Bioplastic Polyhydroxybutyrate Research Articles

Biofabrication of polyhydroxybutyrate (PHB) in engineered Cupriavidus necator H16 from waste molasses

BackgroundCupriavidus necator H16, a native polyhydroxybutyrate (PHB) producer, has emerged as a promising candidate for sustainable bioproduction to replace conventional plastics. However, most fermentation processes still rely on expensive substrates. Therefore, developing an eco-friendly bioprocess for PHB using waste materials is urgent and essential. MethodAn engineered strain Lgg-H16, incorporating galactose permease (galP) and glucokinase (glk), was employed to boost glucose uptake and phosphorylation. Then, biomass and PHB production were improved by fine-tuning the carbon, nitrogen, and phosphorus ratios. Yeast extract was supplemented as an additional nutrient, and a tailored feeding strategy was applied to maximize PHB output. To lower the manufacturing costs, waste molasses from the sugar industry, was utilized for fed-batch fermentation. The physical properties of the PHB, such as molecular weight and crystallinity, were analyzed using differential scanning calorimeter (DSC) and gel permeation chromatography (GPC), respectively. Significant resultsEngineered Lgg-H16 strain exhibited a 2.15-fold increase in specific growth rate compared to the wild type with a C:N:P ratio of 20:1:18, as well as supplemented 2 g/L yeast extract in the medium. PHB yield reached 17.4 g/L with 70 % content from waste molasses in fed-batch fermentation with an atomizer to increase gas dispersion, costing only 1 % of pharmaceutical-grade glucose. All physical properties of the PHB produced by Lgg-H16 were comparable to commercial product, supporting this promising bioprocess by using the low-cost feedstocks for high-value bioplastic PHB.

Lignin valorization to bioplastics with an aromatic hub metabolite-based autoregulation system

Exploring microorganisms with downstream synthetic advantages in lignin valorization is an effective strategy to increase target product diversity and yield. This study ingeniously engineers the non-lignin-degrading bacterium Ralstonia eutropha H16 (also known as Cupriavidus necator H16) to convert lignin, a typically underutilized by-product of biorefinery, into valuable bioplastic polyhydroxybutyrate (PHB). The aromatic metabolism capacities of R. eutropha H16 for different lignin-derived aromatics (LDAs) are systematically characterized and complemented by integrating robust functional modules including O-demethylation, aromatic aldehyde metabolism and the mitigation of by-product inhibition. A pivotal discovery is the regulatory element PcaQ, which is highly responsive to the aromatic hub metabolite protocatechuic acid during lignin degradation. Based on the computer-aided design of PcaQ, we develop a hub metabolite-based autoregulation (HMA) system. This system can control the functional genes expression in response to heterologous LDAs and enhance metabolism efficiency. Multi-module genome integration and directed evolution further fortify the strain’s stability and lignin conversion capacities, leading to PHB production titer of 2.38 g/L using heterologous LDAs as sole carbon source. This work not only marks a leap in bioplastic production from lignin components but also provides a strategy to redesign the non-LDAs-degrading microbes for efficient lignin valorization.

Precision control of ammonium release in Azotobacter vinelandii.

The capture and reduction of atmospheric dinitrogen gas to ammonium can be accomplished through the enzyme nitrogenase in a process known as biological nitrogen fixation (BNF), by a class of microbes known as diazotrophs. The diazotroph Azotobacter vinelandii is a model organism for the study of aerobic nitrogen fixation, and in recent years has been promoted as a potential producer of biofertilizers. Prior reports have demonstrated the potential to partially deregulate BNF in A. vinelandii, resulting in accumulation and extracellular release of ammonium. In many cases, deregulation requires the introduction of transgenic genes or elements to yield the desired phenotype, and the long-term stability of these strains has been reported to be somewhat problematic. In this work, we constructed two strains of A. vinelandii where regulation can be precisely controlled without the addition of any foreign genes or genetic markers. Regulation is maintained through native promoters found in A. vinelandii that can be induced through the addition of extraneous galactose. These strains result in varied degrees of regulation of BNF, and as a result, the release of extracellular ammonium is controlled in a precise, and galactose concentration-dependent manner. In addition, these strains yield high biomass levels, similar to the wild-type A. vinelandii strain and are further able to produce high percentages of the bioplastic polyhydroxybutyrate.

