Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesters poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) was investigated with special regard to intermediate products, kinetics, and yields. During the degradation of PHBV acetate, propionate, n-butyrate, and n-valerate were detected. Additionally, 3-hydroxybutyrate and 3-hydroxyvalerate and four dimeric esters of these two molecules were identified by GC-MS measurements. Three different test systems for the anaerobic degradation of polyesters were studied. It was not possible to get reproducible results by means of the Anaerobic Sturm-test, a simple system based on carbon dioxide measurement. Secondly, a system based on the GC measurement of accumulated organic acids was investigated. A degradation of 90% in two days was calculated by a carbon balance. Best results were reached with the third test system based on the measurement of methane with a gas meter. A degradation of 99% was observed within 30 days.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9.

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

The biodegradation of poly-3-hydroxyalkanoates,PHAs, with long alkyl substitutents byPseudomonas maculicola

Utilizing a quantitative clear zone technique, the activity of an extracellular depolymerase system fromPseudomonas maculicola was investigated. Polymer degradation was influenced by the amount and availability of secondary carbon sources, with a simultaneous utilization of both sources. The initial carbon source in the liquid preculture also affected the eventual colony growth and polymer degradation. The enzyme solution was determined to readily degrade poly-3-hydroxyalkanoates (PHAs) with relatively long alkyl substituents at the 3 position: poly-3-hydroxyoctanoate (PHO), poly-3-hydroxynonanoate (PHN), and their copolymers (P[HO-co-HN]) and poly-3-hydroxyundecanoate (PHU). However, the system was unable to degrade either PHAs with shorter alkyl groups, including poly-3-hydroxybutyrate (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-co-HV]) or PHAs with unusual substituents such as poly(3-hydroxy-5-phenylvaleric acid) (PHPV). It is proposed that degradation of these more bulky side chain polymers was prevented by the inability of the bacteria to assimilate their monomeric components, which inhibited the successful utilization of secondary carbon sources and thus inhibited colony growth.     

Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from activated sludge

Poly-3-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) was produced using a co-culture of activated sludge. When butyric acid was used as sole carbon source, PHB was produced. When valeric acid was added to the medium, PHBV was produced. The 3-hydroxyvalerate (3HV) mole fraction in the PHBV reached a maximum of 54% when valeric acid was used as sole carbon source. When the 3HV units in the co-polymer increased from 0.0 to 54.0 mol%, the melting temperature ( T m ) decreased from 178 to 99°C. The composition, and hence the mechanical properties, of the co-polymer produced by activated sludge can be controlled by adjusting the medium composition.     

Isolation and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading actinomycetes and purification of PHBV depolymerase from newly isolatedStreptoverticillium kashmirense AF1

Streptoverticillium kashmirense AF1 with the ability to degrade a natural polymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was isolated from municipal sewage sludge by soil burial technique. The PHBV film was degraded by the action of extracellular enzymes secreted by the microorganisms. Degradation of PHBV was evident by the formation of clear zones of hydrolysis on the polymer containing mineral salt agar plates. The extent of PHBV degradation increased up to 30 days of incubation. Maximum production of PHBV depolymerase was observed both at pH 8 and pH 7, 45 °C, 1% substrate concentration and in the presence of lactose as an additional carbon source. Two types of extracellular PHBV depolymerases were purified fromS. kashmirense AF1 by gel permeation chromatography using Sephadex G-75. The molecular weights of the two proteins were found to be 35 and 45 kDa approximately, as determined by SDS-PAGE. The results of the Sturm test also showed more CO₂ production as a result of PHBV degradation, in the test as compared to control. The present findings indicated the degradation capabilities ofS. kashmirense AF1.     

Comparative oxo-biodegradation study of poly-3-hydroxybutyrate-co-3-hydroxyvalerate/polypropylene blend in controlled environments

The potential use of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) copolymer as a biodegradable additive in polypropylene (PP) has been explored. The melt blending technique was used to produce the blend of PHBV/PP (PB10). The degradation studies of PB10 were done in the field as well as in controlled laboratory conditions. The structural changes were studied using scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and thermogravimetric analysis (TGA). SEM micrographs showed the formation of agglomerates, pits, grooves, and holes on the treated films as a result of microbial activity. FTIR spectra indicated clear evidences of oxo-biodegradation of polymer chains due to an increase in carbonyl peak index. Thermogravimetric analysis confirmed that the thermal stability of PB10 was increased after soil burial. This study contributed toward the prospective commercial applications of PHBV for use in the food packaging industry.     

Methanogenic Degradation of Poly(3-Hydroxyalkanoates)

Poly(3-hydroxybutyrate) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) were fermented to methane and carbon dioxide within 16 days by an anaerobic sewage sludge consortium. The cultures adapted quickly to metabolize these polymeric compounds, and between 83 and 96% of the substrate carbon was transformed to methane and carbon dioxide.     

