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.     

Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in compost

The microbial degradation of tensile test pieces made of poly(3-hydroxybutyrate) [P(3HB)] or copolymers with 10% [P(3HB-co-10%3HV)] and 20% [P(3HB-co-20%3HV)] 3-hydroxyvaleric acid was studied in small household compost heaps. Degradation was measured through loss of weight (surface erosion) and changes in molecular weight and mechanical strength. It was concluded, on the basis of weight loss and loss of mechanical properties, that P(3HB) and P(3HB-co-3HV) plastics were degraded in compost by the action of microorganisms. No decrease inM _w could be detected during the degradation process. The P(3HB-co-20%3HV) copolymer was degraded much faster than the homopolymer and P(3HB-co-10%3HV). One hundred nine microbial strains capable of degrading the polymersin vitro were isolated from the samples used in the biodegradation studies, as well as from two other composts, and identified. They consisted of 61 Gram-negative bacteria (e.g.,Acidovorax facilis), 10 Gram-positive bacteria (mainlyBacillus megaterium), 35Streptomyces strains, and 3 molds.     

Accumulation of Polyhydroxyalkanoates by Rhizobacteria Underneath Nickel-Hyperaccumulators from Serpentine Ecosystem

Nickel-resistant bacteria isolated from underneath Ni-hyperaccumulators growing on serpentine soils were screened for production of polyhydroxyalkanoates. These rhizobacteria accumulated poly-3-hydroxybutyric acid [P(3HB)] accounting 3.9–67.7% of cell dry weight during growth in gluconate and/or glucose. Cupriavidus pauculus KPS 201 utilized only gluconate and accumulated about 67.7% P(3HB) while, Bacillus firmus AND 408 utilized both carbon sources for polymer synthesis. The isolates being resistant to Ni also accumulated substantial amount of P(3HB) when grown in presence of the heavy metal and this was revealed by transmission electron microscopic studies. Although B. firmus AND 408 produced only P(3HB) at higher concentrations of gluconate, C. pauculus KPS 201 synthesized copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) [P(3HB-co-3HV)]. In presence of 0.8% gluconate and 4 mM Ni, KPS 201 cells produced PHA amounting 81% CDW, which contained 76 and 24 mol% 3HB and 3HV monomers, respectively.     

Optimization of Carbon Dioxide and Valeric Acid Utilization for Polyhydroxyalkanoates Synthesis by Cupriavidus necator

The utilization of captured CO₂ as a part of the CO₂ capture and storage system to produce biopolymers could address current environmental issues such as global warming and depletion of resources. In this study, the effect of feeding strategies of CO₂ and valeric acid on cell growth and synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] in Cupriavidus necator was investigated to determine the optimal conditions for microbial growth and biopolymer accumulation. Among the studied CO₂ concentrations (1–20 %), microbial growth and poly(3-hydroxybutyrate) accumulation were optimal at 1 % CO₂ using a gas mixture at H₂:O₂:N₂ = 7:1:91 % (v/v). When valeric acid was fed together with 1 % CO₂, (R)-3-hydroxyvalerate synthesis increased with increasing valeric acid concentration up to 0.1 %, but (R)-3-hydroxybutyrate synthesis was inhibited at >0.05 % valeric acid. Sequential addition of valeric acid (0.05 % at Day 0 followed by 0.025 % at Day 2) showed an increase in 3HV fraction without inhibitory effects on 3HB synthesis during 4 d accumulation period. The resulting P(3HB-co-3HV) with 17–32 mol  % of 3HV is likely to be biocompatible. The optimal concentrations and feeding strategies of CO₂ and valeric acid determined in this study for microbial P(3HB-co-3HV) synthesis can be used to produce biocompatible P(3HB-co-3HV).     