Biosynthesis of bioplastic polyhydroxybutyrate (PHB) from microbes isolated from paddy/sugarcane fields and fabrication of biodegradable thin film

Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA) family microbial polyester. Three Gram-negative bacteria that produce PHB, Acinetobacter baumannii, Enterobacter hormaechei, and Pseudomonas xanthomarina were isolated and cultured in nitrogen-limited minimal salt media. The pH and culture media were further optimized with various glucose and peptone as carbon and nitrogen sources to improve PHB production. FTIR, UV–vis, NMR, TGA, and XRD were used to characterize the PHB produced. The optimal pH for PHB production by Acinetobacter baumannii, Enterobacter hormaechei, and Pseudomonas xanthomarina was discovered to be 6.5, 7.5, and 7.5, respectively. P. xanthomarina produced the highest PHB (7.5 gm/L) in the optimized culture media. Furthermore, PHB-starch thin films were prepared and characterized by XRD before their biodegradability was investigated. The degradation study reveals that 25% of PHB-starch blended thin film has been degraded within 30 days.

Cost-effective production of bioplastic polyhydroxybutyrate via introducing heterogeneous constitutive promoter and elevating acetyl-Coenzyme A pool of rapidly growing cyanobacteria

Bioplastic production using cyanobacteria can be an effective strategy to cope with environmental problems caused by using petroleum-based plastics. Synechococcus elongatus UTEX 2973 with heterogeneous phaCAB can produce bioplastic polyhydroxybutyrate (PHB) with a high CO2 uptake rate. For cost-effective production of PHB in S. elongatus UTEX 2973, phaCAB was expressed by the constitutive Pcpc560, resulting in the production of 226 mg/L of PHB by only photoautotrophic cultivation without the addition of inducer. Several culture conditions were applied to increase PHB productivity, and when acetate was supplied at a concentration of 1 g/L as an organic carbon source, productivity significantly increased resulting in 607.2 mg/L of PHB and additive cost reduction of more than 300 times was achieved compared to IPTG. Consequently, these results suggest the possibility of cyanobacteria as an agent that can economically produce PHB and as a solution to the problem of petroleum-based plastics.

Calibrating the Composition of a Copolymer

In collaboration with Isao Noda, I have been using Raman spectroscopy to study the properties of the bioplastic polyhydroxybutyrate hexanoate (PHBHx), which depend on the percentage of hexanoate (which produces propyl side branches to the polymer chain). The percentages of hexanoate determine the maximum crystallinity that the polymer can experience, and that determines its physical and chemical properties, which include its optical clarity, dyability, flexibility, and thermal properties (melting and glass transition temperature). It is useful to have an easy way to determine the composition; in this column, we describe how Raman spectroscopy was used to show that this is feasible at an accuracy greater than expected.

An efficient and reusable N,N-dimethylacetamide/LiCl solvent system for the extraction of high-purity polyhydroxybutyrate from bacterial biomass

Multiple methods have been developed for the extraction of the bioplastic polyhydroxybutyrate (PHB) from bacteria. Many of these approaches suffer from low efficiency, are toxic, non-reusable, complex, and costly, which prompts the development of alternative processes. The N,N-dimethylacetamide/LiCl (DMAc/LiCl) solvent system is a widely employed, reusable, and simple method for the solubilization of cellulose and other biological macromolecules. Since bacteria are mostly constituted of polysaccharides, proteins, and other polymers, it may be possible to employ DMAc/LiCl to dissolve them and extract bioplastics. Here, the DMAc/LiCl system was adapted for the breakage of PHB-producing Cupriavidus necator cells combined with the extraction of the polyester. After screening for optimized conditions, the extraction was carried out by incubating 200 mg of dry biomass at 110 ℃ for three hours in DMAc/LiCl. With these parameters, the recovery rate was 95.3% with an excellent PHB purity of 99.0%. Furthermore, the solvent could be reused five times with a minimal loss of efficiency. Extracted PHB characterization indicated that it exhibited physicochemical properties comparable to commercial PHB including its molecular weight, thermal stability, and crystallinity. Thus, the DMAc/LiCl-based extraction of high-purity PHB has the potential to become a widespread method because of its efficiency, recyclability, and convenience.

Acetogenic production of 3-Hydroxybutyrate using a native 3-Hydroxybutyryl-CoA Dehydrogenase.

