Production of copolymer poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) through a one-step cultivation process

A one-step cultivation process for the production of biodegradable polymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] by Cupriavidus sp. USMAA2-4 was carried out using various carbon sources. It was found that Cupriavidus sp. USMAA2-4 could produce approximately 44 wt.% copolymer of P(3HB-co-4HB) with 27 mol% 4HB composition when the combination of oleic acid and 1,4-butanediol are used as carbon sources in 60 h cultivation. The manipulation of carbon-to-nitrogen ratio (C/N) resulted in the increase of dry cell weight, PHA content as well as 4HB composition. A new strategy of introducing oleic acid and 1,4-butanediol together and separately at different concentration demonstrated different yield in PHA content ranging from 47 to 58 wt.%. The molecular weight obtained was 234 kDa (by adding 1,4-butanediol and oleic acid together) and 212 kDa (by adding 1,4-butanediol separately). The copolymer of P(3HB-co-4HB) produced by Cupriavidus sp. USMAA2-4 was detected statistically as a random copolymer when analysed by nuclear magnetic resonance (NMR) spectroscopy.     

Biosynthesis of Poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) terpolymer by <Emphasis Type="Italic">Cupriavidus</Emphasis> sp. USMAA2-4 through two-step cultivation process

A locally isolated Gram-negative bacterium, Cupriavidus sp. USMAA2-4 was found capable of producing terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] using γ-butyrolactone or 1,4-butanediol with either valeric acid or 1-pentanol as the carbon source. The present of 3HB, 3HV and 4HB monomers were confirmed by gas chromatography (GC) and nuclear magnetic resonance (NMR) analysis. PHA concentration of 1.9 g/l was the highest value obtained using the combination of 1,4-butanediol and 1-pentanol through one-step cultivation process. PHA concentration obtained through two-step cultivation process was higher for all the combinations and the highest value achieved was 2.5 g/l using γ-butyrolactone and 1-pentanol as carbon source. Various molar fractions of 4HB and 3HV ranging from 6 to 14 mol% and 39 to 87 mol%, respectively were produced through two-step cultivation process by manipulating the concentration of γ-butyrolactone. As the culture aeration was reduced, the molar fraction of 3HV and 4HB increased from 40 to 67 mol% and 10 to 24 mol%, respectively while the dry cell weight and PHA content decreased. The terpolymer produced was characterized using gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The number-average molecular weight (M _n) and the melting temperature (T _m)) of the terpolymer were in the range of 177–484 kDa and 160–164°C, respectively.     

Characteristics of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production byRalstonia eutropha NCIMB 11599 and ATCC 17699

Ralstonia eutropha NCIMB 11599 and ATCC 17699 were grown, and their productions of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] compared. In flask cultures ofR. eutropha NCIMB 11599, cell concentration, P(3HB-co-4HB) concentration and polymer content decreased considerably with increases in the γ-butyrolactone concentration, and the 4HB fraction was also very low (maximum 1.74 mol%). In fed-batch cultures ofR. eutropha NCIMB 11599, glucose and γ-butyrolactone were fed as the carbon sources, under a phosphate limitation strategy. When glucose was fed as the sole carbon source, with its concentration controlled using an on-line glucose analyzer, 86% of the P(3HB) homopolymer was obtained from 201 g/L of cells. In a two-stage fed-batch culture, where the cell concentration was increased to 104 g/L, with glucose fed in the first step and constant feeding of γ-butyrolactone, at 6 g/h, in the second, final cell concentration at 67 h was 106 g/L, with a polymer content of 82%, while the 4HB fraction was only 0.7 mol%. When the same feeding strategy was applied to the fedbatch culture ofR. eutropha ATCC 17699, where the cell concentration was increased to 42 g/L, by feeding fructose in the first step and γ-butyrolactone (1.5 g/h) in the second, the final cell concentration, polymer content and 4HB fraction at 74 h were 51 g/L, 35% and 32 mol%, respectively. In summary,R. eutropha ATCC 17699 was better thanR. eutropha NCIMB 11599 in terms of P(3HB-co-4HB) production with various 4HB fractions.     