Purification and characterization of an extracellular poly(3-hydroxybutyrate-co-3-hydroxyvalerate) depolymerase from Acidovorax sp. HB01

The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading strain Acidovorax sp. HB01 was isolated from an activated sludge sample. A novel PHBV depolymerase with a molecular weight of 43.4 kDa was purified to homogeneity from the culture supernatant of the HB01 strain. The optimum pH and temperature of the PHBV depolymerase were 7.0 and 50 °C, respectively. The PHBV depolymerase can also degrade polyhydroxybutyrate, poly (3-hydroxybutyrate-co-4-hydroxybutyrate), and poly(caprolactone); however, the PHBV degradation activity of the depolymerase is higher than its activity against the other polymers. Effect of metal ions and various inhibitors on the PHBV depolymerase activity was examined. The addition of Na(+), K(+), and Ca(2+) markedly increased the hydrolysis rate, whereas the enzyme activity was inhibited by Zn(2+), Mg(2+), Mn(2+), and particularly by Cu(2+) and Fe(2+). Ethylenediaminetetraacetic acid was found to have a significant inhibitory effect. The main degradation product of depolymerase was identified as the 3-hydroxybutyric acid monomer and 3-hydroxyvaleric acid monomers via mass spectrometry.     

Degradation of microbial polyester poly(3-hydroxybutyrate) in environmental samples and in culture

Poly(3-hydroxybutyrate) [P(3HB)] test-pieces prepared from the polymer produced by Azotobacter chroococcum were degraded in natural environments like soil, water, compost and sewage sludge incubated under laboratory conditions. Degradation in terms of % weight loss of the polymer was maximum (45%) in sewage sludge after 200 days of incubation at 30°C. The P(3HB)-degrading bacterial cultures (36) isolated from degraded test-pieces showed different degrees of degradation in polymer overlayer method. The extent of P(3HB) degradation increases up to 12 days of incubation and was maximum at 30°C for majority of the cultures. For most efficient cultures the optimum concentration of P(3HB) for degradation was 0.3% (w/v). Supplementation of soluble carbon sources like glucose, fructose and arabinose reduced the degradation while it was almost unaffected with lactose. Though the cultures degraded P(3HB) significantly, they were comparatively less efficient in utilizing copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(3HB-co-3HV)].     

Degradation of poly(3-hydroxybutyrate) by soil streptomycetes

The ability of 64 soil streptomycetes to degrade poly(3-hydroxybutyrate) [P(3HB)] was evaluated on Pridham and Lyons mineral salts agar medium overlayered with the same medium containing 0.2% P(3HB). The streptomycete isolates were grown on this overlayered medium and the degradation was detected by the formation of clear zone surrounding the growth. Four potent degrader isolates identified as species of Streptomyces were selected. Degradation of P(3HB) by these isolates was studied for a period of 8 days. The rate of degradation increased with increase in concentration of P(3HB) in the medium while it decreased with the supplementation of readily utili- zable carbon sources like glucose, fructose and sucrose. All four isolates also degraded the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(3HB–co–3HV)] in solid medium but to a lesser extent. However, the isolates were equally efficient in degrading P(3HB) in liquid medium.     

Biosynthesis of Poly(3-Hydroxybutyrate) and Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) and Its Regulation in Bacteria

Recent data on the biosynthesis of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and its regulation in bacteria are reviewed, with special emphasis on the properties and regulation of the relevant enzymes and their genes. Some conditions promoting the synthesis of PHB and PHBV by natural, mutant, and recombinant producers are considered.     

Identity and potential functions of heterotrophic bacterial isolates from a continuous-upflow fixed-bed reactor for denitrification of drinking water with bacterial polyester as source of carbon and electron donor

A collection of 186 heterotrophic bacteria, isolated directly from a continuous-upflow fixed-bed reactor for the denitrification of drinking water, in which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) granules acted as biofilm carrier, carbon source and electron donor, was studied with regard to taxonomic affiliation and degradation and denitrification characteristics. Two granule samples were taken from a fully operating reactor for enumeration and isolation of heterotrophic bacteria. One sample was drawn from the lower part of the reactor, near the oxic zone, and the other sample from the upper, anoxic part of the fixed bed. Dominant colonies were isolated and the cultures were identified using fatty acid analysis and 16S rDNA sequencing. Their ability to degrade the polymer and 3-hydroxybutyrate and to denitrify in pure culture was assessed. The results show that high numbers of heterotrophic bacteria were present in the biofilms on the polymer granules, with marked differences in taxonomic composition and potential functions between the lower and upper part of the fixed bed. The majority of the isolates were Gram negative bacteria, and most of them were able to reduce nitrate to nitrite or to denitrify, and to utilize 3-hydroxybutyrate as sole source of carbon. Only two groups, one identified as Acidovorax facilis and the other phylogenetically related to Brevundimonas intermedia, could combine denitrification and utilization of poly(3-hydroxybutyrate) (PHB), and were found only in the upper sample. The other groups occurred either in the lower or upper part, or in both samples. They were assigned to Brevundimonas, Pseudomonas, Agrobacterium, Achromobacter, or Phyllobacterium, or were phylogenetically related to Afipia or Stenotrophomonas.     