Biosynthesis and Characterization of Poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) from Bacillus cereus S10

The biodegradable and biocompatible copolymer poly-(3-hydroxybutyrate-co-5 mol% 3-hydroxyvalerate), poly-(3HB-co-5 mol% 3HV), was synthesized by Bacillus cereus S10 and the highest yield was determined as 69.91 % at pH 7 and 30 °C after 48 h of incubation using a glucose as the sole carbon source. Poly-(3HB-co-5 mol% 3HV) was purified from bacterial biomass using chloroform. FTIR analysis showed absorption bands at 1,723, 1,274, 1,373, 1,453, 2,932 cm^?1 corresponding to C=O, C–O stretching, CH₃, –CH₂ and –CH groups, respectively. ¹H-NMR and ¹³C-NMR analysis confirmed that the copolymer was composed of 95 mol% of 3-hydroxybutrate and 5 mol% of 3-HV monomeric units. Poly-(3-HB-co-5 mol% 3HV) was used for nanoparticles preparation. The diameter of nanoparticles was 202 nm.     

Polymers,the Light at the End of Dark Fermentation: Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by a Photoheterotrophic Consortium

Journal of Polymers and the Environment - In this study, the photoheterotrophic consortium C4 was used to produce the copolymer [P(3HB-co-3HV)]. PHA production was enhanced by using response...     

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.     

Accumulation of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid-co-4-hydroxyvaleric acid) by mutants and recombinant strains ofAlcaligenes eutrophus

Alcaligenes eutrophus accumulated a terpolyester of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV), and 4-hydroxyvaleric acid (4HV) during cultivation with 4HV as carbon and energy source under nitrogen starvation. The polyester accumulated by wild-type strains under these conditions contained 4HV at a molar fraction of approximately 5 mol% only. A catabolic pathway of 4HV was postulated, which included the activation of 4HV to 4HV-CoA and a conversion of 4HV-CoA to 3HV-CoA. Tn5::mob-induced mutants were isolated fromA. eutrophus HF39, which were affected in 4HV and/or valeric acid catabolism. Among 83 mutants were 27 4HV-negative or 4HV-leaky mutants; two mutants were identified which accumulated a terpolyester with a molar fraction of 10.1 to 22.7 mol% 4HV. In addition, a further increase in the molar fraction of 4HV in poly(3HB-co-3HV-co-4HV) and a two- to fourfold increase in the PHA synthase activity were monitored in these mutants or others and also in HF39, if the cells were complemented with the hybrid plasmid pHP1014::PP1, which contained the PHA biosynthesis genes ofA. eutrophus H16. Application of mutagenesis plus recombinant DNA techniques resulted in the accumulation of a terpolyester with up to 30 mol% 4HV and with approximately equimolar fractions of 3HB, 3HV, and 4HV.     

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.     

Biodegradation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by a Tropical Marine Bacterium, Pseudoalteromonas sp. NRRL B-30083

Pseudoalteromonas sp. NRRL B-30083 was isolated as the predominant PHBV-degrading organism from a tropical marine environment. In complex medium, the isolate grew well at temperatures between 23°C and 33°C, with an optimal doubling time of about 30 min. NaCl was required at concentrations between 0.2 N and 0.8 N. Optimal pH levels for growth were between pH 6.5 and pH 8.5. Liquid cultures grew modestly on PHBV as a sole carbon source under optimal conditions, although PHBV depolymerase activity was not detected.     

Anaerobic microbial degradation of poly (3-hydroxyalkanoates) with various terminal electron acceptors

The microbial degradation of poly (3-hydroxyalkanoates) (PHAs) under anaerobic conditions with various terminal electron acceptors was examined. Nitrate-reducing consortia were established using activated sludge, and PHAs were shown to be biodegradable under these conditions. A positive correlation between carbon dioxide production and nitrate reduction was demonstrated. Nitrous oxide accumulated as the main N-containing product of nitrate reduction. The amount of PHAs in activated sludge cultures decreased approximately 20% within 40 days of incubation. Attempts were made to establish iron- and sulfate-reducing consortia from spring water, yet it could not be demonstrated that the mixed cultures were capable of degrading PHAs. Pure cultures of iron- and sulfate-reducing bacteria could not utilize PHAs as sole carbon sources. Methanogenic environments sampled included pond sediment and rumen fluid. PHAs were fermented to methane and carbon dioxide after 10 weeks by a sediment consortium, with 43 to 57% of the substrate carbon transformed to methane. Although it could not be demonstrated that PHAs were biodegraded by a rumen fluid consortium, a facultative anaerobic bacterium, identified as aStaphylococcus sp., that could grow on PHAs was isolated from rumen fluid.     