Isolation of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) producer from Malaysian environment using γ-butyrolactone as carbon source

Samples from various natural environments in Peninsular Malaysia were screened for microorganisms that are capable of producing poly(3-hydroxybutyrate-co-4-hydroxybutyrate). A total of 663 isolates were isolated and 119 out of these isolates were identified as possible PHA producers based on Nile red staining methods. All these potential producers emitted pink fluorescence when grown on solid mineral salts medium (MSM) containing Nile red and exposed to UV light. The isolates obtained in this study were cultivated in MSM containing γ-butyrolactone as the carbon source. Gas chromatography (GC) analysis confirmed that 95 out of the 119 isolates were PHA producers. Among the 95 positive isolates, 77 isolates produced only P(3HB) homopolymer and 18 isolates produced PHA containing 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) monomers. Of these 18 isolates, USMAA1020 was screened as the best P(3HB-co-4HB) producer based on GC analysis. For further confirmation, PHA was extracted from the isolate and analyzed by GC as well as nuclear magnetic resonance (NMR). Results from both analyses confirmed that this isolate was capable of producing PHA containing 3HB and 4HB. Based on, biochemical characterization, 16S rRNA sequencing, DNA base composition, cellular fatty acids analysis and DNA–DNA hybridization, it is clearly indicated that this isolate belongs to the genus Cupriavidus. Poly(3HB-co-4HB) was synthesized by this bacterium in one-stage, two-stage and three-stage cultivation using γ-butyrolactone as the carbon source. The highest 4HB composition of 82 mol% was obtained through three-stage cultivation.     

Purification and Characterization of Fungal Poly(3-hydroxybutyrate) Depolymerase from Paecilomyces lilacinus F4-5 and Enzymatic Degradation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Film

Summary Poly(3-hydroxybutyrate) [P(3HB)] depolymerase was purified from a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]-degrading fungus, Paecilomyces lilacinus F4-5 by hydrophobic and ion exchange column chromatography, and showed a molecular mass of 45 kDa. The optimum temperature and pH of the P(3HB) depolymerase were 50 °C and 7.0, respectively. The enzyme was stable for at least 30 min at temperatures below 40 °C, while the activity abruptly decreased over 55 °C. Enzymatic P(3HB-co-3HV) degradation showed a similar degradation pattern to that of film overlaid by fungal hyphae. It reflects that the fungal degradation of P(3HB-co-3HV) in soil is mainly caused by extracellular depolymerases.     

Improved production of poly(3-hydroxybutyrate-co-4-hydroxbutyrate) copolymer using a combination of 1,4-butanediol and γ-butyrolactone

A locally isolated Gram-negative bacterium, Cupriavidus sp. USMAA2-4 was able to synthesize poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] when fed with the precursor carbon 1,4-butanediol using a two-stage cultivation process. When 1% (w/v) of 1,4-butanediol was used, 31 wt.% of P(3HB-co-4HB) copolymer with 41 mol.% of 4HB molar fraction was produced. Both the PHA content and 4HB composition of the copolymer increased as the concentration of 1,4-butanediol increased but the cell biomass did not show any significant changes. However, the 4HB fraction could be further increased using a combination of γ-butyrolactone and 1,4-butanediol. As high as 84 mol.% of 4HB composition was achieved with a combination of 0.35% (w/v) 1,4-butanediol and 1.4% (w/v) γ-butyrolactone. Nevertheless, it was found that Cupriavidus sp. USMAA2-4 cells were inhibited by high concentration of γ-butyrolactone. P(3HB-co-4HB) copolymer was also successfully synthesized using a simplified aerated tank.     

Characterization of new isolated <Emphasis Type="Italic">Ralstonia</Emphasis><Emphasis Type="Italic">eutropha</Emphasis> strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) production

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and γ–hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of γ–hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments. The nucleotide sequence 1,378 bp reported in this study will appear in the GenBank nucleotide sequence database under accession number EF988626.     