Production in Escherichia coli of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with differing monomer compositions from unrelated carbon sources

The industrial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been hindered by high cost and a complex control strategy caused by the addition of propionate. In this study, based on analysis of the PHBV biosynthesis process, we developed a PHBV biosynthetic pathway from a single unrelated carbon source via threonine biosynthesis in Escherichia coli. To accomplish this, we (i) overexpressed threonine deaminase, which is the key factor for providing propionyl-coenzyme A (propionyl-CoA), from different host bacteria, (ii) removed the feedback inhibition of threonine by mutating and overexpressing the thrABC operon in E. coli, and (iii) knocked out the competitive pathways of catalytic conversion of propionyl-CoA to 3-hydroxyvaleryl-CoA. Finally, we constructed a series of strains and mutants which were able to produce the PHBV copolymer with differing monomer compositions in a modified M9 medium supplemented with 20 g/liter xylose. The largest 3-hydroxyvalerate fraction obtained in the copolymer was 17.5 mol%.     

Fungal contribution to in situ biodegradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) film in soil

The contribution of fungi to the microbial degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films in soil was studied. Various traces, cavities, and grooves observed on the dented surface of PHBV films demonstrated that the degradation was a concerted effect of a microbial consortium colonizing the film surface, including fungi, bacteria, and actinomycetes. The succession of microbial consortia in the soil around the PHBV films during the degradation showed a distinctive increase in the fungal population, resulting in its dominance. Comparison of the degradation ability of microbial strains isolated from soil where PHBV films were degraded, revealed that fungi showed the highest contribution to PHBV degradation, growing very rapidly along the film surface with their high degradation ability and then expanding their hyphae in a three-dimensional manner.     

Degradation of polyesters by a novel marine Nocardiopsis aegyptia sp. nov.: application of Plackett-Burman experimental design for the improvement of PHB depolymerase activity

This is the first report on the degradation of poly(3-hydroxybutyrate) (PHB), and its copolymers poly(3-hydroxyvalerate) P(3HB-co-10-20% HV) by Nocardiopsis aegyptia, a new species isolated from marine seashore sediments. The strain excreted an extracellular PHB depolymerase and grew efficiently on PHB or its copolymers as the sole carbon sources. The degradation activity was detectable by the formation of a transparent clearing zone around the colony on an agar Petri plate after 25 days, or a clearing depth under the colony in test tubes within 3 weeks. The previous techniques proved that the bacterium was able to assimilate the monomeric components of the shorter alkyl groups of the polymers. Nocardiopsis aegyptia hydrolyzed copolymers 10-20% PHBV more rapidly than the homopolymer PHB. The bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate), and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). The samples were degraded at the surface and proceeded to the inner part of the materials. Clear morphological alterations of the polymers were noticed, indicating the degradative capability of the bacterium. Plackett-Burman statistical experimental design has been employed to optimize culture conditions for maximal enzyme activity. The main factors that had significant positive effects on PHB depolymerase activity of Nocardiopsis aegyptia were sodium gluconate, volume of medium/flask and age of inoculum. On the other hand, MgSO4.7H2O, KH2PO4, K2HPO4 and NH4NO3 exhibited negative effects. Under optimized culture conditions, the highest activity (0.664 U/mg protein) was achieved in a medium predicted to be near optimum containing (in g/L): PHB, 0.5; C6H11O7Na, 7.5; MgSO4.7H2O, 0.35; K2HPO4, 0.35; NH4NO3, 0.5; KH2PO4, 0.35; malt extract, 0.5 and prepared with 50% seawater. The medium was inoculated with 1% (v/v) spore suspension of 7 days old culture. Complete clarity of the medium was achieved after 3 days at 30 degrees C.     

