Production of poly(3-hydroxybutyrate) from inexpensive substrates

Two inexpensive substrates, starch and whey were used to produce poly(3-hydroxybutyrate) (PHB) in fed-batch cultures of Azotobacter chroococcum and recombinant Escherichia coli, respectively. Oxygen limitation increased PHB contents in both fermentations. In fed-batch culture of A. chroococcum, cell concentration of 54 g l⁻¹ with 46% PHB was obtained with oxygen limitation, whereas 71 g l⁻¹ of cell with 20% PHB was obtained without oxygen limitation. The timing of PHB biosynthesis in recombinant E. coli was controlled using the agitation speed of a stirred tank fermentor. A PHB content of 80% could be obtained with oxygen limitation by increasing the agitation speed up to only 500 rpm.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha from soybean oil

High-yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha KCTC 2662 was investigated using soybean oil and γ-butyrolactone as carbon sources. In flask culture, it was shown that R. eutropha KCTC 2662 accumulated PHAs during the growth phase. The optimum carbon to nitrogen ratio (C/N ratio) giving the highest cell and PHA yield was 20 g-soybean oil/g-(NH(4))(2)SO(4). The 4-hydroxybutyrate (4HB) fraction in the copolymer was not strongly affected by the C/N ratio. In a 2.5-L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from soybean oil as the sole carbon source by batch and fed-batch cultures of R. eutropha with dry cell weights of 15-32 g/L, PHA contents of 78-83 wt% and yields of 0.80-0.82 g-PHA/g-soybean oil used. By co-feeding soybean oil and γ-butyrolactone as carbon sources, a copolymer of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] could be produced with dry cell weights of 10-21 g/L, yields of 0.45-0.56 g-PHA/g-soybean oil used (0.39-0.50g-PHA/g-carbon sources used) and 4HB fractions of 6-10 mol%. Higher supplementation of γ-butyrolactone increased the 4HB fraction in the copolymer, but decreased cell and PHA yield.     

Utilization of agricultural residues for poly(3-hydroxybutyrate) production by Halomonas boliviensis LC1

Aims: Utilization of cheap and readily available agricultural residues as cheap carbon sources for poly(3‐hydroxybutyrate) (PHB) production by Halomonas boliviensis. Methods and Results: Wheat bran was hydrolysed by a crude enzyme preparation from Aspergillus oryzae NM1 to provide a mixture of reducing sugars composed mainly of glucose, mannose, xylose and arabinose. Growth of H. boliviensis using a mixture of glucose (0·75% w/v) and xylose (0·25% w/v) in the medium led to a PHB content and concentration of 45 wt% and 1 g l^?1, respectively, after 30 h. A similar PHB concentration was attained when H. boliviensis was grown on wheat bran hydrolysate but with a lower PHB content, 34 wt%. In a batch cultivation mode in a fermentor, using 1·8% (w/v) reducing sugars, the maximum PHB accumulation by H. boliviensis was attained in 20 h, but was reduced to about 30 wt%. By adding butyric acid (0·8% v/v), sodium acetate (0·8% w/v) and decreasing the reducing sugars concentration to 1·0% w/v in the medium, PHB accumulation and concentration were increased to 50 wt% and 4 g l^?1, respectively, after 20 h. Butyric acid and sodium acetate for PHB production could also be provided by anaerobic digestion of solid potato waste. Conclusions: Cheap and readily available agricultural residues can be used as substrates to produce PHB. The production of PHB by H. boliviensis using wheat bran hydrolysate as source of carbon is expected to reduce the production cost and motivates further studies. Significance and Impact of the Study: Large‐scale commercial utilization of PHB is mainly hampered by its high production cost. Carbon source for PHB production accounts up to 50% of the total production costs. Thus, the use of waste agricultural residues can substantially reduce the substrate cost (and in turn even provide value to the waste), and can downsize the production costs. This improves the market competitiveness. Studies on PHB production by moderate halophiles were recently initiated with H. boliviensis and findings show that it has potential for commercial exploitation. PHB production by H. boliviensis using wheat bran and potato waste is hence interesting.     

Production of poly (3-hydroxybutyrate) from starch by Azotobacter chroococcum

Production of poly (3-hydroxybutyrate) (PHB) from starch was investigated in flask, batch, and fed-batch cultures of Azotobacter chroococcum. In flask culture, PHB content increased up to 74% of dry cell wt with increasing culture volume. In batch culture, PHB content increased to 44% with O2 limitation. In fed-batch culture, cell concentration of 71 g/l with 20% PHB was obtained without O2 limitation, whereas cell concentration of 54 g/l with 46% PHB was obtained with O2 limitation.     

