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.     

Physico-chemical properties of polyhydroxyalkanoate produced by mixed-culture nitrogen-fixing bacteria

Ultra-high molecular weight polyhydroxyalkanoates (PHAs) with low polydispersity index (PDI = 1.3) were produced in a novel, pilot scale application of mixed cultures of nitrogen-fixing bacteria. The number average molecular weight (M _n) of the poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was determined to be 2.4 × 10⁶ and 2.5 × 10⁶ g mol⁻¹, respectively. Using two types of carbon sources, biomass contents of the P(3HB) and P(3HB-co-3HV) were 18% and 30% (PHA in dry biomass), respectively. The extracted polymers were analysed for their physical properties using analytical techniques such as nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC) and gel permeation chromatography (GPC). NMR confirmed the formation of homopolymer and copolymer. DSC showed a single melting endotherm peak for both polymers, with enthalpies that indicated crystallinity indices of 44% and 37% for P(3HB) and P(3HB-co-3HV), respectively. GPC showed a sharp unimodal trace for both polymers, reflecting the homogeneity of the polymer chains. The work described here emphasises the potential of mixed colony nitrogen-fixing bacteria cultures for producing biodegradable polymers which have properties that are very similar to those from their pure-culture counterparts and therefore making a more economically viable route for obtaining biopolyesters.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator from waste rapeseed oil using propanol as a precursor of 3-hydroxyvalerate

Waste rapeseed oil is a useful substrate for polyhydroxyalkanoates (PHA) production employing Cupriavidus necator H16. In fed-batch mode, we obtained biomass and PHA yields of 138 and 105 g l⁻¹, respectively. Yield coefficient and volumetric productivity were 0.83 g PHA per g oil and 1.46 g l⁻¹ h⁻¹, respectively. Propanol at 1% (v/v) enhanced both PHA and biomass formation significantly and, furthermore, resulted in incorporation of 3-hydroxyvalerate units into PHA structure. Thus, propanol can be used as an effective precursor of 3-hydroxyvalarete for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. During the fed-batch cultivation, propanol concentration was maintained at 1% which resulted in 8% content of 3-hydroxyvalerate in copolymer.     

The synthesis of short- and medium-chain-length poly(hydroxyalkanoate) mixtures from glucose- or alkanoic acid-grown <Emphasis Type="Italic">Pseudomonas oleovorans</Emphasis>

Pseudomonas oleovorans NRRL B-778 accumulated mixtures of poly-3-hydroxybutyrate (PHB) and medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) when grown on glucose, octanoic acid or oleic acid, whereas growth on nonanoic acid or undecanoic acid resulted in copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV). Acetone fractionation verified the presence of PHB/mcl-PHA mixtures. The acetone-insoluble (AIS) fractions of the polymers derived from glucose (PHA-glucose), octanoic acid (PHA-octanoic) and oleic acid (PHA-oleic) were exclusively PHB while the acetone-soluble (AS) fractions contained mcl-PHA composed of differing ratios of 3-hydroxy-acid monomer units, which ranged in chain length from 6 to 14 carbon atoms. In contrast, both the AIS and AS fractions from the polymers derived from nonanoic acid (PHA-nonanoic) and undecanoic acid (PHA-undecanoic) were composed of comparable ratios of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). The unfractionated PHA-glucose, PHA-octanoic and PHA-oleic polymers had melting temperatures (T _m) between 177 and 179°C, enthalpies of fusion (ΔH _f) of 20 cal/g and glass transition temperatures (T _g) of 3–4°C. This was due to the large PHB content in the polymer mixtures. On the other hand, the PHA-nonanoic and PHA-undecanoic polymers had thermal properties that supported their copolymer nature. In both cases, the T _m values were 161°C, ΔH _f values were 7cal/g and T _g values were −3°C. Journal of Industrial Microbiology & Biotechnology (2002) 28, 147–153 DOI: 10.1038/sj/jim/7000231 Received 30 July 2001/ Accepted in revised form 04 November 2001     

Synthesis of biodegradable polyesters by Gram negative bacterium isolated from Malaysian environment