Increased 3HV Concentration in the Bacterial Production of 3-Hydroxybutyrate (3HB) and 3-Hydroxyvalerate (3HV) Copolymer with Acid-Digested Rice Straw Waste

Bacterial synthesis of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) copolymer [P(3HB-co-3HV)] using the hydrolysate of rice straw waste as a carbon source was affected by the composition of the hydrolysate, which depends highly on the rice straw pretreatment condition. Acid digestion with 2 % sulfuric acid generated larger production of P(3HB-co-3HV) than 6 % sulfuric acid, but 3HV concentration in the copolymer produced with 2 % acid hydrolysate was only 8.8 % compared to 18.1 % with 6 % acid hydrolysate. To obtain a higher 3HV mole fraction for enhanced flexibility of the copolymer, an additional heating was conducted with the 2 % acid hydrolysate after removal of residual rice straw. As the additional heating time increased a higher concentration of levulinic acid was generated, and consequently, the mole fraction of 3HV in P(3HB-co-3HV) increased. Among the conditions tested (i.e., 20-, 40-, 60-min), 60-min additional heating following 2 % sulfuric acid digestion achieved the highest 3HV mole fraction of 22.9 %. However, a longer heating time decreased the P(3HB-co-3HV) productivity, probably due to the increased intermediates concentrations acting as inhibitors in the hydrolysates. Therefore, the use of additional heating needs to consider both the increase in the 3HV mole fraction and the decrease in the P(3HB-co-3HV) productivity.     

Recovery of solid fuel from municipal solid waste by hydrothermal treatment using subcritical water

Hydrothermal treatments using subcritical water (HTSW) such as that at 234 °C and 3 MPa (LT condition) and 295 °C and 8 MPa (HT condition) were investigated to recover solid fuel from municipal solid waste (MSW). Printing paper, dog food (DF), wooden chopsticks, and mixed plastic film and sheets of polyethylene, polypropylene, and polystyrene were prepared as model MSW components, in which polyvinylchloride (PVC) powder and sodium chloride were used to simulate Cl sources.While more than 75% of carbon in paper, DF, and wood was recovered as char under both LT and HT conditions, plastics did not degrade under either LT or HT conditions. The heating value (HV) of obtained char was 13,886-27,544 kJ/kg and was comparable to that of brown coal and lignite. Higher formation of fixed carbon and greater oxygen dissociation during HTSW were thought to improve the HV of char.Cl atoms added as PVC powder and sodium chloride to raw material remained in char after HTSW. However, most Cl originating from PVC was found to converse into soluble Cl compounds during HTSW under the HT condition and could be removed by washing.From these results, the merit of HTSW as a method of recovering solid fuel from MSW is considered to produce char with minimal carbon loss without a drying process prior to HTSW. In addition, Cl originating from PVC decomposes into soluble Cl compound under the HT condition. The combination of HTSW under the HT condition and char washing might improve the quality of char as alternative fuel.     

Thermal Properties and Biodegradability Studies of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

For investigating the relationship between thermal properties and biodegradability of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), several films of PHBV containing different polyhydroxyvalerate (HV) fractions were subjected to degradation in different conditions for up to 49 days. Differential scanning calorimetry (DSC), thermogravimetry (TG), specimen weight loss and scanning electron microscopy (SEM) were performed to characterize the thermal properties and enzymatic biodegradability of PHBV. The experimental results suggest that the degradation rates of PHBV films increase with decreasing crystallinity; the degradability of PHBV occurring from the surface is very significant under enzymatic hydrolysis; the crystallinity of PHBV decreased with the increase of HV fraction in PHBV; and no decrease in molecular weight was observed in the partially-degraded polymer.     