Fabrication and characterization of biodegradable poly(3-hydroxybutyrate) composite containing bioglass

Bacterially derived poly(3-hydroxybutyrate) (P(3HB)) has been used to produce composite films by incorporating Bioglass particles (<5 microm) in 5 and 20 wt % concentrations. P(3HB) was produced using a large scale fermentation technique. The polymer was extracted using the Soxhlet technique and was found to have similar thermal and structural properties to the commercially available P(3HB). The effects of adding Bioglass on the microstructure surface and thermal and mechanical properties were examined using differential scanning calorimetry, dynamic mechanical analysis (DMA), X-ray diffraction, surface interferometry, electron microscopy, and nanoindentation. The addition of increasing concentrations of Bioglass in the polymer matrix reduced the degree of crystallinity of the polymer as well as caused an increase in the glass transition temperature as determined by DMA. The presence of Bioglass particulates reduced the Young's modulus of the composite. The storage modulus and the loss modulus, however, increased with the addition of 20 wt % Bioglass. A short period (28 days) in vitro bioactivity study in simulated body fluid confirmed the bioactivity of the composites, demonstrated by the formation of hydroxyapatite crystals on the composites' surface.     

Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1

The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T₁ has two disulfide bonds, one of which appears to be necessary for the full enzyme activity. This depolymerase hydrolyzed not only hydrophobic poly(3-hydroxybutyrate) but also water-soluble trimer and larger oligomers of D-(−)-3-hydroxybutyrate, regardless of their solubilities in water. Kinetic analyses with oligomers of various sizes indicated that the substrate cleaving site of the enzyme consisted of four subsites with individual affinities for monomer units of the substrate. Analyses of the hydrolytic products of oligomers, which had labeled D-(−)-3-hydroxybutyrate at the hydroxy terminus, showed that the enzyme cleaved only the second ester linkage from the hydroxy terminus of the trimer and tetramer, and acted as an endo-type hydrolase toward the pentamer and higher oligomers. The enzyme appeared to have a hydrophobic site which interacted with poly(3-hydroxybutyrate) and determined the affinity of the enzyme toward the hydrophobic substrate.     

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) byPseudomonas acidovorans

Summary Terpolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvarelate (3HV) were produced byPseudomonas acidovorans in nitrogen-free culture solutions of 1,4-butanediol and pentanol. When 1,4-butanediol was used as the sole carbon source, a polyester with an unusually high 4HB fraction of 99 mol% was produced.     

Acid-tolerant poly(3-hydroxybutyrate) hydrolases from moulds

Abstract We have isolated some mould strains that can grow under acid conditions with poly(3-hydroxybutyrate) (PHB) as sole carbon source, and secrete PHB hydrolases active at pH values at least down to 3. An improved assay method for such enzymes using a pH stat has been developed, and used to determine the dependence of reaction rate on enzyme and polymer concentrations. The implications of these kinetic properties of the PHB hydrolase for its mode of action are discussed.     

Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate)-based biopolymer systems

Biodegradable biopolymers attract much attention in biology and medicine due to its wide application. The present review considers a biodegradable and biocompatible polymer of bacterial origin, poly(3-hydroxybutyrate), which has wide perspectives in medicine and pharmaceutics. It highlights basic properties of biopolymer (biodegradability and biocompatibility) and also biopolymer systems: various materials, devices and compositions based on the biopolymer. Application of poly(3-hydroxybutyrate)-based biopolymer systems in medicine as surgical implants, in bioengineering as cell culture scaffolds, and in pharmacy as novel drug dosage forms and drug systems are also considered.     

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Alcaligenes latus

Summary Copolymers of (R)-3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) were efficiently produced by Alcaligenes latus in the culture solution containing sucrose and -butyrolactone. The conversion yield of -butyrolactone into 4HB unit of copolymer by A. latus was as high as 60%.     

Production of green biocellulose nanofibers by Gluconacetobacter xylinus through utilizing the renewable resources of agriculture residues

The present study demonstrates the ability to produce green biocellulose nanofibers using the renewable resources of agriculture residues. Locally grown wheat straws (WS) were hydrolyzed under different conditions. Their hydrolysates were utilized to produce the nanofibers in separate hydrolysis fermentation process by Gluconacetobacter xylinus strain bacterium. Highest biocellulose production of ~10.6 g/L was achieved with samples that were enzymatically hydrolyzed. Moreover, acidic hydrolyzed WS produced up to 9.7 g/L, with total sugar concentrations in culture media of 43 g/L. Generally, enzymatic hydrolysis of WS resulted in more total sugar concentration than the acidic hydrolysis (i.e., 52.12 g/L), while water hydrolysis produced the least. This can be related to utilizing Xylanase in addition to Cellulase and Beta-glucosidase that helps to hydrolyse WS dry basis of cellulose and hemicelluloses. Sugar mixtures produced under all hydrolysis conditions were mainly composed of glucose and xylose with average percentages of 56 and 28 %, respectively. Acidic hydrolysis at higher acid concentration, as well as soaking WS in the acidic solution for longer time, improved the total sugar concentration in the culture media by 18 %. Conducting thermal treatment at more intense conditions of higher temperature or heating time improved the total sugar produced with acidic hydrolysis. These conditions, however, resulted in further production of furfural, which considerably affected bacterial cells proliferation. This resulted in lowest sugar consumption in the range of 62–64 % that affected final BC production.     