3-Hydroxybutyrate Oligomer Hydrolase and 3-Hydroxybutyrate Dehydrogenase Participate in Intracellular Polyhydroxybutyrate and Polyhydroxyvalerate Degradation in Paracoccus denitrificans

Genes encoding 3-hydroxybutyrate oligomer hydrolase (PhaZc) and 3-hydroxybutyrate dehydrogenase (Hbd) were isolated from Paracoccus denitrificans. PhaZc and Hbd were overproduced as His-tagged proteins in Escherichia coli and purified by affinity and gel filtration chromatography. Purified His-tagged proteins had molecular masses of 31 kDa and 120 kDa (a tetramer of 29-kDa subunits). The His-tagged PhaZc hydrolyzed not only 3-hydroxybutyrate oligomers but also 3-hydroxyvalerate oligomers. The His-tagged Hbd catalyzed the dehydrogenation of 3-hydroxyvalerate as well as 3-hydroxybutyrate. When both enzymes were included in the same enzymatic reaction system with 3-hydroxyvalerate dimer, sequential reactions occurred, suggesting that PhaZc and Hbd play an important role in the intracellular degradation of poly(3-hydroxyvalerate). When the phaZc gene was disrupted in P. denitrificans by insertional inactivation, the mutant strain lost PhaZc activity. When the phaZc-disrupted P. denitrificans was complemented with phaZc, PhaZc activity was restored. These results suggest that P. denitrificans carries a single phaZc gene. Disruption of the phaZc gene in P. denitrificans affected the degradation rate of PHA.     

Production of targeted poly(3-hydroxyalkanoates) copolymers by glycogen accumulating organisms using acetate as sole carbon source

One of the main limitations in bacterial polyhydroxyalkanoate (PHA) production with mixed cultures is the fact that primarily polyhydroxybutyrate (PHB) homopolymers are generated from acetate as the main carbon source, which is brittle and quite fragile. The incorporation of different 3-hydroxyalkanoate (HA) components into the polymers requires the addition of additional carbon sources, leading to extra costs and complexity. In this study, the production of poly(3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)-co-3-hydroxy-2-methylvalerate (3HMV)), with 7-35C-mol% of 3HV fractions from acetate as the only carbon source was achieved with the use of glycogen accumulating organisms (GAOs). An enriched GAO culture was obtained in a lab-scale reactor operated under alternating anaerobic and aerobic conditions with acetate fed at the beginning of the anaerobic period. The production of PHAs utilizing the enriched GAO culture was investigated under both aerobic and anaerobic conditions. A polymer content of 14-41% of dry cell weight was obtained. The PHA product accumulated by GAOs under anaerobic conditions contained a relatively constant proportion of non-3HB monomers (30+/-5C-mol%), irrespective of the amount of acetate assimilated. In contrast, under aerobic conditions, GAOs only produced 3HB monomers from acetate causing a gradually decreasing 3HV fraction during this aerobic feeding period. The PHAs were characterized by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The data demonstrated that the copolymers possessed similar characteristics to those of commercially available poly(3HB-co-3HV) (PHBV) products. The PHAs produced under solely anaerobic conditions possessed lower melting points and crystallinity, higher molecular weights, and narrower molecular-weight distributions, compared to the aerobically produced polymers. This paper hence demonstrates the significant potential of GAOs to produce high quality polymers from a simple and cheap carbon source, contributing considerably to the growing research body on bacterial PHA production by mixed cultures.     

Effect of organosoluble salts on the nanofibrous structure of electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Electrospinning of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in chloroform was investigated to develop non-woven biodegradable nanofibrous structures for tissue engineering. Ultrafine PHBV fibers were obtained by electrospinning of 20 wt.% PHBV solution in chloroform and the resulting fiber diameters were in the range of 1.0-4.0 microm. When small amounts of benzyl trialkylammonium chlorides were added to the PHBV solution, the average diameter was decreased to 1.0 microm and the fibers were amounted in a straight shape. Conductivity of the PHBV solution was a major parameter affecting the morphology and diameter of the electrospun PHBV fibers. PHBV non-woven structures electrospun with salt exhibited a higher degradation rate than those prepared without salt probably due to the increase of surface area of PHBV fibers.     

Hydrolytic Degradation of Poly(3-Hydroxybutyrate) and Its Copolymer with 3-Hydroxyvalerate of Different Molecular Weights in vitro

The hydrolytic degradation of polymer films of poly(3-hydroxybutyrate) of different molecular weights and its copolymers with 3-hydroxyvalerate (9 mol % 3-hydroxyvalerate in the poly(3-hydroxybutyrate) chain) of different molecular weights was studied in model conditions in vitro. The changes in the physicochemical properties of the polymers were investigated using different analytical techniques: viscometry, differential scanning calorimetry, gravimetrical method, and water contact angle measurement for polymers. The data showed that in a period of 6 months the weight of polymer films decreased insignificantly. The molecular weight of the samples was reduced significantly; the largest decline (up to 80% of the initial molecular weight of the polymer) was observed in the high-molecular-weight poly(3-hydroxybutyrate). The surface of all investigated polymers became more hydrophilic. In this work, we focus on a mathematical model that can be used for the analysis of the kinetics of hydrolytic degradation of poly(3-hydroxyaklannoate)s by noncatalytic and autocatalytic hydrolysis mechanisms. It was also shown that the degree of crystallinity of some polymers changes differently during degradation in vitro. Thus, the studied polymers can be used to develop biodegradable medical devices such that they can perform their functions for a long period of time.