of Comamonas acidovorans YM1609. From these results it was concluded that the isolated PPL-degrading enzyme belongs to the class of PHB depolymerases. A conserved amino acid sequence, Gly-X₁-Ser-X₂-Gly (lipase box), was found at the N-terminal side of the amino acid sequence. Site-directed mutagenesis of the TP4 enzyme confirmed that ²⁰Ser in the lipase box was essential for the enzyme activity. This is the first report of the isolation a PHB depolymerase from Acidovorax.     

Enhanced production of poly(3-hydroxybutyrate) by filamentation-suppressed recombinantEscherichia coli in a defined medium

Fed-batch cultures of recombinantEscherichia coli strains were carried out for the production of poly(3-hydroxybutyric acid) (PHB) in a chemically defined medium. TheE. coli strains used were XL1-Blue, harboring pSYL105, a stable high-copy number plasmid containing theAlcaligenes eutrophus polyhydroxyalkanoate (PHA) genes, and XL1-Blue, harboring pSYL107, which is pSYL105 containing theE. coli ftsZ gene to suppress filamentation. With XL1-Blue(pSYL105) the final cell mass and PHB concentration obtained in 62 h were 102 and 22.5 g/L, respectively. Fed-batch culture of XL1-Blue(pSYL107) under identical conditions resulted in a final cell mass and PHB concentration of 127.5 and 48.2 g/L, respectively. The PHB contents obtained with XL1-Blue(pSYL105) and XL1-Blue(pSYL107) were 22.1 and 37.8%, respectively. Therefore, PHB was more efficiently produced in a defined medium by employing filamentation-suppressed recombinantE. coli.     

Aromatic/Aliphatic Polyester Blends

Blends of poly(3-hydroxybutyrate) (PHB) and poly(ethylene terephthalate-co-1,4-cyclohexenedimethanol terephthalate) (PETG) were prepared in a batch mixer and in a twin screw extruder and characterized by differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), field emission scanning electron microscopy (FE SEM), flexural tests, biodegradation tests in soil compost and in an enzymatic medium. The torque data showed that the addition of PETG to PHB improved its processability. DSC, DMA and FE SEM showed that the polymers are immiscible with morphology dependent on the processing conditions. A fine dispersion of PETG in the PHB matrix was observed for extruded and injection molded blends. Flexural modulus for blends was higher for blends in comparison with PHB, while the impact resistance of blends containing 20 wt% and 30 wt% of PETG is comparable to the value for PHB. PHB is biodegradable, while PETG did not degrade either in simulated soil or in the α-amylase medium. On the other hand, the PHB phase of the blends degrades under these aging conditions. Thus, the addition of PETG to PHB results in advantage such as improving of processability and Young′s modulus without significant changes in the impact resistance while keeping the biodegradability of PHB.     

Purification and Properties of a Poly (β-hydroxybutyrate) Depolymerase From Penicillium sp.

An extracellular poly (β-hydroxybutyrate) (PHB) depolymerase was purified from a Penicillium sp. DS9701-09a by centrifugation, ultrafiltration, precipitation and gel filtration chromatography. The specific activity of the purified enzyme was 37.9-folds higher than that of the culture supernatant and the recovery yield was 11.8%. The PHB deploymerase molecular mass was 44.8 kDa from analysis of both Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometer. The isoelectric point of 6.7 for the enzyme was determined by a two-dimensional electrophoresis. The optimum enzyme activity was observed at a temperature of 50 °C and pH 5.0. The apparent K _m of the enzyme was found to be 1.35 mg/mL. The PHB depolymerase consisted of 16 kinds of normal amino acids. The secondary structure of the enzyme was determined by CD spectrum. α-helix and β-turn were found to be 66% and 34% for the enzyme without ammonium sulphite. Chemical inhibition on the PHB depolymerase activity was examined and EDTA was found to have a significantly inhibitory effect.     