A locally isolated Gram negative bacterium, Cupriavidus sp. USMAA9-39 was able to produce various types of biodegradable polyesters through a two-step cultivation process. These are copolymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)]. These polymers were synthesized by this bacterium when grown with a combination of some carbon sources. The biosynthesis of P(3HB-co-4HB) was achieved by using carbon sources such as γ-butyrolactone or 1,4-butanediol or by a combination of oleic acid with either γ-butyrolactone or 1,4-butanediol. Meanwhile, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was produced using 1-pentanol or valeric acid or by a combination of oleic acid with either 1-pentanol or valeric acid. When γ-butyrolactone or 1,4-butanediol with either valeric acid or 1-pentanol were used as mixed carbon sources, P(3HB-co-3HV-co-4HB) terpolymer were produced. The presence of 3HB, 3HV or/and 4HB monomers were confirmed by gas chromatography and nuclear magnetic resonance (NMR) spectroscopy.     

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.     

The Terpolymer Produced by Azotobacter Chroococcum 7B: Effect of Surface Properties on Cell Attachment

The copolymerization of poly(3-hydroxybutyrate) (PHB) is a promising trend in bioengineering to improve biomedical properties, e.g. biocompatibility, of this biodegradable polymer. We used strain Azotobacter chroococcum 7B, an effective producer of PHB, for biosynthesis of not only homopolymer and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also novel terpolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-poly(ethylene glycol) (PHB-HV-PEG), using sucrose as the primary carbon source and valeric acid and poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-HV-PEG was confirmed by ¹H nuclear-magnetic resonance analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) of produced biopolymer, the protein adsorption to the terpolymer, and cell growth on biopolymer films were studied. Despite of low EG-monomers content in bacterial-origin PHB-HV-PEG polymer, the terpolymer demonstrated significant improvement in biocompatibility in vitro in contrast to PHB and PHB-HV polymers, which may be coupled with increased protein adsorption, hydrophilicity and surface roughness of PEG-containing copolymer.     

Microbial production of polyhydroxyalkanoate block copolymer by recombinant Pseudomonas putida

Polyhydroxyalkanoate (PHA) synthesis genes phaPCJ _Ac cloned from Aeromonas caviae were transformed into Pseudomonas putida KTOY06ΔC, a mutant of P. putida KT2442, resulting in the ability of the recombinant P. putida KTOY06ΔC (phaPCJ _A.c ) to produce a short-chain-length and medium-chain-length PHA block copolymer consisting of poly-3-hydroxybutyrate (PHB) as one block and random copolymer of 3-hydroxyvalerate (3HV) and 3-hydroxyheptanoate (3HHp) as another block. The novel block polymer was studied by differential scanning calorimetry (DSC), nuclear magnetic resonance, and rheology measurements. DSC studies showed the polymer to possess two glass transition temperatures (T _g), one melting temperature (T _m) and one cool crystallization temperature (T _c). Rheology studies clearly indicated a polymer chain re-arrangement in the copolymer; these studies confirmed the polymer to be a block copolymer, with over 70 mol% homopolymer (PHB) of 3-hydroxybutyrate (3HB) as one block and around 30 mol% random copolymers of 3HV and 3HHp as the second block. The block copolymer was shown to have the highest tensile strength and Young’s modulus compared with a random copolymer with similar ratio and a blend of homopolymers PHB and PHVHHp with similar ratio. Compared with other commercially available PHA including PHB, PHBV, PHBHHx, and P3HB4HB, the short-chain- and medium-chain-length block copolymer PHB-b-PHVHHp showed differences in terms of mechanical properties and should draw more attentions from the PHA research community.     

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)].     

盐单胞菌利用短链脂肪酸合成聚羟基脂肪酸酯

盐单胞菌(Halomonas)能够利用多种底物为碳源生长,由于其能在高盐条件下进行不灭菌的开放发酵,已被开发用作下一代生物技术的底盘细胞.包括乙酸、丙酸和丁酸在内的短链挥发性脂肪酸能够以生物质为原料制备,有望成为用于微生物发酵的新型碳源.利用10-50g/L浓度的丁酸为碳源对Halomonas sp.TD01和TD08...     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer by <Emphasis Type="Italic">Azotobacter chroococcum</Emphasis> strain 7B

