Revisiting the Single Cell Protein Application of Cupriavidus necator H16 and Recovering Bioplastic Granules Simultaneously

Cupriavidus necator H16 (formerly known as Hydrogenomonas eutropha) was famous as a potential single cell protein (SCP) in the 1970s. The drawback however was the undesirably efficient accumulation of non-nutritive polyhydroxybutyrate (PHB) storage compound in the cytoplasm of this bacterium. Eventually, competition from soy-based protein resulted in SCP not receiving much attention. Nevertheless, C. necator H16 remained in the limelight as a producer of PHB, which is a material that resembles commodity plastics such as polypropylene. PHB is a 100% biobased and biodegradable polyester. Although tremendous achievements have been attained in the past 3 decades in the efficient production of PHB, this bioplastic is still costly. One of the main problems has been the recovery of PHB from the cell cytoplasm. In this study, we showed for the first time that kilogram quantities of PHB can be easily recovered in the laboratory without the use of any solvents and chemicals, just by using the cells as SCP. In addition, the present study also demonstrated the safety and tolerability of animal model used, Sprague Dawley given lyophilized cells of C. necator H16. The test animals readily produced fecal pellets that were whitish in color, as would be expected of PHB granules. The pellets were determined to contain about 82-97 wt% PHB and possessed molecular mass of around 930 kg/mol. The PHB granules recovered biologically possessed similar molecular mass compared to chloroform extracted PHB [950 kg/mol]. This method now allows the production and purification of substantial quantities of PHB for various experimental trials. The method reported here is easy, does not require expensive instrumentation, scalable and does not involve extensive use of solvents and strong chemicals.     

Thermodynamic limitations of PHB production from formate and fructose in Cupriavidus necator

The chemolithotroph Cupriavidus necator H16 is known as a natural producer of the bioplastic-polymer PHB, as well as for its metabolic versatility to utilize different substrates, including formate as the sole carbon and energy source. Depending on the entry point of the substrate, this versatility requires adjustment of the thermodynamic landscape to maintain sufficiently high driving forces for biological processes. Here we employed a model of the core metabolism of C. necator H16 to analyze the thermodynamic driving forces and PHB yields from formate for different metabolic engineering strategies. For this, we enumerated elementary flux modes (EFMs) of the network and evaluated their PHB yields as well as thermodynamics via Max-min driving force (MDF) analysis and random sampling of driving forces. A heterologous ATP:citrate lyase reaction was predicted to increase driving force for producing acetyl-CoA. A heterologous phosphoketolase reaction was predicted to increase maximal PHB yields as well as driving forces. These enzymes were then verified experimentally to enhance PHB titers between 60 and 300% in select conditions. The EFM analysis also revealed that PHB production from formate may be limited by low driving forces through citrate lyase and aconitase, as well as cofactor balancing, and identified additional reactions associated with low and high PHB yield. Proteomics analysis of the engineered strains confirmed an increased abundance of aconitase and cofactor balancing. The findings of this study aid in understanding metabolic adaptation. Furthermore, the outlined approach will be useful in designing metabolic engineering strategies in other non-model bacteria.     

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.     

Effect of nitrogen and/or oxygen concentration on poly(3-hydroxybutyrate) accumulation by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis>

The behaviour of Halomonas boliviensis during growth in fed-batch culture under different kind of nutrient restrictions was examined. The metabolic switch between growth and accumulation phase is determined by the limitation in one or more essential nutrient for bacterial growth. The aim of this study was to test the effect of applying limitations of a essential nutrient, such as nitrogen, and the influence of different O₂ concentrations on poly(3-hydroxybutyrate) (PHB) production during the accumulation phase. Single limitations of nitrogen and oxygen provoke PHB accumulations of 45 and 37 % (g g^?1), respectively, while N limitation with low O₂ supply causes the highest PHB accumulation of 73 %. The characterization of the PHB production with the strain H. boliviensis would allow a better optimization of the process and enrich the knowledge about the PHB production from strains different than Cupriavidus necator.     

