Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol

Polyhydroxyalkanoates (PHAs) have been recognized as good substitutes for the non-biodegradable petrochemically produced polymers. However, their high (real or estimated) current production cost limits their industrial applications. This work exploits two strategies to enhance PHAs substitution potential: the increase in PHA volumetric productivity in high density cultures and the use of waste glycerol (GRP), a by-product from the biodiesel industry, as primary carbon source for cell growth and polymer synthesis. Cupriavidus necator DSM 545 was used to accumulate poly(3-hydroxybutyrate) (P(3HB)) from GRP and from commercial glycerol (PG) as control substrate. On PG, productivities between 0.6 g_PHB L^?1 h^?1 and 1.5 g_PHB L^?1 h^?1 were attained. The maximum cell DW was 82.5 g_DW L^?1, the P(3HB) content being 62%. When GRP was used, 68.8 g_DW L^?1 with a P(3HB) accumulation of 38% resulting in a final productivity of 0.84 g_PHB L^?1 h^?1 was obtained. By decreasing the biomass concentration at which accumulation was triggered, a productivity of 1.1 g_PHB L^?1 h^?1 (50% P(3HB), w/w) was attained using GRP. P(3HB) molecular weights (M_w) ranged from 7.9 × 10⁵ to 9.6 × 10⁵ Da.     

Use of controlled exogenous stress for improvement of poly(3-hydroxybutyrate) production in Cupriavidus necator

The PHB production by Cupriavidus necator H16 depends on the type and concentration of stress factors and on the time of stress application. Hydrogen peroxide and ethanol significantly enhanced PHB accumulation in C. necator cells. Improved yields (10.9 g/L PHB) were observed after exposure of bacterial culture to 0.5 mmol/L H₂O₂ at the beginning of cultivation and to additional peroxide stress (5 mmol/L H₂O₂) after 60 h of cultivation (beginning of the stationary phase). Production was then ≈28 % higher than in control (8.50 g/L PHB). The highest yields (11.2 g/L PHB) were observed when ethanol (0.5 %) was applied at the beginning of stationary phase. An application of exogenous stress could thus be used as a simple strategy for a significant improvement of PHB production in C. necator.     

Biosynthesis of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16 from jatropha oil as carbon source

Poly(3-hydroxybutyrate) (PHB) is a biodegradable polymer that can be synthesized through bacterial fermentation. In this study, Cupriavidus necator H16 is used to synthesize PHB by using Jatropha oil as its sole carbon source. Different variables mainly jatropha oil and urea concentrations, and agitation rate were investigated to determine the optimum condition for microbial fermentation in batch culture. Based on the results, the highest cell dry weight and PHB concentrations of 20.1 and 15.5 g/L, respectively, were obtained when 20 g/L of jatropha oil was used. Ethanol was used as external stress factor and the addition of 1.5 % ethanol at 38 h had a positive effect with a high PHB yield of 0.987 g PHB/g jatropha oil. The kinetic studies for cell growth rate and PHB production were conducted and the data were fitted with Logistic and Leudeking–Piret models. The rate constants were evaluated and the theoretical values were in accordance with the experimental data obtained.     

The use of LipidGreen2 for visualization and quantification of intracellular Poly(3-hydroxybutyrate) in Cupriavidus necator

Numerous studies have been conducted to develop a rapid protocol for the quantification of poly(3-hydroxybutyrate) during bacterial fermentation as an alternative to time-consuming gravimetric or analytical methods. Fluorescence spectroscopy is one of the most promising approaches. In this study, it could be demonstrated that the novel fluorescent probe LipidGreen2 is able to stain selectively poly(3-hydroxybutyrate) in Cupriavidus necator. Optimal excitation and emission wavelengths were evaluated using 3D-Excitation-Emission-Matrix, displaying the best intensities between 440-460 nm and 490–520 nm for excitation and emission, respectively. The lipophilic fluorophore LipidGreen2 showed a high long-term stability even when incubated under ambient lighting. Due to a strong linear relationship between side scatter and biomass concentration, the influence of the inner filter effects could be incorporated, and adjusting the sample to a specific OD is thus superfluous. The developed method allows a very accurate quantification of poly(3-hydroxybutyrate) in just 15 min, following a comprehensible and simple protocol. It is also excellently suited for bioimaging of intracellular poly(3-hydroxybutyrate) granules.     