Bacterial Poly(Hydroxyalkanoate) Polymer Production from the Biodiesel Co-product Stream

A co-product stream from soy-based biodiesel production (CSBP) containing glycerol, fatty acid soaps, and residual fatty acid methyl esters (FAME) was utilized as a fermentation feedstock for the bacterial synthesis of poly(3-hydroxybutyrate) (PHB) and medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) polymers. Pseudomonas oleovorans NRRL B-14682 and P. corrugata 388 grew and synthesized PHB and mcl-PHA, respectively, when cultivated in up to 5% (w/v) CSBP. In shake flask culture, P. oleovorans grew to 1.3 ± 0.1 g/L (PHA cellular productivity = 13–27% of the bacterial cell dry weight; CDW) regardless of the initial CSBP concentration, whereas P. corrugata reached maximum cell yields of 2.1 g/L at 1% CSBP, which tapered off to 1.7 g/L as the CSBP media concentration was increased to 5% (maximum PHA cellular productivity = 42% of the CDW at 3% CSBP). While P. oleovorans synthesized PHB from CSBP, P. corrugata produced mcl-PHA consisting primarily of 3-hydroxyoctanoic acid (C_8:0; 39 ± 2 mol%), 3-hydroxydecanoic acid (C_10:0; 26 ± 2 mol%) and 3-hydroxytetradecadienoic acid (C_14:2; 15 ± 1 mol%). The molar mass (M_n) of the PHB polymer decreased by 53% as the initial CSBP culture concentration was increased from 1% to 5% (w/v). In contrast, the M_n of the mcl-PHA polymer produced by P. corrugata remained constant over the range of CSBP concentrations used.     

Pseudomonas lemoignei has five poly(hydroxyalkanoic acid) (PHA) depolymerase genes: A comparative study of bacterial and eukaryotic PHA depolymerases

Four polyhydroxyalkanoate (PHA) depolymerases were purified from the culture fluid ofPseudomonas lemoignei: poly(3-hydroxybutyrate) (PHB), depolymerase A (M _r , 55,000), and PHB depolymerase B (M _r , 67,000) were specific for PHB and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) as substrates. The third depolymerase additionally hydrolyzed poly(3-hydroxyvalerate) (PHV) at high rates (PHV depolymerase;M _r , 54,000). The N-terminal amino acid sequences of the three purified proteins, of a fourth partially purified depolymerase (PHB depolymerase C), and of the PHB depolymerases ofComamonas sp. were determined. Four PHA depolymerase genes ofP. lemoignei (phaZ1,phaZ2,phaZ3, andphaZ4) have been cloned inEscherichia coli, and the nucleotide sequence ofphaZ1 has been determined recently (D. Jendrossek, B. Müller, and H. G. Schlegel,Eur. J. Biochem. 218, 701–710, 1993). In this study the nucleotide sequences ofphaZ2 andphaZ3 were determined.PhaZ1,phaZ2, andphaZ4 were identified to encode PHB depolymerase C, PHB depolymerase B, and PHV depolymerase, respectively.PhaZ3 coded for a novel PHB depolymerase ofP. lemoignei, named PHB depolymerase D. None of the four genes harbored the PHB depolymerase A gene, which is predicted to be encoded by a fifth depolymerase gene ofP. lemoignei (phaZ5) and which has not been cloned yet. The deduced amino acid sequences ofphaZ1–phaZ3 revealed high homologies to each other (68–72%) and medium homologies to the PHB depolymerase gene ofAlcaligenes faecalis T₁ (25–34%). Typical leader peptide amino acid sequences, lipase consensus sequences (Gly-Xaa-Ser-Xaa-Gly), and unusually high proportions of threonine near the C terminus were found in PhaZ1, PhaZ2, and PhaZ3. Considering the biochemical data of the purified proteins and the amino acid sequences, PHA depolymerases ofP. lemoignei are most probably serine hydrolases containing a catalytical triad of Asp, His, and Ser similar to that of lipases. A comparison of biochemical and genetic data of various eubacterial and one eukaryotic PHA depolymerases is provided also.Paper presented at the Bio/Environmentally Degradable Polymer Society—Second National Meeting, August 19–21, 1993, Chicago, Illinois.     

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.     

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.     

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.     

Electron Microscope Observation of Biodegradation of Polymers

Biodegradable polymers generally decompose in the various media in our environments. These environments contain soils, seawater, and activated sludge. If biodegradable materials waste is discarded, they decompose in these media. The biodegradation process of biodegradable polymers was investigated by scanning electron microscopy. Polycaprolactone, polybutylene succinate, and P(3HB-co-3HV) were tested. The shapes of holes on the decomposing surfaces are different according to the biodegradation media. Semispherical holes are observed on the surfaces of polybutylene succinate films degraded in activated sludge and cracks are observed on the surfaces of polycaprolactone films degraded in soil.