Enhanced production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) copolymer with manipulated variables and its properties

Cupriavidus sp. USMAA1020, a local isolate was able to biosynthesis poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer with various 4HB precursors as the sole carbon source. Manipulation of the culture conditions such as cell concentration, phosphate ratio and culture aeration significantly affected the synthesis of P(3HB-co-4HB) copolymer and 4HB composition. P(3HB-co-4HB) copolymer with 4HB compositions ranging from 23 to 75 mol% 4HB with various mechanical and thermal properties were successfully produced by varying the medium aeration. The physical and mechanical properties of P(3HB-co-4HB) copolymers were characterized by NMR spectroscopy, gel-permeation chromatography, tensile test, and differential scanning calorimetry. The number-average molecular weights (M _n) of copolymers ranged from 260 × 10³ to 590 × 10³Da, and the polydispersities (M _w/M _n) were between 1.8 and 3.0. Increases in the 4HB composition lowered the molecular weight of these copolymers. In addition, the increase in 4HB composition affected the randomness of copolymer, melting temperature (T _m), glass transition temperature (T _g), tensile strength, and elongation to break. Enzymatic degradation of P(3HB-co-4HB) films with an extracellular depolymerase from Ochrobactrum sp. DP5 showed that the degradation rate increased proportionally with time as the 4HB fraction increased from 17 to 50 mol% but were much lower with higher 4HB fraction. Degradation of P(3HB-co-4HB) films with lipase from Chromobacterium viscosum exhibited highest degradation rate at 75 mol% 4HB. The biocompatibility of P(3HB-co-4HB) copolymers were evaluated and these copolymers have been shown to support the growth and proliferation of fibroblast cells.     

A high throughput Nile red fluorescence method for rapid quantification of intracellular bacterial polyhydroxyalkanoates

A rapid quantitative measurement of accumulated polyhydroxyalkanoate (PHA) is essential for rapid monitoring of PHA production by microorganisms. In the present study, a 96-well microplate was used as a high throughput means to measure the fluorescence intensity of the Nile red stained cells containing PHA. The linear correlation obtained between intracellular PHA concentration and the fluorescence intensity represents the potential of the Nile red method employment to determine PHA concentration. The optimal ranges of excitation and emission wavelengths were determined using bacterial cells containing different types of PHAs, of different co-monomers and compositions. Interestingly, in spite of different co-monomers compositions in each PHA, all tested PHAs fluoresced maximally at excitation wavelength between 520 and 550 nm, and emission wavelength between 590 and 630 nm. The developed staining method also had successfully demonstrated a good correlation between the amount of accumulated PHA based on the fluorescence intensity measurements and that from chromatographic analysis to evaluate poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)], using the same calibration curve, despite of different co-monomers that the PHA consist. Strongly supported by these experimental results, it can therefore be concluded that the developed staining method can be efficiently applied for rapid monitoring of PHA production.     

Biocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolyesters produced byAlcaligenes sp. MT-16

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), copolyesters, with 3-hydroxyvalerate (3HV) contents ranging from 17 to 60 mol%, were produced byAlcaligenes sp. MT-16, and their biocompatibility evaluated by the growth of Chinese hamster ovary (CHO) cells and the adsorption of blood proteins and platelets onto their film surfaces. The number of CHO cells that adhered to and grew on these films was higher with increasing 3HV content. In contrast, the tendency for blood proteins and platelets to adhere to the copolyester surfaces significantly decreased with increasing 3HV content. Examination of the surface morphology using atomic force microscopy revealed that the surface roughness was an important factor in determining the biocompatibility of theses copolyesters. The results obtained in this study suggest that poly(3HB-co-3HV) copolyesters, with >30 mol% 3HV, may be useful in biocompatible biomedical applications.     

Biosynthesis and Characterization of Poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) from Palm Oil Products in a Wautersia eutropha Mutant

Palm kernel oil, palm olein, crude palm oil and palm acid oil were used for the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by a mutant strain of Wautersia eutropha (formerly Ralstonia eutropha) harboring the Aeromonas caviae polyhydroxyalkanoate (PHA) synthase gene. Palm kernel oil was an excellent carbon source for the production of cell biomass and P(3HB-co-3HHx). About 87% (w/w) of the cell dry weight as P(3HB-co-3HHx) was obtained using 5 g palm kernel oil/l. Gravimetric and microscopic analyses further confirmed the high PHA content in the recombinant cells. The molar fraction of 3HHx remained constant at 5 mol % regardless of the type and concentration of palm oil products used. The small amount of 3HHx units was confirmed by ¹³C NMR analysis. The number average molecular weight (M_n) of the PHA copolymer produced from the various palm oil products ranged from 27 0000 to 46 0000 Da. The polydispersity was in the range of 2.6–3.9.     