Use of enzymes in extraction of polyhydroxyalkanoates produced by Cupriavidus necator

Poly(3‐hydroxybutyrate) (P(3HB)) and its copolymer poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (P(3HB‐co‐3HV), are biodegradable thermoplastic polymers. They are members of the polyhydroxyalkanoate (PHA) family, synthesized and accumulated as a carbon and energy reserve by a variety of microorganisms. The aim of this study was to evaluate the use of the proteases Corolase^® L10, Alcalase^® 2.4L, Corolase^® 7089 and Protemax^® FC and glycosidases Celumax^® BC, Rohament^® CL and Rohalase^® Barley for the recovery of P(3HB) and P(3HB‐co‐3HV) synthesized by Cupriavidus necator. The enzyme Celumax^® BC provided better lysis of the bacterial cell membrane and the results for the optimization of the operating conditions showed that this enzyme is most stable in acetate buffer at pH 4.0, bath at 60°C, hydrolysis time of 1 h and concentration of 0.02% (w/w). The optimization of the operating conditions showed that the enzyme Celumax^® BC provided better lysis of the bacterial cell in acetate buffer at pH 4.0, bath at 60°C, hydrolysis time of 1 h and concentration of 0.02% (w/w). These conditions resulted in lysis of the membrane of the bacteria with a recovery of 93.2% P(3HB‐co‐3HV) with 94% purity. The results showed that the use of enzymes for the polymer extraction is an efficient process that assists in the cell disruption of Cupriavidus necator. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

The effect of carbon source supplementation on the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator

The objective of the present study was to investigate the ability of Cupriavidus necator to produce poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) on various carbon sources in batch cultivation. These results show that C. necator produces poly-3-hydroxybutyrate from single carbon sources. The highest poly-3-hydroxybutyrate (P3HB) content was achieved at growth on fructose in the exponential growth phase. The maximum yield of the P3HV content was obtained when fructose was mixed with acetate. The highest content P3HB-co-3HV was also achieved by C. necator when we supplied C-excess and N- and P-normal conditions. These results indicate that C. necator accumulates high polyhydroxyalkanoates (PHA) content by depleting these elements in the culture medium. Nitrogen and phosphorus limitation has no significant effect on the PHA production, whereas C-excess leads to an increase in PHA formation of up to 92% PHAs of cell dry weight after growth on 5 g/L acetate and 40 g/L fructose.     

Preparation,statistical optimization and characterization of poly(3-hydroxybutyrate) fermented by Cupriavidus necator utilizing various hydrolysates of alligator weed (Alternanthera philoxeroides) as a sole carbon source

Alligator weed (Alternanthera philoxeroides) is a stoloniferous, amphibious and perennial herb which has invaded many parts of the world and led to serious environmental and ecological problems. In order to exploit cheap carbon source for poly(3-hydroxybutyrate) (PHB) production, alligator weed hydrolysates were prepared by acid and enzyme treatment and used for PHB production via Cupriavidus necator. The bacterium utilized alligator weed enzymatic hydrolysate and produced the PHB concentration of 3.8 ± 0.2 g/L at the conditions of pH 7.0, 27.5°C, 1.5 g/L of nitrogen source, and 25 g/L of carbon source, this exceeded the value of 2.1 ± 0.1 g/L from acid hydrolysate media at the same conditions. In order to obtain the optimum conditions of PHB production, response surface methodology was employed which improved PHB content. The optimum conditions for PHB production are as follows: carbon source, 34 g/L; nitrogen source, 2 g/L; pH, 7; temperature, 28°C. After 72 hr of incubation, the bacterium produced 8.5 g/L of dry cell weight and 4.8 g/L of PHB. The PHB was subjected to Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and Molecular weight analysis and found the melting temperature, number average molecular mass, and polydispersity were 168.20°C, 185 kDa, and 2.1, respectively.     

Mathematical modelling and process optimization of a continuous 5-stage bioreactor cascade for production of poly[-(R)-3-hydroxybutyrate] by Cupriavidus necator

A multistage system for poly(hydroxyalkanoate) (PHA) production consisting of five continuous stirred tank reactors in series (5-CSTR) with Cupriavidus necator DSM 545 as production strain was modelled using formal kinetic relations. Partially growth-associated production of PHA under nitrogen limited growth was chosen as modelling strategy, thus the Luedeking-Piret’s model of partial growth-associated product synthesis was applied as working hypothesis. Specific growth rate relations adjusted for double substrate (C and N source) limited growth according to Megee et al. and Mankad-Bungay relation were tested. The first stage of the reactor cascade was modelled according to the principle of nutrient balanced continuous biomass production system, the second one as two substrate controlled process, while the three subsequent reactors were adjusted to produce PHB under continuous C source fed and nitrogen deficiency. Simulated results of production obtained by the applied mathematical models and computational optimization indicate that PHB productivity of the whole system could be significantly increased (from experimentally achieved 2.14 g L^?1 h^?1 to simulated 9.95 g L^?1 h^?1) if certain experimental conditions would have been applied (overall dilution rate, C and N source feed concentration). Additionally, supplemental feeding strategy for switching from batch to continuous mode of cultivation was proposed to avoid substrate inhibition.     

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.     

Structure and properties of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) produced by Alcaligenes latus

Summary Random copolymers of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) with a wide range of compositions varying from 0 to 83 mol% 4HB were produced by Alcaligenes latus from the mixed carbon substrates of 3-hydroxybutyric and 4-hydroxybutyric acids. The structure and physical properties of P(3HB-co-4HB) were characterized by¹H and¹³C NMR spectroscopy, gel-permeation chromatography, and differential scanning calorimetry. The isothermal radial growth rates of spherulites of P(3HB-co-4HB) were much slower than the rate of P(3HB) homopolymer. The enzymatic degradation rates of P(3HB-co-4HB) films by a PHB depolymerase were strongly influenced by the copolymer composition.     