3-Hydroxybutyrate (3HB) is a product of interest as it is a precursor to the commercially produced bioplastic polyhydroxybutyrate. It can also serve as a platform for fine chemicals, medicines, and biofuels, making it a value-added product and feedstock. Acetogens non-photosynthetically fix CO2 into acetyl-CoA and have been previously engineered to convert acetyl-CoA into 3HB. However, as acetogen metabolism is poorly understood, those engineering efforts have had varying levels of success. 3HB, using acetyl-CoA as a precursor, can be synthesized by a variety of different pathways. Here we systematically compare various pathways to produce 3HB in acetogens and discover a native (S)-3-hydroxybutyryl-CoA dehydrogenase, hbd2, responsible for endogenous 3HB production. In conjunction with the heterologous thiolase atoB and CoA transferase ctfAB, hbd2 overexpression improves yields of 3HB on both sugar and syngas (CO/H2/CO2), outperforming the other tested pathways. These results uncovered a previously unknown 3HB production pathway, inform data from prior metabolic engineering efforts, and have implications for future physiological and biotechnological anaerobic research.

Engineering nonphotosynthetic carbon fixation for production of bioplastics by methanogenic archaea

The conversion of CO2 to value-added products allows both capture and recycling of greenhouse gas emissions. While plants and other photosynthetic organisms play a key role in closing the global carbon cycle, their dependence on light to drive carbon fixation can be limiting for industrial chemical synthesis. Methanogenic archaea provide an alternative platform as an autotrophic microbial species capable of non-photosynthetic CO2 fixation, providing a potential route to engineered microbial fermentation to synthesize chemicals from CO2 without the need for light irradiation. One major challenge in this goal is to connect upstream carbon-fixation pathways with downstream biosynthetic pathways, given the distinct differences in metabolism between archaea and typical heterotrophs. We engineered the model methanogen, Methanococcus maripaludis, to divert acetyl-coenzyme A toward biosynthesis of value-added chemicals, including the bioplastic polyhydroxybutyrate (PHB). A number of studies implicated limitations in the redox pool, with NAD(P)(H) pools in M. maripaludis measured to be <15% of that of Escherichia coli, likely since methanogenic archaea utilize F420 and ferredoxins instead. Multiple engineering strategies were used to precisely target and increase the cofactor pool, including heterologous expression of a synthetic nicotinamide salvage pathway as well as an NAD+-dependent formate dehydrogenase from Candida boidinii. Engineered strains of M. maripaludis with improved NADH pools produced up to 171 ± 4 mg/L PHB and 24.0 ± 1.9% of dry cell weight. The metabolic engineering strategies presented in this study broaden the utility of M. maripaludis for sustainable chemical synthesis using CO2 and may be transferable to related archaeal species.

Improved polyhydroxybutyrate production by Cupriavidus necator and the photocatalyst graphitic carbon nitride from fructose under low light intensity

The photocatalyst graphitic carbon nitride (g-C3N4) is known to photostimulate the production of the bioplastic polyhydroxybutyrate (PHB) by Cupriavidus necator. In previous studies, the combination of C. necator and g-C3N4 increased PHB yield from either an organic or inorganic carbon substrate under a light intensity of 4200 lx. Here, different parameters including light intensity, pH, temperature, nitrogen and carbon concentrations, aeration, and inoculum size were explored to maximize PHB production by hybrid photosynthesis from fructose and visible light. A g-C3N4/C. necator culture grown with a lower light intensity of 2100 lx, an inoculum size of 128.30 × 106 CFU ml−1, and constant aeration produced 7.16 g l−1 d−1 PHB with a product yield from fructose of 60.94%. Furthermore, the ratio of incident photons harvested by g-C3N4 converted into NADPH+H+ by C. necator for PHB production was improved to 19.74% after the process optimization. In comparison, the PHB production rate of a non-optimized g-C3N4/C. necator system exposed to 4200 lx was only 2.94 g l−1 d−1 with a product yield from fructose of 33.29%. These results demonstrate that hybrid photosynthesis productivity can be significantly augmented by decreasing light intensity and adjusting other parameters, which is promising for future bioproduction applications.