Accumulation of PHA and its copolyesters by Methylobacterium sp. KCTC 0048

Summary Methylobacterium sp. KCTC 0048 isolated from soil, could synthesize a variety of copolyesters when secondary carbon substrates were added to nitrogen-limited cultures containing methanol as a major carbon and energy source. The copolyester of 3-hydroxy-butyrate and 3-hydroxyvalerate, P(3HB-co-3HV) accumulated when valeric acid, pentanol or heptanoic acid was added to the nitrogen-limited medium containing methanol. The copolyester of 3-hydroxybutyrate and 4-hydroxybutyrate, P(3HB-co-4HB) was synthesized from 4-hydroxybutyrate, 1,4-butanediol, or -butyrolactone, and the copolyester of 3-hydroxybutyrate and 3-hydroxypropionate (P(3HB-co-3HP)), from 3-hydroxypropionate as the secondary carbon substrates, respectively.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with high molar fractions of 3-hydroxyvalerate by a threonine-overproducing mutant of Alcaligenes sp. SH-69

A threonine overproducing mutant of Alcaligenes sp. SH-69 was isolated and its ability to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), was investigated. The 3HV fraction in poly(3HB-co-3HV) produced from glucose as the sole carbon source exceeded 22 mol%, which is approximately six times higher than that achieved by the wild type under the same culture conditions. Furthermore, the addition of a relatively low concentration (10 mM) of propionic acid, valeric acid or levulinic acid to the glucose medium greatly increased the molar fraction of 3HV in the copolyester, to 38–77 mol%. The results suggest that metabolic engineering of the biosynthetic pathways supplying polyhydroxyalkanoate monomers, such as the threonine biosynthetic pathway, can lead to new poly(3HB-co-3HV)-producing strains.     

Fermentative production of P(3HB-co-3HV) from propionic acid by Alcaligenes eutrophus in fed-batch culture with pH-stat continuous substrate feeding method

The feeding of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by Alcaligenes eutrophus ATCC17697 was optimized using a fed-batch culture system. The concentration of propionic acid was maintained at 3 g l^–1 as growth was inhibited by propionic acid in the broth. A pH-stat substrate feeding system was used in which propionic acid was fed automatically to maintain a pH of the culture broth at 7.0. By feeding a substrate solution containing 20% (w/v) propionic acid, 4.9% (w/v) ammonia water [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10] in cell growth phase, the concentration of propionic acid in the broth was maintained at 3 g l^–1 giving a specific growth rate of 0.4 h^–1. To promote P(3HB-co-3HV) production, two stage fed-batch culture which consisted of the stage for the cell growth and the stage for the P(3HB-co-3HV) accumulation was carried out. When the substrate solution whose C/N molar ratio was 50 was fed in P(3HB-co-3HV) accumulation phase, the cell concentration and the P(3HB-co-3HV) content in the cells reached 64 g l^–1 and 58% (w/w) in 55.5 h, respectively.     

Biosynthesis and characterization of poly(d-lactate-co-glycolate-co-4-hydroxybutyrate)