Production of biodegradable copolyesters of 3-hydroxybutyrate and 4-hydroxybutyrate by Alcaligenes eutrophus

Summary New copolyesters of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) were produced by Alcaligenes eutrophus from various carbon sources of 4-hydroxybutyric acid, 4-chlorobutyric acid, 1,4-butanediol, and -butyrolactone. The composition of copolyesters varied from 0 to 37 mol% 4HB, depending on the carbon sources supplied. The biosynthetic pathway of copolyesters has been discussed. The copolyester film was biodegradable in soil and activated sludge. The rate of biodegradation was enhanced by the presence of 4HB units.     

Production of Poly(3-hydroxybutyrate) from waste potato starch

There has been a considerable interest in using low cost carbon substrates for the production of Poly(3-hydroxybutyrate) (PHB). We have shown that saccharified waste potato starch can be used as a viable alternative carbon source in high cell density PHB production. Using Ralstonia eutropha NCIMB 11599 with phosphate limitation, 179 g/l biomass, 94 g/l PHB, Y(biomass/starch) = 0.46 g/g, Y(PHB/starch) = 0.22 g/g, and PHB productivity = 1.47 g/(l*h) were achieved. Residual maltose accumulated in the fed-batch reactor but caused no noticeable inhibition. Performance with saccharified starch was virtually identical to that with glucose.     

Carboxylate-induced degradation of poly(3-hydroxybutyrate)s

This communication shows that thermal degradation of poly(3-hydroxybutyrate)s (PHBs) is induced by carboxylate groups via a newly proposed E1cB mechanism. In PHBs with end groups in the form of carboxylic acid salts with Na+, K+, and Bu4N+ counterions, the proposed mechanism explains the dependence of thermal stability on the size of the counterion. The degradation via intermolecular alpha-deprotonation by carboxylate is suggested to be the main PHB decomposition pathway at moderate temperatures. The results of the present study show the ability to control the degradation and stability of poly(3-hydroxybutyrate)s as well as of their blends via chemical structure and concentration of the carboxylate polymer end groups.     

Production of poly(3-hydroxybutyrate) from whey by cell recycle fed-batch culture of recombinant Escherichia coli

A new fermentation strategy using cell recycle membrane system was developed for the efficient production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes. By cell recycle, fed-batch cultivation employing an external membrane module, the working volume of fermentation could be constantly maintained at 2.3 l. The final cell concentration, PHB concentration and PHB content of 194 g l^–1, 168 g l^–1 and 87%, respectively, were obtained in 36.5 h by the pH-stat cell recycle fed-batch culture using whey solution concentrated to contain 280 g lactose l^–1 as a feeding solution, resulting in a high productivity of 4.6 g PHB l^–1 h^–1.     

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli

A metabolically engineered Escherichia coli has been constructed for the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from unrelated carbon sources. Genes involved in succinate degradation in Clostridium kluyveri and P(3HB) accumulation pathway of Ralstonia eutropha were co-expressed for the synthesis of the above copolyester. E. coli native succinate semialdehyde dehydrogenase genes sad and gabD were both deleted for eliminating succinate formation from succinate semialdehyde, which functioned to enhance the carbon flux to 4HB biosynthesis. The metabolically engineered E. coli produced 9.4 g l^?1 cell dry weight containing 65.5% P(3HB-co-11.1 mol% 4HB) using glucose as carbon source in a 48 h shake flask growth. The presence of 1.5–2 g l^?1 α-ketoglutarate or 1.0 g l^?1 citrate enhanced the 4HB monomer content from 11.1% to more than 20%. In a 6 l fermentor study, a 23.5 g l^?1 cell dry weight containing 62.7% P(3HB-co-12.5 mol% 4HB) was obtained after 29 h of cultivation. To the best of our knowledge, this study reports the highest 4HB monomer content in P(3HB-co-4HB) produced from unrelated carbon sources.     

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

[This corrects the article on p. 3236 in vol. 59.].     

Microbial production of poly(lactate-co-3-hydroxybutyrate) from hybrid Miscanthus-derived sugars

P[(R)-lactate-co-(R)-3-hydroxybutyrate] [P(LA-co-3HB)] was produced in engineered Escherichia coli using lignocellulose-derived hydrolysates from Miscanthus × giganteus (hybrid Miscanthus) and rice straw. Hybrid Miscanthus-derived hydrolysate exhibited no negative effect on polymer production, LA fraction, and molecular weight of the polymer, whereas rice straw-derived hydrolysate reduced LA fraction. These results revealed that P(LA-co-3HB) was successfully produced from hybrid Miscanthus-derived sugars.