Potential one-step strategy for PET degradation and PHB biosynthesis through co-cultivation of two engineered microorganisms

The management and recycling of plastic waste is a challenging global issue. Polyethylene terephthalate (PET), one of the most widely used synthetic plastics, can be hydrolyzed by a series of enzymes. However, upcycling the resulting monomers is also a problem. In this study, we designed a co-cultivation system, in which PET degradation was coupled with polyhydroxybutyrate (PHB) production. First, PETase from Ideonalla sakaiensis was expressed in Yarrowia lipolytica Po1f with a signal peptide from lipase. The engineered PETase-producing Y. lipolytica was confirmed to hydrolyze bis(2-hydroxyethyl) terephthalate (BHET) and PET powder into the monomers terephthalate (TPA) and ethylene glycol (EG). Simultaneously, a TPA-degrading Pseudomonas stutzeri strain isolated from PET waste was transformed with a recombinant plasmid containing the phbCAB operon from Ralstonia eutropha, which encodes enzymes for the biosynthesis of PHB. The two co-cultivated engineered microbes could directly hydrolyze BHET to produce the bioplastic PHB in one fermentation step. During this process, 5.16 g/L BHET was hydrolyzed in 12 h, and 3.66 wt% PHB (3.54 g/L cell dry weight) accumulated in 54 h. A total of 0.31g/L TPA was produced from the hydrolyzation of PET in 228 h. Although PHB could not be synthesized directly from PET because of the low hydrolyzing efficiency of PETase, this study provides a new strategy for the biodegradation and upcycling of PET waste by artificial microflora.

Photo-augmented PHB production from CO2 or fructose by Cupriavidus necator and shape-optimized CdS nanorods

Particulate photocatalysts developed for the solar energy-driven reduction of the greenhouse gas CO2 have a small product range and low specificity. Hybrid photosynthesis expands the number of products with photocatalysts harvesting sunlight and transferring charges to microbes harboring versatile metabolisms for bioproduction. Besides CO2, abiotic photocatalysts have been employed to increase microbial production yields of reduced compounds from organic carbon substrates. Most single-reactor hybrid photosynthesis systems comprise CdS assembled in situ by microbial activity. This approach limits optimization of the morphology, crystal structure, and crystallinity of CdS for higher performance, which is usually done via synthesis methods incompatible with life. Here, shape and activity optimized CdS nanorods were hydrothermally produced and subsequently applied to Cupriavidus necator for the heterotrophic and autotrophic production of the bioplastic polyhydroxybutyrate (PHB). C. necator with CdS NR under light produced 1.5 times more PHB when compared to the same bacterium with suboptimal commercially-available CdS. Illuminated C. necator with CdS NR synthesized 1.41 g PHB from fructose over 120 h and 28 mg PHB from CO2 over 48 h. Interestingly, the beneficial effect of CdS NR was specific to C. necator as the metabolism of other microbes often employed for bioproduction including yeast and bacteria was negatively impacted. These results demonstrate that hybrid photosynthesis is more productive when the photocatalyst characteristics are optimized via a separated synthesis process prior to being coupled with microbes. Furthermore, bioproduction improvement by CdS-based photocatalyst requires specific microbial species highlighting the importance of screening efforts for the development of performant hybrid photosynthesis.

Nonmetallic Abiotic-Biological Hybrid Photocatalyst for Visible Water Splitting and Carbon Dioxide Reduction.

SummaryBoth artificial photosystems and natural photosynthesis have not reached their full potential for the sustainable conversion of solar energy into specific chemicals. A promising approach is hybrid photosynthesis combining efficient, non-toxic, and low-cost abiotic photocatalysts capable of water splitting with metabolically versatile non-photosynthetic microbes. Here, we report the development of a water-splitting enzymatic photocatalyst made of graphitic carbon nitride (g-C3N4) coupled with H2O2-degrading catalase and its utilization for hybrid photosynthesis with the non-photosynthetic bacterium Ralstonia eutropha for bioplastic production. The g-C3N4-catalase system has an excellent solar-to-hydrogen efficiency of 3.4% with a H2 evolution rate up to 55.72 μmol h−1 while evolving O2 stoichiometrically. The hybrid photosynthesis system built with the water-spitting g-C3N4-catalase photocatalyst doubles the production of the bioplastic polyhydroxybutyrate by R. eutropha from CO2 and increases it by 1.84-fold from fructose. These results illustrate how synergy between abiotic non-metallic photocatalyst, enzyme, and bacteria can augment solar-to-multicarbon chemical conversion.

