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Recombinant Cupriavidus necator H16/pMPJAS03, expressing a P. putida KT2440 enoyl-CoA hydratase (phaJ), was able to synthesize short-chain-length/ medium-chain-length (scl-mcl) PHA copolymers with a high content of mcl subunits using its native poly(3-hydroxyalkanoate) synthase. The cells were cultivated on fructose with canola oil or canola oil/decanoic acid (DA) mixtures in fed-batch fermentations. The recombinant C. necator H16 (without any synthase modification) produced a polymer composed of 3-hydroxybutyrate (3HB) with mcl-subunits, including 3-hydroxyhexanoate (3HHx), and about 300-fold more 3-hydroxyoctanoate (3HO) than the yields reported in previous studies, as well as a significant amount of 3-hydroxydecanoate (3HD). Increasing the DA content in the feed from 0% to 15% v/v increased the molar content of the 3HD subunits from 1.2 to 2.1 mol%. The presence of larger monomers, such as 3HO and 3HD, decreased the crystallinity and melting temperature and modified the mechanical properties of the polymers. Thus, replacing either of the two gene products (phaJ or phaC1) required to produce PHA from CoA-3-hydroxy fatty acids with broader spectrum enzymes, is suitable for the production of commercially useful scl-mcl-PHA.

International audience; Plasmid expression level heterogeneity in Cupriavidus necator was studied in response to stringent culture conditions, supposed to enhance plasmid instability, through plasmid curing strategies. Two plasmid curing strategies were compared based on their efficiency at generating heterogeneity in batch: rifampicin addition and temperature increase. A temperature increase from 30 degrees to 37 degrees C was the most efficient plasmid curing strategy. To generate a heterogeneous population in terms of plasmid expression levels, successive batches at supra optimal culture temperature (i.e. 37 degrees C) were initially conducted. Three distinct fluorescent subpopulations P-0 (not fluorescent), P-1 (low fluorescence intensity, median = 1 10(3)) and P-2 (high fluorescence intensity, median = 6 10(3)) were obtained. From there, the chemostat culture was implemented to study the long-term stress response under well-controlled environment at defined dilution rates. For dilution rates comprised between 0.05 and 0.10 h(-1), the subpopulation P-2 (62% vs 90%) was favored compared to P1 cells (54% vs 1%), especially when growth rate increased. Our biosensor was efficient at discriminating subpopulation presenting different expression levels under stringent culture conditions. Plus, we showed that controlling growth kinetics had a stabilizing impact on plasmid expression levels, even under heterogeneous expression conditions.

The cost of polyhydroxyalkanoates (PHAs) can be reduced by improving their productivity and recovery. In this study, we attempted to obtain a high cell density culture from a 13 L bioreactor and subsequently improved the recently developed biological recovery process using mealworms to obtain the PHA granules. A cell dry weight of 161 g/L containing 68-70 wt% P(3HB) was obtained. The freeze-dried cells contained a significant amount of mineral salts from the culture medium which reduced the cells' palatability for the mealworms. A simple washing procedure with water was sufficient to remove the residual mineral salts and this improved the cells' consumption by up to 12.5% of the mealworms' body weight. As a result, one kilogram of mealworms consumed 125 g of the washed cells daily and 87.2 g of feacal pellets were recovered, which was almost twice the weight of the unwashed cells. In addition, it also improved the purity of the PHA in the faecal pellets to a value90% upon washing with water to remove the water-soluble compounds. This study has demonstrated a significant improvement in the production and recovery of PHA. In addition, the resulting mealworms showed a significant increase in protein content up to 79% and a decrease in fat content down to 8.3% of its dry weight.