The ability of Azotobacter chroococcum strain 7B, producer of poly(3-hydroxybutyrate) (PHB), to synthesize its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was studied. It was demonstrated, for the first time, that A. chroococcum strain 7B was able to synthesize P(3HB-co-3HV) with various molar rates of HV in the polymer chain when cultivated on medium with sucrose and carboxylic acids as precursors of HV elements in the PHB chain, namely, valeric (13.1–21.6 mol %), propanoic (3.1 mol %), and hexanoic (2.1 mol %) acids. Qualitative and functional differences between PHB and P(3HB-co-3HV) were demonstrated by example of the release kinetic of methyl red from films made of synthesized polymers. Maximal HV incorporation into the polymer chain (28.8mol %) was recorded when the nutrient medium was supplemented with 0.1% peptone on the background of 20 mM valerate. These results suggest that that the studied strain can be regarded as a potential producer of not only PHB but also P(3HB-co-3HV).     

Production of copolymer consisting of 3-hydroxybutyrate and 3-hydroxyvalerate by fed-batch culture of Alcaligenes SP. SH-69

Summary Production of copolymer consisting of 3-hydroxybutyrate and 3-hydroxyvalerate [poly(3HB-co-3HV)] by fed-batch culture of Alcaligenes sp. SH-69 was investigated using glucose as a sole carbon source. Synthesis of poly(3HB-co-3HV) during the polymer accumulation stage was favored under dissolved oxygen tension at 20% and C/N ratio (mol glucose/mol ammonium) of 23.1. When conditions were optimal, 36 g liter^-1 of poly(3HB-co-3HV) containing 3.0 mol% of 3HV was produced. Decreasing C/N ratio resulted in an increase of 3HV fraction in the copolymer to a maximum level of 6.3 mol%.     

Production of short-side-chain polyhydroxyalkanoates by various bacteria from the rRNA superfamily III

 A group of 13 bacterial species from the rRNA superfamily III were tested for their ability to produce the biodegradable polyesters poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3-HB-co-4-HB)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate [P(3HB-co-3-HV)]. Screening for polyhydroxyalkanoate production was performed on cultures obtained from solid media, rolling cultures and shaking flasks. All 13 species were able to store a poly(3-hydroxybutyrate) homopolymer, 12 species could produce P(3-HB-co-3-HV) copolymers, but only 9 species accumulated P(3-HB-co-4-HB) copolymers. Similarities in polyhydroxyalkanoate-accumulation behaviour between closely related strains were noted. Received: 18 December 1995/Received revision: 3 April 1996/Accepted: 28 May 1996     

Chemical recycling of polyhydroxyalkanoates as a method towards sustainable development

Chemical recycling of bio-based polymers polyhydroxyalkanoates (PHAs) by thermal degradation was investigated from the viewpoint of biorefinery. The thermal degradation resulted in successful transformation of PHAs into vinyl monomers using alkali earth compound (AEC) catalysts. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s (PHBVs) were smoothly and selectively depolymerized into crotonic (CA) and 2-pentenoic (2-PA) acids at lower degradation temperatures in the presence of CaO and Mg(OH)₂ as catalysts. Obtained CA from 3-hydroxybutyrate sequences in PHBV was copolymerized with acrylic acid to produce useful water-soluble copolymers, poly(crotonic acid-co-acrylic acid) that have high glass-transition temperatures. The copolymerization of CA derived from PHA pyrolysis is an example of cascade utilization of PHAs, which meets the idea of sustainable development.     

Degradation of aliphatic polyester films by commercially available lipases with special reference to rapid and complete degradation of poly(L-lactide) film by lipase PL derived from Alcaligenes sp

Commercial lipases were examined for their degradation efficiency of aliphatic polyester films. In 100 days immersion of polyester films in lipase solutions at37 °C at pH 7.0,Lipase Asahi derived from Chromobacterium viscosum degraded polybutylene succinate-co-adipate (PBSA), poly (-caprolactone) (PCL) and polybutylene succinate (PBS), and Lipase F derived from Rhizopus niveus degraded PBSA and PCL during 4–17 days. Lipase F-AP15 derived fromRhizopus orizae could degrade PBSA in 22 days. In these cases, PBS and PBSA were mainly degraded to dimers, whereas PCL was mainly degraded to monomers. Only poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHB/V) and poly (L-lactide) (PLA) were not degraded in the experiments. However, PLA degraded completely at 55 °C, pH 8.5 with Lipase PL during 20 days. This result could be explained with the sequential reactions of the chemical hydrolysis of the polymer to oligomers at higher pH and temperature, and the succeeding enzymatic hydrolysis of oligomers to the monomers.     

Biosynthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) polymers

In support of programs to identify polyhydroxyalkanoates with improved materials properties, we report on our efforts to characterize the mechanical and thermal properties of copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). The copolyesters, having molar fraction of 3HHx ranging from 2.5 to 35 mol % and average molecular weights ranging from 1.15 x 10(5) to 6.65 x 10(5), were produced by fermentation using Aeromonas hydrophila and a recombinant strain of Pseudomonas putida GPp104. The polymers were chloroform extracted and characterized by solution-state and solid-state nuclear magnetic resonance (NMR) spectroscopy and a variety of mechanical and thermal tests. Solution-state (1)H NMR data were used to determine polymer composition-of-matter, while solution-state (13)C NMR data provided polymer-sequence information. Solvent fractionation and NMR spectroscopic characterization of these polymers showed that polymers containing up to 9.5 mol % 3HHx had a Bernoullian compositional distribution. By contrast, polymers containing more than 9.5 mol % 3HHx had a bimodal polymer composition. Solvent fractionation of these 3HHx-rich polyesters produced two polymer fractions, each of which was again consistent with Bernoullian polymerization statistics. Solid-state NMR relaxation experiments provided insight into aging in poly(3HB-co-3HHx) copolymers, demonstrating increased polymer-chain motion with increasing 3HHx content. The elongation-to-break ratio in the polyesters increased with increasing molar fraction of 3HHx monomers. Aging properties of the poly(3HB-co-3HHx) copolymers were very similar to copolymers of 3HB and 3-hydroxyvalerate (3HV). However, poly(3HB-co-3HHx) exhibited increased activation energy to thermal degradation with increasing 3HHx content.     

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch cultures of Pseudomonas sp EL-2

Pseudomonas sp EL-2 was cultivated to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from a structurally unrelated carbon source, glucose, by a fed-batch culture technique. Variation of the carbon to nitrogen (C/N) ratio of the medium produced optimal P(3HB-co-3HV) production at a C/N ratio of 95. Production of P(3HB-co-3HV) was favored by a dissolved oxygen tension of 40%. A maximum biomass concentration of 38 g L⁻¹ containing 53% P(3HB-co-3HV) was achieved after 45 h of cultivation. This corresponds to a volumetric productivity of 0.84 g L⁻¹ h⁻¹. The copolymer contained 7.5 mol% 3-hydroxyvalerate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 36–40. Received 28 January 1999/ Accepted in revised form 11 September 1999     

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.     

Incorporation of polyethylene glycol in polyhydroxyalkanoic acids accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 (MTCC 3853), a free-living nitrogen-fixing bacterium accumulates poly(3-hydroxybutyric acid) [PHB, 69% of cell dry weight (CDW)] when grown on glucose and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBV with 19.2 mol% 3HV] when grown on glucose and valerate. Use of ethylene glycol (EG) and/or polyethylene glycols (PEGs) of low molecular weight as sole carbon source were detrimental to A. chroococcum growth and polymer yields. PEG-200, however, in the presence of glucose was incorporated into the polyhydroxyalkanoate (PHA) polymer. Addition of PEG-200 (150 mM) to culture medium during mid-log phase growth favored increased incorporation of EG units (12.48 mol%) into the PHB polymer. In two-step culture experiments, where valerate and PEG simultaneously were used in fresh medium, EG was incorporated most effectively in the absence of glucose, leading to the formation of a copolymer containing 18.05 mol% 3HV and 14.78 mol% EG. The physico-mechanical properties of PEG-containing copolymer (PHBV–PEG) were compared with those of the PHB homopolymer and the PHBV copolymer. The PHBV–PEG copolymer appeared to have less crystallinity and greater flexibility than the short-chain-length (SCL) PHA polymers.