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.     

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.     

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.     

Production of poly(3-hydroxybutyrate) by solid-state fermentation with <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g^–1 medium on soy cake in 36 h, and 0.7 mg g^–1 medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g^–1 medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.Revisions requested 31 August 2004; Revisions received 12 October 2004     

Biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer by Cupriavidus sp. USMAA1020 isolated from Lake Kulim, Malaysia

Cupriavidus sp. USMAA1020 was isolated from Malaysian environment and able to synthesize poly(3-hydroxybutyrate-co-4-hydroxybutyrate), [P(3HB-co-4HB)] when grown on gamma-butyrolactone as the sole carbon source. The polyester was purified from freeze-dried cells and analyzed by nuclear magnetic resonance (NMR) spectroscopy. 1H and 13C NMR results confirmed the presence of 3HB and 4HB monomers. In a one-step cultivation process, P(3HB-co-4HB) accumulation by Cupriavidus sp. USMAA1020 was affected by carbon to nitrogen ratio (C/N). A two-step cultivation process accumulated P(3HB-co-4HB) copolyester with a higher 4HB fraction (53 mol%) in nitrogen-free mineral medium containing gamma-butyrolactone. The biosynthesis of P(3HB-co-4HB) was also achieved by using 4-hydroxybutyric acid and alkanediol as 1,4-butanediol. The composition of copolyesters varied from 32 to 51 mol% 4HB, depending on the carbon sources supplied. The copolyester produced by Cupriavidus sp. USMAA1020 has a random sequence distribution of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) units when analyzed by nuclear magnetic resonance (NMR) spectroscopy. When gamma-butyrolactone was used as the sole carbon source, the 4HB fraction in copolyester increased from 25 to 60 mol% as the concentration of gamma-butyrolactone in the culture medium increased from 2.5 g/L to 20.0 g/L.     

Enhancing the 3-hydroxyvalerate component in bioplastic PHBV production by Cupriavidus necator

In the current context of global warming, the substitution of conventional plastics with bioplastics is a challenge. To take up this challenge, we must meet different technical and economic constraints. In the case of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the technical properties can be modulated by varying the 3-hydroxyvalerate content. 3-Hydroxyvalerate (3-HV) enhancement is an issue; therefore, simultaneous evaluation of several 3-hydroxyvalerate-enhancing substrates through fractional factorial design of experiments is described. Eight substrates citric, valeric, propionic, and levulinic acids; propanol; pentanol; and sodium propionate were studied for 3-HV enhancement, and sodium glutamate was studied for biomass and polyhydroxyalkanoate (PHA) enhancement. The most efficient 3-hydroxyvalerate-enhancing factors were levulinic acid, sodium propionate, and pentanol; however, pentanol, at a concentration of 1 g/L, had an extremely negative influence on biomass production and the PHA content of cells. The effect of the inoculum nutrient composition on the final 3-HVcontent was also evaluated. These results showed that the most efficient combination for the production of high 3-HVcontent in PHBV was primary inoculum growth on mineral medium followed by fermentation for 48 h with levulinic acid and sodium propionate (at 1 g/L) as the only carbon sources. This allowed us to produce PHBV with a 3-HVcontent of 80 mol % and overall volumetric and specific productivities of 2 mg/L/h and 3.9 mg/g(CDW) /h, respectively, with the addition of only 2 g/L of inducing substances.     

Staining and quantification of poly-3-hydroxybutyrate in Saccharomyces cerevisiae and Cupriavidus necator cell populations using automated flow cytometry.

BACKGROUND: Poly [(R)-3-hydroxybutyric acid] (PHB) is a prokaryote storage material for carbon and energy that accumulates in cells under unbalanced growth conditions. Because this class of biopolymers has plastic-like properties, it has attracted considerable interest for biomedical applications and as a biodegradable commodity plastic. Current flow cytometric techniques to quantify intracellular PHB are based on Nile red. Here, an improved cytometric technique for cellular PHB quantification utilizing BODIPY 493/503 staining was developed. This technique was then automated using an automated flow cytometry system. MATERIALS: Using flow cytometry, the fluorescence of Saccharomyces cerevisiae and Cupriavidus necator with varying PHB content after staining with BODIPY 493/503 and Nile red was compared, and automated staining techniques were developed for both cultures. RESULTS: BODIPY 493/503 staining had less background staining, higher sensitivity and specificity to PHB, and higher saturation values than did Nile red staining. The developed automated staining procedure was capable of analyzing the PHB content of a bioreactor sample every 25 min and measured the average PHB content with accuracy comparable to offline GC analysis. CONCLUSION: BODIPY 493/503 produced an overall better staining for PHB than did Nile red. When combined with the automated system, this technique provides a new method for the online monitoring and control of bioreactors.