Over the past decade, formic acid and acetic acid have gained increasing attention as alternative feedstocks for poly-3-hydroxybutyrate (PHB) production as these potentially CO

This study investigates the effect of strategies on poly(3-hydroxybutyrate) [P(3HB)] production in bioreactor. In the production of P(3HB), urea and glucose feeding streams were developed to characterize the fed-batch culture conditions for new Cupriavidus necator NSDG-GG mutant. Feeding urea in repeated fed-batch stage (RFB-I) at 6, and 12 h in cultivation led to insignificant kinetic effect on the cell dry mass (CDM) and P(3HB) accumulation. Feeding glucose in repeated fed-batch stage (RFB-II) demonstrated that the incremental feeding approach of glucose after urea in fill-and-draw (F/D) mode at 24, 30, 36, 42, and 48 h in fermentation increased CDM and P(3HB) concentration. In the 1st cycle in RFB-II, the cumulative CDM reached the value of 26.22 g/L and then it increased with the successive repeated fed-batches to attain biomass of 145 g/L at the end of 5th cycle of RFB-II. The final cumulative P(3HB) concentration at the end of 5th cycle of RFB-II reached 111 g/L with the overall yield of 0.50 g P(3HB) g gluc− 1; the CDM productivity from the RFB-II cycles was in the range of 0.84–1.3 g/(L·h). The RFB-II of glucose in an increment mode produced nearly 2.2 times more increase in CDM and P(3HB) productivities compared to the decrement RFB-II mode. Repeated cultivation had also the advantage of avoiding extra time required for innoculum preparation, and sterilization of bioreactor during batch, thereby it increased the overall industrial importance of the process.

Cupriavidus necator H16 gains increasing attention in microbial research and biotechnological application due to its diverse metabolic features. Here we present a tightly controlled gene expression system for C. necator including the pBBR1-vector that contains hybrid promoters originating from C. necator native tolC-promoter in combination with a synthetic tetO-operator. The expression of the reporter gene from these plasmids relies on the addition of the exogenous inducer doxycycline (dc). The novel expression system offers a combination of advantageous features as; (i) high and dose-dependent recombinant protein production, (ii) tight control with a high dynamic range (On/Off ratio), which makes it applicable for harmful pathways or for toxic protein production, (iii) comparable cheap inducer (doxycycline, dc), (iv) effective at low inducer concentration, that makes it useful for large scale application, (v) rapid, diffusion controlled induction, and (vi) the inducer does not interfere within the cell metabolism. As applications of the expression system in C. necator H16, the growth ability on glycerol was enhanced by constitutively expressing the E. coli glpk gene-encoding for glycerol kinase. Likewise, we used the system to overcome the expression toxicity of mevalonate pathway in C. necator H16. With this system, the mevalonate-genes were successfully introduced in the host and the recombinant strains could produce about 200 mg/l mevalonate.

Waste paper, a major fraction of municipal solid waste, has a potential to serve as renewable feedstock for the biorefineries of fuels, chemicals and materials due to rich in cellulose and abundant at low cost. This study evaluates the possibility of waste office paper (WOP) to serve as a potential feedstock for the biorefinery production of poly (3-hydroxybutyrate). In this study, the WOP was pretreated, enzymatically saccharified and the hydrolysate was used for PHB production. The hydrolysate mainly consists of glucose (22.70 g/L) and xylose (1.78 g/L) and the corresponding sugar yield was about 816 mg/g. Ammonium sulphate and C/N ratio 20 were identified as most favorable for high yield of PHB. The batch fermentation of Cupriavidus necator using the pretreated WOP hydrolysate resulted in cell biomass, PHB production and PHB content of 7.74 g/L, 4.45 g/L and 57.52%, respectively. The volumetric productivity and yield achieved were 0.061 g/L/h and 0.210 g/g sugar, respectively. The results suggested that WOP could be a potential alternative feedstock for the biorefinery production of bioplastics.