Poly(d -lactate-co-glycolate-co-4-hydroxybutyrate) [poly(d -LA-co-GA-co-4HB)] and poly(d -lactate-co-glycolate-co-4-hydroxybutyrate-co-d -2-hydroxybutyrate) [poly(d -LA-co-GA-co-4HB-co-d -2HB)] are of interest for their potential applications as new biomedical polymers. Here we report their enhanced production by metabolically engineered Escherichia coli. To examine the polymer properties, poly(d -LA-co-GA-co-4HB) polymers having various monomer compositions (3.4–41.0mol% of 4HB) were produced by culturing the engineered E. coli strain expressing xylBC from Caulobacter crescentus, evolved phaC1 from Pseudomonas sp. MBEL 6-19 (phaC1437), and evolved pct from Clostridium propionicum (pct540) in a medium supplemented with sodium 4HB at various concentrations. To produce these polymers without 4HB feeding, the 4HB biosynthetic pathway was additionally constructed by expressing Clostridium kluyveri sucD and 4hbD. The engineered E. coli expressing xylBC, phaC1437, pct540, sucD, and 4hbD successfully produced poly(d -LA-co-GA-co-4HB-co-d -2HB) and poly(d -LA-co-GA-co-4HB) from glucose and xylose. Through modulating the expression levels of the heterologous genes and performing fed-batch cultures, the polymer content and titer could be increased to 65.76wt% and 6.19g/L, respectively, while the monomer fractions in the polymers could be altered as desired. The polymers produced, in particular, the 4HB-rich polymers showed viscous and sticky properties suggesting that they might be used as medical adhesives.     

Polyhydroxyalkanoate copolymers from forest biomass

The potential for the use of woody biomass in poly-β-hydroxyalkanoate (PHA) biosynthesis is reviewed. Based on previously cited work indicating incorporation of xylose or levulinic acid (LA) into PHAs by several bacterial strains, we have initiated a study for exploring bioconversion of forest resources to technically relevant copolymers. Initially, PHA was synthesized in shake-flask cultures of Burkholderia cepacia grown on 2.2% (w/v) xylose, periodically amended with varying concentrations of levulinic acid [0.07–0.67% (w/v)]. Yields of poly(β-hydroxybutyrate-co-β-hydroxyvalerate) [P(3HB-co-3HV)] from 1.3 to 4.2 g/l were obtained and could be modulated to contain from 1.0 to 61 mol% 3-hydroxyvalerate (3HV), as determined by ¹H and ¹³C NMR analyses. No evidence for either the 3HB or 4HV monomers was found. Characterization of these P(3HB-co-3HV) samples, which ranged in molecular mass (viscometric, M _v) from 511–919 kDa, by differential scanning calorimetry and thermogravimetric analyses (TGA) provided data which were in agreement for previously reported P(3HB-co-3HV) copolymers. For these samples, it was noted that melting temperature (T _m) and glass transition temperature (T _g) decreased as a function of 3HVcontent, with T _m demonstrating a pseudoeutectic profile as a function of mol% 3HV content. In order to extend these findings to the use of hemicellulosic process streams as an inexpensive carbon source, a detoxification procedure involving sequential overliming and activated charcoal treatments was developed. Two such detoxified process hydrolysates (NREL CF: aspen and CESF: maple) were each fermented with appropriate LA supplementation. For the NREL CF hydrolysate-based cultures amended with 0.25–0.5% LA, P(3HB-co-3HV) yields, PHA contents (PHA as percent of dry biomass), and mol% 3HV compositions of 2.0 g/l, 40% (w/w), and 16–52 mol% were obtained, respectively. Similarly, the CESF hydrolysate-based shake-flask cultures yielded 1.6 g/l PHA, 39% (w/w) PHA contents, and 4–67 mol% 3HV compositions. These data are comparable to copolymer yields and cellular contents reported for hexose plus levulinic acid-based shake-flask cultures, as reported using Alcaligenes eutrophus and Pseudomonas putida. However, our findings presage a conceivable alternative, forestry-based biorefinery approach for the production of value-added biodegradable PHA polymers. Specifically, this review describes the current and potential utilization of lignocellulosic process streams as platform precursors to PHA polymers including hemicellulosic hydrolysates, residual cellulose-derived levulinic acid, tall oil fatty acids (Kraft pulping residual), and lignin-derived aromatics.     