Production of green biodegradable plastics of poly(3-hydroxybutyrate) from renewable resources of agricultural residues

This work describes potential opportunities for utilization of agro-industrial residues to produce green biodegradable plastics of poly(3-hydroxybutyrate) (PHB). Wheat straws were examined with good efficacy of carbon substrates using Cupriavidus necator. Production was examined in separate hydrolysis and fermentation (SHF) in the presence and absence of WS hydrolysis enzymes, and in simultaneous saccharification and fermentation (SSF) with enzymes. Results showed that production of PHB in SSF was more efficient in terms of viable cell count, cell dry weight, and PHB production and yield compared to those of SHF and glucose-control cultures. While glucose control experiment produced 4.6 g/L PHB; SSF produced 10.0 g/L compared to 7.1 g/L in SHF when utilizing enzymes during WS hydrolysis. Results showed that most of sugars produced during the hydrolysis were consumed in SHF (~98 %) compared to 89.2 % in SSF. Results also demonstrated that a combination of glucose and xylose can compensate for the excess carbon required for enhancing PHB production by C. necator. However, higher concentration of sugars at the beginning of fermentation in SHF can lead to cell inhibition and consequently catabolite repressions. Accordingly, results demonstrated that the gradual release of sugars in SSF enhanced PHB production. Moreover, the presence of sugars other than glucose and xylose can eliminate PHB degradation in medium of low carbon substrate concentrations in SSF.     

Detection of polyhydroxyalkanoate synthase activity on a polyacrylamide gel

This study presents a method to detect active polyhydroxyalkanoate (PHA) synthase on a polyacrylamide gel that combines the polyhydroxybutyrate (PHB) polymerization reaction with Sudan Black B staining. After separation of the protein samples on a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the slab gel was submerged in a buffer containing β-hydroxybutyryl-coenzyme A (3-HBCoA) as substrate and incubated at room temperature for in vitro PHB polymerization. The active PHA synthase catalyzed 3-HBCoA into the PHB polymer and was stained with Sudan Black B. The active PHA synthase appeared as a dark blue band. The activity staining was of high sensitivity, capable of detecting 3.9 ng (0.273 mU) of Cupriavidus necator H16 PHA synthase purified from recombinant Escherichia coli. The detection sensitivity of activity staining was comparable to that of Western blotting analysis. Furthermore, the high sensitivity of activity staining enabled specific detection of the active PHA synthase in the crude extract of wild-type strain C. necator H16. This study provides a rapid, sensitive, and highly specific method for detecting active PHA synthase in gel. The method could be applied to detecting PHA synthase from wild-type bacteria and to the process of enzyme purification.     

Microbial synthesis of polyhydroxybutyrate from glycerol: Gluconeogenesis, molecular weight and material properties of biopolyester

Glycerol is considered as an ideal feedstock for producing bioplastics via bacterial fermentation due to its ubiquity, low price, and high degree of reduction substrate. In this work, we study the yield and cause of limitation in poly(3‐hydroxybutyrate) (PHB) production from glycerol. Compared to glucose‐based PHB production, PHB produced by Cupriavidus necator grown on glycerol has a low productivity (0.92 g PHB/L/h) with a comparably low maximum specific growth rate of 0.11 h^?1. We found that C. necator can synthesize glucose from glycerol and that the lithotrophical utilization of glycerol (non‐fermentative substrate) or gluconeogenesis is an essential metabolic pathway for biosynthesis of cellular components. Here, we show that gluconeogenesis affects the reduction of cell mass, the productivity of biopolymer product, and the molecular chain size of intracellular PHB synthesized from glycerol by C. necator. We use NMR spectroscopy to show that the isolated PHB is capped by glycerol. We then characterized the physical properties of the isolated glycerol‐based PHB with differential scanning calorimetry and tensile tests. We found that although the final molecular weight of the glycerol‐based PHB is lower than those of glucose‐based and commercial PHB, the thermal and mechanical properties of the biopolymers are similar. Biotechnol. Bioeng. 2012; 109: 2808–2818. © 2012 Wiley Periodicals, Inc.     

<Superscript>13</Superscript>C-assisted metabolic flux analysis to investigate heterotrophic and mixotrophic metabolism in <Emphasis Type="Italic">Cupriavidus necator</Emphasis> H16

Introduction

Cupriavidus necator H16 is a gram-negative bacterium, capable of lithoautotrophic growth by utilizing hydrogen as an energy source and fixing carbon dioxide (CO₂) through Calvin–Benson–Bassham (CBB) cycle. The potential to utilize synthesis gas (Syngas) and the prospects of rerouting carbon from polyhydroxybutyrate synthesis to value-added compounds makes C. necator an excellent chassis for industrial application.

Objectives

In the context of lack of sufficient quantitative information of the metabolic pathways and to advance in rational metabolic engineering for optimized product synthesis in C. necator H16, we carried out a metabolic flux analysis based on steady-state ¹³C-labelling.