This study evaluates a biorefinery concept for producing poly(3-hydroxybutyrate) (PHB) with the cyanobacterial strain Synechocystis salina. Due to this reason, pigment extraction and cell disruption were investigated as pre-treatment steps for the harvested cyanobacterial biomass. The results demonstrated that at least pigment removal was necessary to obtain PHB with processable quality (weight average molecular weight: 569-988kgmol-1, melting temperature: 177-182°C), which was comparable to heterotrophically produced PHB. The removed pigments could be utilised as additional by-products (chlorophylls 0.27-1.98mgg-1 TS, carotenoids 0.21-1.51mgg-1 TS, phycocyanin 0-127mgg-1 TS), whose concentration depended on the used nutrient source. Since the residual biomass still contained proteins (242mgg-1 TS), carbohydrates (6.1mgg-1 TS) and lipids (14mgg-1 TS), it could be used as animal feed or converted to biomethane (348 mn3 t-1VS) and fertiliser. The obtained results indicate that the combination of photoautotrophic PHB production with pigment extraction and utilisation of residual biomass offer the highest potential, since it contributes to decrease the environmental footprint of the process and because biomass could be used in a cascading way and the nutrient cycle could be closed.

Polyhydroxyalkanoates (PHAs) are produced in microbes as a source of carbon and energy storage. They are biodegradable and have properties similar to synthetic plastics, which make them an interesting alternative to petroleum-based plastics. In this study, a refined method of recovering PHA from Cupriavidus necator biomass was proposed by incorporating the use of the yellow mealworm (the larval phase of the mealworm beetle, Tenebrio molitor) as partial purification machinery, followed by washing of the fecal pellets with distilled water and sodium hydroxide. The PHA contents of the cells used in this study were 55wt% (produced from palm olein) and 60 wt% (produced from waste animal fats). The treatment of distilled water and NaOH further increased the purity of PHA to 94%. In parallel, analysis of the 16S rRNA metagenomic sequencing of the mealworm gut microbiome has revealed remarkable changes in the bacterial diversity, especially between the mealworms fed with cells produced from palm olein and waste animal fats. This biological recovery of PHA from cells is an attempt to move towards a green and sustainable process with the aim of reducing the use of harmful solvents and strong chemicals during polymer purification. The results obtained show that - purities of >90%, without a reduction in the molecular weight, can be obtained through this integrative biological recovery approach. In addition, this study has successfully shown that the cells, regardless of their origins, were readily consumed by the mealworms, and there is a correlation between the feed type and the mealworm gut microbiome.

The biotechnologically important Gram-negative β-proteobacterium Ralstonia eutropha H16 is able to grow lithoautotrophically by utilizing CO2 and H2 as sole carbon and energy sources, respectively. CO2 is fixed by the CBB cycle, which is encoded in duplicate on the genome of R. eutropha H16. The transcription of both cbb operons is controlled by the transcription regulator CbbR dependent on intracellular PEP levels as a response to the carbon-state of the cell. As demonstrated in this study transcription control of both cbb operons appears to be more complex and additionally involves, next to CbbR, the transcription regulator RegA as part of the global transcription regulation system RegA/RegB. The identification of a highly conserved RegA/RegB homologue in R. eutropha H16 and experimental evidence gathered in this study reveal that RegA plays a crucial role in the transcription control of both cbb promoters. RegA is able to induce cbb promoter activity and controls transcription in combination with CbbR dependent on cellular PEP concentrations. These results clearly demonstrate that RegA plays an important role in cbb operon transcription regulation and may also be relevant for the control of other energy-utilizing and energy-generating pathways of R. eutropha H16. In addition to promoting a more complete understanding of the CO2 fixation mechanism of R. eutropha H16 these findings also provide crucial insights for the utilization of this bacterium in biotechnological applications with respect to CO2 fixation.

The increasing global demand for sustainable resources necessitates the complete utilization of feedstock. Wheat bran consists of significant amount of cellulose and hemicellulose which can be used as a renewable resource for production of fermentable sugars. In this study, alkaline pretreated wheat bran was enzymatically hydrolyzed using cellulase of Trichoderma reesei (37 FPU/g) and β - glucosidase of Aspergillus niger (50 CBU/g). Among the nitrogen sources tested, ammonium sulphate was identified as best nitrogen source for the production of polyhydroxybutyrate (PHB). The overall sugar concentration was about 62.91g/L with the corresponding sugar yield of 629.1mg/g wheat bran and the sugars released were mainly composed of glucose (48.35g/L) and xylose (14.56g/L). The PHB producing mutant strain, Ralstonia eutropha NCIMB 11599 grown in wheat bran hydrolysate produced cell density, PHB and yield of 24.5g/L, 62.5%, and 0.319g/g sugar respectively, with a productivity of 0. 0.255g/L/h. Thus, the results suggested that the wheat bran could be a potential alternative feedstock as it does not require any detoxification due to less inhibitory compounds for production of high cell density with significant amount of polyhydroxybutyrate.