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     

Evaluation of unrefined glycerine pitch as an efficient renewable carbon resource for the biosynthesis of novel yellow-pigmented P(3HB-co-4HB) copolymer towards green technology

Discharging the unrefined glycerine, a by-product from biodiesel production is the simplistic solution adopted for its management which has led to its price reduction in the market worldwide and created serious environmental impact. Therefore, we have explored the application of unrefined glycerine pitch as direct fermentative substrate in the biosynthesis of novel yellow-pigmented poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer by Cupriavidus sp. USMAHM13 through onestage cultivation. Utilization of glycerine pitch (10 g/L) together with 1,4-butanediol (5 g/L) had resulted in the highest achievement of 2.91 g/L of P(3HB-co-40%4HB) copolymer which was naturally dyed with the yellow pigment through the co-extraction process. Enhancement of 4HB monomer accumulation was also attained through the addition of ammonium acetate as nitrogen source. It was revealed that utilization of recovered crude glycerine from glycerine pitch was more preferred compared to the other recovered components. Utilization of glycerine pitch in the biosynthesis of P(3HB-co-4HB) copolymer would not only contribute to the efficient waste management but also would promote the development of cost-efficiency microbial fermentation.     

Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas bluephagenesis grown in bioreactors

Bacterial outer membrane (OM) is a self-protective and permeable barrier, while having many non-negligible negative effects in industrial biotechnology. Our previous studies revealed enhanced properties of Halomonas bluephagenesis based on positive cellular properties by OM defects. This study further expands the OM defect on membrane compactness by completely deleting two secondary acyltransferases for lipid A modification in H. bluephagenesis, LpxL and LpxM, and found more significant advantages than that of the previous lpxL mutant. Deletions on LpxL and LpxM accelerated poly(3-hydroxybutyrate) (PHB) production by H. bluephagenesis WZY229, leading to a 37% increase in PHB accumulation and 84-folds reduced endotoxin production. Enhanced membrane permeability accelerates the diffusion of γ-butyrolactone, allowing H. bluephagenesis WZY254 derived from H. bluephagenesis WZY229 to produce 82wt% poly(3-hydroxybutyrate-co-23mol%4-hydroxybutyrate) (P(3HB-co-23mol%4HB)) in shake flasks, showing increases of 102% and 307% in P(3HB-co-4HB) production and 4HB accumulation, respectively. The 4HB molar fraction in copolymer can be elevated to 32 mol% in the presence of more γ-butyrolactone. In a 7-l bioreactor fed-batch fermentation, H. bluephagenesis WZY254 supported a 84 g l⁻¹ dry cell mass with 81wt% P(3HB-co-26mol%4HB), increasing 136% in 4HB molar fraction. This study further demonstrated that OM defects generate a hyperproduction strain for high 4HB containing copolymers.     

Regulating the molar fraction of 4-hydroxybutyrate in Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by biological fermentation and enzymatic degradation

The regulation of 4-hydroxybutyrate (4HB) molar fraction in the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] of a local isolate Cupriavidus sp. USMAA1020 was attempted by employing a feeding strategy through fed-batch fermentation in 100-L fermenter. The growth of Cupriavidus sp. USMAA1020 was enhanced by frequently feeding carbon and nitrogen at a ratio of 5 (C/N 5) using a DO-stat with cascade mode at 20% (v/v) dissolved oxygen (DO). The feeding of C/N 5 and the use of the DO-stat mode were able to regulate the 4HB composition from 0–67 mol% by sequential feeding of γ-butyrolactone and supplementing oleic acid. A high 4HB molar fraction of 67 mol% with a PHA concentration of 5.2 g/L was successfully obtained by employing this feeding strategy. Notably, enzymatic degradation carried out enhanced the 4HB composition of the copolymer synthesized. PHB depolymerase enzyme from Acidovorax sp. was used to degrade this P(3HB-co-70-mol%4HB) copolymer and the 4HB composition could be increased up to 83 mol%. The degradation process was observed by monitoring the time-dependent change in the weight loss of copolymer films. The percentage of weight loss of solvent-cast film increased proportionally up to 19% within 3 h, whereas salt-leached films showed 90% of weight loss within 3 h of incubation and were completely degraded by 4 h. The molecular weight (M _n ) of the films treated with enzyme demonstrated a slight decrease. SEM observation exhibited a rough surface morphology of the copolymer degraded with depolymerase enzyme.