Methods

In this study, steady-state carbon labelling experiments, using either d-[1-¹³C]fructose or [1,2-¹³C]glycerol, were undertaken to investigate the carbon flux through the central carbon metabolism in C. necator H16 under heterotrophic and mixotrophic growth conditions, respectively.

Results

We found that the CBB cycle is active even under heterotrophic condition, and growth is indeed mixotrophic. While Entner–Doudoroff (ED) pathway is shown to be the major route for sugar degradation, tricarboxylic acid (TCA) cycle is highly active in mixotrophic condition. Enhanced flux is observed in reductive pentose phosphate pathway (redPPP) under the mixotrophic condition to supplement the precursor requirement for CBB cycle. The flux distribution was compared to the mRNA abundance of genes encoding enzymes involved in key enzymatic reactions of the central carbon metabolism.

Conclusion

This study leads the way to establishing ¹³C-based quantitative fluxomics for rational pathway engineering in C. necator H16.

     

Synthesis of the building block 2-hydroxyisobutyrate from fructose and butyrate by Cupriavidus necator H16

2-Hydroxyisobutyryl-coenzyme A mutase, originally discovered in the context of methyl tert-butyl ether degradation in Aquincola tertiaricarbonis L108, catalyzes the isomerization of 3-hydroxybutyryl-coenzyme A (3-HB-CoA) to 2-hydroxyisobutyryl-CoA. It thus constitutes the basis for a biotechnological route from practically any renewable carbon to 2-hydroxyisobutyrate (2-HIB) via the common metabolite 3-hydroxybutyrate. At first sight, recombinant Cupriavidus necator H16 expressing the mutase seems to be well suited for such a synthesis process, as a strong overflow metabolism via (R)-3-HB-CoA is easily induced in this bacterium possessing the poly-3-hydroxybutyrate metabolism. However, the recently established stereospecificity of the mutase, dominantly preferring the (S)-enantiomer of 3-HB-CoA, calls for a closer investigation of C. necator as potential 2-HIB production strain and raised the question about the strain’s potential to yield 2-HIB from substrates directly providing (S)-3-HB-CoA. We compared two mutase-expressing C. necator H16 strains for their capability to synthesize 2-HIB from fructose and butyrate, delivering either (R)- or (S)-3-HB-CoA. Our results indicate that due to the enantiospecificity of the mutase, fructose is a weaker substrate for 2-HIB synthesis than butyrate. Production rates achieved with the PHB-negative strain H16 PHB^?4 on butyrate were higher than on fructose. Using the wild-type did not significantly improve the production rates as the latter showed a 34-fold and a 5-fold lower 2-HIB synthesis rate compared to H16 PHB^?4 on fructose and butyrate, respectively. Moreover, both strains showed concomitant excretion of undesired side products, such as pyruvate and 3-hydroxybutyrate, significantly decreasing the 2-HIB yield.     

Study of metabolic network of Cupriavidus necator DSM 545 growing on glycerol by applying elementary flux modes and yield space analysis

A metabolic network consisting of 48 reactions was established to describe intracellular processes during growth and poly-3-hydroxybutyrate (PHB) production for Cupriavidus necator DSM 545. Glycerol acted as the sole carbon source during exponential, steady-state cultivation conditions. Elementary flux modes were obtained by the program Metatool and analyzed by using yield space analysis. Four sets of elementary modes were obtained, depending on whether the pair NAD/NADH or FAD/FADH₂ contributes to the reaction of glycerol-3-phosphate dehydrogenase (GLY-3-P DH), and whether 6-phosphogluconate dehydrogenase (6-PG DH) is present or not. Established metabolic network and the related system of equations provide multiple solutions for the simultaneous synthesis of PHB and biomass; this number of solutions can be further increased if NAD/NADH or FAD/FADH₂ were assumed to contribute in the reaction of GLY-3-P DH. As a major outcome, it was demonstrated that experimentally determined yields for biomass and PHB with respect to glycerol fit well to the values obtained in silico when the Entner–Doudoroff pathway (ED) dominates over the glycolytic pathway; this is also the case if the Embden–Meyerhof–Parnas pathway dominates over the ED.     

Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins,PhaP6 and PhaP7, in Ralstonia eutropha H16

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with “phasin 2 motifs.” To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules.     