Cupriavidus necator H16 has nine genes of poly(3-hydroxybutyrate) (PHB) depolymerases or oligomer hydrolases (intracellular PHB mobilization enzymes). In this study, we evaluated the relation between these genes and the accumulation, consumption, and molecular weight of polyhydroxyalkanoates (PHAs) accumulating in strain H16 and in a recombinant C. necator strain, KNK-005, which harbors an NSDG mutant of the PHA synthase gene (phaCAc) from Aeromonas caviae. PhaZ6 had a significant influence on the molecular weight of PHA when palm kernel oil was used as a carbon source. The 005dZ6 strain (ΔphaZ6 mutant of KNK-005) could produce ultra-high-molecular-weight poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) with weight-average molecular weight (Mw) >3.0×10(6) (approximately double that of KNK-005). Under PHA consumption conditions, deletion of phaZ1 and phaZ2 had a significant and slight attenuating effect, respectively, on the reduction in PHA content of KNK-005 cells. Regardless of the PHA consumption, its Mw did not decrease. Thus, 005dZ126 (the ΔphaZ1ΔphaZ2ΔphaZ6 triple mutant of KNK-005) is a promising strain capable of producing PHBHHx of ultra-high-molecular-weight and barely degrades PHBHHx enzymatically intracellularly. This is the first report examining the relation between intracellular PHB mobilization enzymes and molecular weight of PHAs accumulating in C. necator H16 and the derivatives.

Biocatalysis has significant advantages over organic synthesis in the field of chiral molecule production and several types of stereoselective enzymes are already in use in industrial biotechnology. However, there is still a high demand for new enzymes capable of transforming bulky molecules with sufficient operability. In order to reveal novel high-potential biocatalysts, the complete genome of the β-proteobacterium Ralstonia eutropha H16 was screened for potential short-chain dehydrogenases/reductases (SDRs). We were able to identify two (S)-enantioselective SDRs named A5 and B3. These showed clear preference towards long-chain and aromatic secondary alcohols, aldehydes and ketones, with diaryl diketone benzil as one of the best substrates. In addition the phylogenetic analysis of all enzyme types, which are known to facilitate benzil reduction, revealed at least two separate evolutionary clusters. Our results indicate the biotechnological potential of SDRs A5 and B3 for the production of chiral compounds with potential commercial value.

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with Ralstonia eutropha relies on the addition of propionate during fermentation, and propionate consumption is one of the major factors affecting the cost of PHBV production. In this study, 7 strains were obtained by genetic manipulating the methylcitric acid cycle and the methylmalonyl-CoA pathway in R. eutropha. Disruption of prpC1 and prpC2 genes did not affect cell growth and PHBV accumulation. All 7 strains were able to accumulation high amounts of PHBVs with 3HV fractions of 0.41-29.1 mol% during cultivation in flasks. Fermentation in 7.5-L fermenter showed that genetically engineered Rem-8 was able to yield biomass of 132.8 CDWg/L, of which 68.6% were PHBV with 3HV fraction of 26.0 mol% in the biopolymer, indicating promising potentials of commercialization in the future.