Phasin PhaP1 is involved in polyhydroxybutyrate granules morphology and in controlling early biopolymer accumulation in Azospirillum brasilense Sp7

Phasins are amphiphilic proteins involved in the regulation of the number and size of polyhydroxybutyrate (PHB) granules. The plant growth promoting bacterium Azospirillum brasilense Sp7 accumulates high quantities of bioplastic PHB as carbon and energy source. By analyzing the genome, we identified six genes that code for proteins with a Phasin_2 domain. To understand the role of A. brasilense Sp7 PhaP1 (PhaP1Abs) on PHB synthesis, the phaP1 gene (AMK58_RS17065) was deleted. The morphology of the PHB granules was analyzed by transmission electron microscopy (TEM) and the PHB produced was quantified under three different C:N ratios in cultures subjected to null or low-oxygen transfer. The results showed that PhaP1Abs is involved in PHB granules morphology and in controlling early biopolymer accumulation. Using RT-PCR it was found that phasin genes, except phaP4, are transcribed in accordance with the C:N ratio used for the growth of A. brasilense. phaP1, phaP2 and phaP3 genes were able to respond to the growth conditions tested. This study reports the first analysis of a phasin protein in A. brasilense Sp7.

Enhancement of bioplastic polyhydroxybutyrate P(3HB) production from glucose by newly engineered strain Cupriavidus necator NSDG-GG using response surface methodology

This study aimed to enhance production of polyhydroxybutyrate P(3HB) by a newly engineered strain of Cupriavidus necator NSDG-GG by applying response surface methodology (RSM). From initial experiment of one-factor-at-a-time (OFAT), glucose and urea were found to be the most significant substrates as carbon and nitrogen sources, respectively, for the production of P(3HB). OFAT experiment results showed that the maximum biomass, P(3HB) content, and P(3HB) concentration of 8.95g/L, 76wt%, and 6.80g/L were achieved at 25g/L glucose and 0.54g/L urea with an agitation rate of 200rpm at 30°C after 48h. In this study, RSM was applied to optimize the three key variables (glucose concentration, urea concentration, and agitation speed) at a time to obtain optimal conditions in a multivariable system. Fermentation experiments were conducted in shaking flask by cultivation of C. necator NSDG-GG using various glucose concentrations (10-50g/L), urea concentrations (0.27-0.73g/L), and agitation speeds (150-250rpm). The interaction between the variables studied was analyzed by ANOVA analysis. The RSM results indicated that the optimum cultivation conditions were 37.70g/L glucose, 0.73g/L urea, and 200rpm agitation speed. The validation experiments under optimum conditions produced the highest biomass of 12.84g/L, P(3HB) content of 92.16wt%, and P(3HB) concentration of 11.83g/L. RSM was found to be an efficient method in enhancing the production of biomass, P(3HB) content, and P(3HB) concentration by 43, 21, and 74%, respectively.

Sustainable Bioelectrosynthesis of the Bioplastic Polyhydroxybutyrate: Overcoming Substrate Requirement for NADH Regeneration

One of the main limitations to achieve sustainable synthesis of polyhydroxybutyrate (PHB) is the cost of NADH regeneration, as it requires a side enzymatic reaction usually including a NAD-dependent dehydrogenase enzyme with its substrate or other photo- and electrochemical approaches that create unwanted byproducts and the enzymatically inactive dimer NAD2. Herein, a bioelectrocatalytic method combining both enzymatic and electrochemical approaches was used to regenerate enzymatically active NADH. The method employed a modified glassy carbon electrode that possesses both NADH regeneration and acetoacetyl-CoA (AcAcCoA) reduction features. The modified electrode exhibited an apparent Michaelis constant (KM) value of 814 ± 11 μM and a maximum current density (jmax) of 27.9 ± 1.3 μA cm–2 for NAD+ reduction and a KM value of 47 ± 2 μM and jmax of 0.97 ± 0.03 μA cm–2 for AcAcCoA reduction. The modified electrode was subsequently employed in the bioelectrosynthesis of the bioplastic PHB and yielded 1.6 mg in a ...

Methods for Generating Microbial Cocultures that Grow in the Absence of Fixed Carbon or Nitrogen.