Model-guided dynamic control of essential metabolic nodes boosts acetyl-coenzyme A–dependent bioproduction in rewired Pseudomonas putida

Pseudomonas putida is evolutionarily endowed with features relevant for bioproduction, especially under harsh operating conditions. The rich metabolic versatility of this species, however, comes at the price of limited formation of acetyl-coenzyme A (CoA) from sugar substrates. Since acetyl-CoA is a key metabolic precursor for a number of added-value products, in this work we deployed an in silico-guided rewiring program of central carbon metabolism for upgrading P. putida as a host for acetyl-CoA–dependent bioproduction. An updated kinetic model, integrating fluxomics and metabolomics datasets in addition to manually-curated information of enzyme mechanisms, identified targets that would lead to increased acetyl-CoA levels. Based on these predictions, a set of plasmids based on clustered regularly interspaced short palindromic repeats (CRISPR) and dead CRISPR-associated protein 9 (dCas9) was constructed to silence genes by CRISPR interference (CRISPRi). Dynamic reduction of gene expression of two key targets (gltA, encoding citrate synthase, and the essential accA gene, encoding subunit A of the acetyl-CoA carboxylase complex) mediated an 8-fold increase in the acetyl-CoA content of rewired P. putida. Poly(3-hydroxybutyrate) (PHB) was adopted as a proxy of acetyl-CoA availability, and two synthetic pathways were engineered for biopolymer accumulation. By including cell morphology as an extra target for the CRISPRi approach, fully rewired P. putida strains programmed for PHB accumulation had a 5-fold increase in PHB titers in bioreactor cultures using glucose. Thus, the strategy described herein allowed for rationally redirecting metabolic fluxes in P. putida from central metabolism towards product biosynthesis—especially relevant when deletion of essential pathways is not an option.     

Flux balance analysis of the ammonia-oxidizing bacterium Nitrosomonas europaea ATCC19718 unravels specific metabolic activities while degrading toxic compounds

The ammonia-oxidizing bacterium Nitrosomonas europaea has been widely recognized as an important player in the nitrogen cycle as well as one of the most abundant members in microbial communities for the treatment of industrial or sewage wastewater. Its natural metabolic versatility and extraordinary ability to degrade environmental pollutants (e.g., aromatic hydrocarbons such as benzene and toluene) enable it to thrive under various harsh environmental conditions. Constraint-based metabolic models constructed from genome sequences enable quantitative insight into the central and specialized metabolism within a target organism. These genome-scale models have been utilized to understand, optimize, and design new strategies for improved bioprocesses. Reduced modeling approaches have been used to elucidate Nitrosomonas europaea metabolism at a pathway level. However, genome-scale knowledge about the simultaneous oxidation of ammonia and pollutant metabolism of N. europaea remains limited. Here, we describe the reconstruction, manual curation, and validation of the genome-scale metabolic model for N. europaea, iGC535. This reconstruction is the most accurate metabolic model for a nitrifying organism to date, reaching an average prediction accuracy of over 90% under several growth conditions. The manually curated model can predict phenotypes under chemolithotrophic and chemolithoorganotrophic conditions while oxidating methane and wastewater pollutants. Calculated flux distributions under different trophic conditions show that several key pathways are affected by the type of carbon source available, including central carbon metabolism and energy production.     

Integrating thermodynamic and enzymatic constraints into genome-scale metabolic models

Stoichiometric genome-scale metabolic network models (GEMs) have been widely used to predict metabolic phenotypes. In addition to stoichiometric ratios, other constraints such as enzyme availability and thermodynamic feasibility can also limit the phenotype solution space. Extended GEM models considering either enzymatic or thermodynamic constraints have been shown to improve prediction accuracy. In this paper, we propose a novel method that integrates both enzymatic and thermodynamic constraints in a single Pyomo modeling framework (ETGEMs). We applied this method to construct the EcoETM (E. coli metabolic model with enzymatic and thermodynamic constraints). Using this model, we calculated the optimal pathways for cellular growth and the production of 22 metabolites. When comparing the results with those of iML1515 and models with one of the two constraints, we observed that many thermodynamically unfavorable and/or high enzyme cost pathways were excluded from EcoETM. For example, the synthesis pathway of carbamoyl-phosphate (Cbp) from iML1515 is both thermodynamically unfavorable and enzymatically costly. After introducing the new constraints, the production pathways and yields of several Cbp-derived products (e.g. L-arginine, orotate) calculated using EcoETM were more realistic. The results of this study demonstrate the great application potential of metabolic models with multiple constraints for pathway analysis and phenotype prediction.     