Algae Biomass Based Media for Poly(3-hydroxybutyrate) (PHB) Production by Escherichia coli

In addition to biodiesel production from algae, the production of other valuable bioproducts facilitates the development of an algae-based biorefinery platform. The goal of this study was to utilize the aqueous fraction from a novel algal wet lipid extraction procedure as the medium for the production of a bio product, poly(3-hydroxybutyrate) (PHB), via the growth of recombinant Escherichia coli. PHB yield was measured at 34 % of the E. coli dry cell mass, and was increased to 51 % when the algae aqueous medium was supplemented with glucose. While the addition of inorganic nutrients to the aqueous phase did not increase PHB production or growth of E. coli, growth of E. coli was observed to increase with the supplementation of carbon substrate (glucose). The addition of carbon rich waste to the aqueous fraction of wastewater-derived algae may in the future provide a sustainable alternative. Future research will be directed at evaluating this concept to develop a sustainable process for the production of bioplastics through an algae-based biorefinery platform.     

Formation of polyester blends by a recombinant strain ofPseudomonas oleovorans: Different poly(3-hydroxyalkanoates) are stored in separate granules

WhenPseudomonas oleovorans (GPo1) is grown on sodium octanoate under ammonium limiting conditions, it is able to accumulate a copolyester consisting of medium chain length 3-hydroxyalkanoic acids (PHA_m). 3-Hydroxybutyrate is only incorporated in trace amounts. WhenP. oleovorans is equipped with the PHB biosynthetic genes ofAlcaligenes eutrophus (GPo1[pVK101::PP1]), it forms a polyester containing major amounts of 3-hydroxybutyrate. The resulting polymer however is a blend of PHA_m and PHB, rather than a copolymer of 3-hydroxybutyrate and medium chain length 3-hydroxyalkanoic acids [11]. To establish whether PHA_m and PHB molecules are stored in the same or separate granules by this recombinantP. oleovorans strain, we studied polymer forming cells by freeze-fracture electron microscopy. This approach is possible because previous freeze-fracture electron microscopy studies on PHA_m and PHB accumulating strains have shown that PHA_m and PHB granules can be distinguished from each other: PHA_m granules from mushroom-like structures, whereas PHB granules from needle structures during freeze-fracturing. In this paper we show that stationary phase cells of GPo1[pVK101::PP1] contained both mushroom and needle-like structures, indicating that PHA_m and PHB chains were stored in separate granules. To be able to determine whether the separation of PHA_m and PHB is complete, the respective granules were separated on sucrose gradients. A total cell extract of GPo1[pVK101::PP1] which was subjected to sucrose gradient centrifugation revealed two white bands of different densities: the upper band with a density of 1.05 g/mL consisted exclusively of PHA_m granules, while the lower band with a density of 1.19 g/mL consisted of PHB granules only. Thus, when bacteria synthesize both PHA_m and PHB, the resulting polymer chains are segregated completely and stored in separate granules.     

Kinetic Study of Biopolymer (PHB) Synthesis in <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. in Submerged Fermentation Process Using TEM

Poly-β-hydroxybuyrate (PHB) is a carbon—energy storage material which is accumulated as intracellular granule in variety of microorganism under nutrient starved conditions. Solid PHB is a biodegradable thermoplastic polymer and is utilizable in various ways similar to many conventional plastics. Ralstonia eutropha (Alcaligenes sp.), a gram negative bacteria accumulates PHB as insoluble granules inside the cells when nutrients other than carbon are limited. In this report effort has been made to analyze PHB granule synthesis inside Alcaligenes sp. NCIM 5085 by transmission electron microscopy and qualitative estimation of PHB was carried out by fourier transform infrared spectroscopy which provide better precision compared to other conventional techniques previously applied for PHB determination. Maximum PHB concentration of 2.20 ± 0.40 g/L and cell biomass of 3.42 ± 0.20 g/L was obtained after 48.0 h of fermentation. Leudking-Piret equation deduced mixed growth associated product formation which varies from earlier reports.     

Microbial Degradation of Poly(3-Hydroxybutyrate-co-4-Hydroxybutyrate) in Soil

The microbial degradation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3/4HB) copolymers with different 4HB molar fraction were investigated in soil for 60?days. In the degradation process, changes in weight loss and molecular weight were periodically measured to determine the biodegradability; the surface morphology also was observed using scanning electron microscopy and polarizing optical microscopy. The results showed that the rate of degradation of P3/4HB depends strongly on its crystallinity and surface morphology. Enzymatic degradation, which proceeded via surface erosion mechanisms, was observed mainly during the degradation period in soil. The amorphous interspherulitic regions and crystal center were prone to degrade.