This work is motivated by both (1) the desire to make interesting products without the reliance on fixed carbon or fixed nitrogen using microbial cocultures, and (2) the desire to develop new methods for growing microbial communities. The bioplastic polyhydroxybutyrate (PHB), for example, is considered prohibitively expensive to make from sugar (compared to polypropylene). Utilizing and building on engineered strains of Synechococcus elongatus and Azotobacter vinelandii, we have combined a nitrogen-fixing organism with a carbon-fixing organism to make PHB from air, water, sunlight, and trace minerals. Our observations of coculture growth in batch culture led us to develop an improved system based on manipulating the osmotic pressure within a hydrogel. The methods we used to develop this coculture are described in detail in the following chapter, including notes detailing some of our additional observations or thoughts on this system.

Direct conversion of cellulose and hemicellulose to fermentable sugars by a microbially-driven Fenton reaction

The aim of this work was to develop a microbially-driven Fenton reaction that fragments cellulose and hemicellulose, degrades cellodextrins and xylodextrins, and produces short-chain oligosaccharides and monomeric sugars in a single bioreactor. The lignocellulose degradation system operates at neutral pH and does not require addition of conventional lignocellulose-degrading enzymes, thus avoiding problems associated with enzyme accessibility and specificity. The ability to produce useful bioproducts was demonstrated by production of the bioplastic polyhydroxybutyrate with the xylan degradation products as starting substrate.

Systems biology and metabolic modelling unveils limitations to polyhydroxybutyrate accumulation in sugarcane leaves; lessons for C4 engineering.

In planta production of the bioplastic polyhydroxybutyrate (PHB) is one important way in which plant biotechnology can address environmental problems and emerging issues related to peak oil. However, high biomass C4 plants such as maize, switch grass and sugarcane develop adverse phenotypes including stunting, chlorosis and reduced biomass as PHB levels in leaves increase. In this study, we explore limitations to PHB accumulation in sugarcane chloroplasts using a systems biology approach, coupled with a metabolic model of C4 photosynthesis. Decreased assimilation was evident in high PHB-producing sugarcane plants, which also showed a dramatic decrease in sucrose and starch content of leaves. A subtle decrease in the C/N ratio was found which was not associated with a decrease in total protein content. An increase in amino acids used for nitrogen recapture was also observed. Based on the accumulation of substrates of ATP-dependent reactions, we hypothesized ATP starvation in bundle sheath chloroplasts. This was supported by mRNA differential expression patterns. The disruption in ATP supply in bundle sheath cells appears to be linked to the physical presence of the PHB polymer which may disrupt photosynthesis by scattering photosynthetically active radiation and/or physically disrupting thylakoid membranes.

Statistical physical and nutrient optimization of bioplastic polyhydroxybutyrate production by Cupriavidus necator

Polyhydroxyalkanoates are biodegradable polymer materials that accumulate in numerous bacteria. The polyhydroxybutyrate is the most common type of polyhydroxyalkanoates, which potentially serves as precursor for bioplastic production. The most extensively studied polyhydroxybutyrate producing bacteria is Cupriavidus necator due to its capability to accumulate large amounts of this biopolymer in simple culture medium. Accumulation of polyhydroxyalkanoates granules in the cytoplasm of C. necator significantly depended on pH, aeration, carbon sources, nitrogen sources, and minerals in the culture medium. In the present study, the effect of both nutritional and physical variables on polyhydroxybutyrate production was investigated in order to optimize these conditions. At first, on the basis of one-factor-at-a-time experiments, fructose and ammonium chloride were found to be the most suitable sources of carbon and nitrogen for biopolymer production. Then the most significant factors affecting granules accumulation were recognized as fructose, agitation speed, KH2PO4, and initial pH using the Plackett–Burman and central composite design. ANOVA analysis showed significant interaction between fructose and agitation speed. After optimization of the medium, compositions for polyhydroxybutyrate production were determined as follows: fructose 35 g/L, KH2PO4 1.75 g/L, MgSO4·7H2O 1.2 g/L, citric acid 1.7 g/L, trace element 10 mL/L, initial pH = 7, and agitation speed 175 rpm. Under this optimal culture conditions, the maximum yield of PHB was 7.48 g/L. The present strategies included in this study could be used for PHB production by this bacterium. These results are the highest values of PHB ever obtained from batch culture of C. necator reported so far.