Reaction engineering studies for the production of 2-hydroxyisobutyric acid with recombinant Cupriavidus necator H 16

Recombinant Cupriavidus necator H 16 with a novel metabolic pathway using a cobalamin-dependent mutase was exploited to produce 2-hydroxyisobutyric acid (2-HIBA) from renewable resources through microbial fermentation. 2-HIBA production capacities of different strains of C. necator H 16 deficient in the PHB synthase gene and genetically engineered to enable the production of 2-HIBA from the intracellular PHB precursor (R)-3-hydroxybutyryl-CoA were evaluated in 48 parallel milliliter-scale stirred tank bioreactors (V = 11 mL). The effects of media composition, limitations, pH, and feed rate were studied with respect to the overall process performances of the different recombinant strains. 2-HIBA production was at a maximum at nitrogen limiting conditions and if the pH was controlled between 6.8 and 7.2 under fed-batch operating conditions (intermittent fructose addition). The final concentration of 2-HIBA was 7.4 g L⁻¹ on a milliliter scale. Best reaction conditions identified on the milliliter scale were transferred to a laboratory-scale fed-batch process in a stirred tank bioreactor (V = 2 L). Two different process modes for the production of 2-HIBA, a single-phase and a dual-phase fermentation procedure, were evaluated and compared on a liter scale. The final concentration of 2-HIBA was 6.4 g L⁻¹ on a liter scale after 2 days of cultivation.     

Engineering Cupriavidus necator H16 for the autotrophic production of (R)-1,3-butanediol

Butanediols are widely used in the synthesis of polymers, specialty chemicals and important chemical intermediates. Optically pure R-form of 1,3-butanediol (1,3-BDO) is required for the synthesis of several industrial compounds and as a key intermediate of β-lactam antibiotic production. The (R)-1,3-BDO can only be produced by application of a biocatalytic process. Cupriavidus necator H16 is an established production host for biosynthesis of biodegradable polymer poly-3-hydroxybutryate (PHB) via acetyl-CoA intermediate. Therefore, the utilisation of acetyl-CoA or its upstream precursors offers a promising strategy for engineering biosynthesis of value-added products such as (R)-1,3-BDO in this bacterium. Notably, C. necator H16 is known for its natural capacity to fix carbon dioxide (CO₂) using hydrogen as an electron donor. Here, we report engineering of this facultative lithoautotrophic bacterium for heterotrophic and autotrophic production of (R)-1,3-BDO. Implementation of (R)-3-hydroxybutyraldehyde-CoA- and pyruvate-dependent biosynthetic pathways in combination with abolishing PHB biosynthesis and reducing flux through the tricarboxylic acid cycle enabled to engineer strain, which produced 2.97 g/L of (R)-1,3-BDO and achieved production rate of nearly 0.4 Cmol Cmol⁻¹ h⁻¹ autotrophically. This is first report of (R)-1,3-BDO production from CO₂.     

Synergistic experimental and computational approach identifies novel strategies for polyhydroxybutyrate overproduction

Polyhydroxybutyrate (PHB) is a sustainable bioplastic produced by bacteria that is a potential replacement for conventional plastics. This study delivers an integrated experimental and computational modeling approach to decipher metabolic factors controlling PHB production and offers engineering design strategies to boost production. In the metabolically robust Rhodopseudomonas palustris CGA009, PHB production significantly increased when grown on the carbon- and electron-rich lignin breakdown product p-coumarate (C₉H₈O₃) compared to virtually no PHB titer from acetate (C₂H₃NaO₂). The maximum yield did not improve further when grown on coniferyl alcohol (C₁₀H₁₂O₃), but comparison of the PHB profiles showed that coniferyl alcohol's higher carbon content resulted in a higher rate of PHB production. Combined experimental results revealed that cytoplasmic space may be a limiting factor for maximum PHB titer. In order to obtain a systems-level understanding of factors driving PHB yield, a model-driven investigation was performed. The model yielded several engineering design strategies including utilizing reduced, high molecular weight substrates that bypass the thiolase reaction (phaA). Based on these strategies, utilization of butyrate was predicted and subsequently validated to produce PHB. Model analysis also explained why nitrogen starvation was not essential for PHB production and revealed that renewable and abundant lignin aromatics are ideal candidates for PHB production. Most importantly, the generality of the derived design rules allows them to be applied to any PHB-producing microbe with similar metabolic features.     

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.