Online monitoring process for the production of polyhydroxyalkanoates (PHA), using cooking oil (UCO) as the sole carbon source and Cupriavidus necator , was developed. A batch reactor was operated and hydroxybutyrate homopolymer was obtained. The biomass reached a maximum concentration of 11.6 ± 1.7 g L −1 with a polymer content of 63 ± 10.7% (w/w). The yield of product on substrate was 0.77 ± 0.04 g g −1 . Near-infrared (NIR) spectroscopy was used for online monitoring of the fermentation, using a transflectance probe. Partial least squares regression was applied to relate NIR spectra with biomass, UCO and PHA concentrations in the broth. The NIR predictions were compared with values obtained by offline reference methods. Prediction errors to these parameters were 1.18, 2.37 and 1.58 g L −1 for biomass, UCO and PHA, respectively, which indicate the suitability of the NIR spectroscopy method for online monitoring and as a method to assist bioreactor control.

Acetic acid, a potential growth inhibitor, commonly occurs in lignocellulosic hydrolysates. The growth of Cupriavidus necator DSM 545 and production of poly(3-hydroxybutyrate) (PHB) by this bacterium in a glucose-based medium supplemented with various initial concentrations of acetic acid are reported. The bacterium could use both glucose and acetic acid to grow and produce PHB, but acetic acid inhibited growth once its initial concentration exceeded 0.5 g/L. As acetic acid is an unavoidable contaminant in hydrolysates used as sugar sources in commercial fermentations, a mathematical model was developed to describe its impact on growth and the production of PHB. The model was shown to satisfactorily apply to growth and PHB production data obtained in media made with acetic-acid-containing hydrolysates of Napier grass and oil palm trunk as carbon substrates.

The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.

Ralstonia eutropha H16 is capable of utilizing 3,3′-thiodipropionic acid (TDP) and 3,3’-dithiodipropionic acid (DTDP) as precursor substrates for biosynthesis of a polythioester (PTE) heteropolymer consisting of 3-hydroxybutyric acid (3HB) and 3-mercaptopropionic acid (3MP). To elucidate the hitherto unknown catabolic pathways of TDP and DTDP in R. eutropha H16, 19 defined deletion mutants were generated based on extensive functional genome analyses. Deletions of two ABC-type transporter clusters (H16_A0357-0359, H16_A3658-3660) resulted in an alteration of poly(3HB- co -3MP) composition with TDP as precursor to only 10.2 ± 1.9 mol% 3MP in comparison to 15.1 ± 5.5 mol% in the wild type. A mutant strain of H16 lacking Bordetella uptake gene-like substrate binding proteins (H16_A2779, H16_A0337) incorporated only 7.4 ± 3.8 mol% 3MP into PTE heteropolymers with DTDP as precursor in comparison to 24.5 ± 14.5 mol% in the wild type. Therefore, both gene products are probably involved in transport processes of this compound into the cells. However, the most significant reduction in 3MP contents of the heteropolymers with DTDP as precursor occurred upon the deletion of a gene encoding the putative thiol-disulfide interchange protein DsbD (H16_A3455, 3.9 ± 2.6 mol% 3MP). DsbD is proposed to be involved in the reduction of DTDP into two molecules of 3MP, the common cleavage product of TDP and DTDP.

Glycerol was utilized by Cupriavidus necator DSM 545 for production of poly-3-hydroxybutyrate (PHB) in fed-batch fermentation. Maximal specific growth rates (0.12 and 0.3h(-1)) and maximal specific non-growth PHB production rate (0.16 g g(-1)h(-1)) were determined from two experiments (inocula from exponential and stationary phase). Saturation constants for nitrogen (0.107 and 0.016 g L(-1)), glycerol (0.05 g L(-1)), non-growth related PHB synthesis (0.011 g L(-1)) and nitrogen/PHB related inhibition constant (0.405 g L(-1)), were estimated. Five relations for specific growth rate were tested using mathematical models. In silico performed optimization procedures (varied glycerol/nitrogen ratio and feeding) has resulted in a PHB content of 70.9%, shorter cultivation time (23 h) and better PHB yield (0.347 g g(-1)). Initial concentration of biomass 16.8 g L(-1) and glycerol concentration in broth between 3 and 5 g L(-1) were decisive factors for increasing of productivity.

Bacterial polyhydroxyalkanoates (PHA) are expensive partly due to the recovery and purification processes. Thus, many studies have been carried out in order to minimize the cost. Here we report on the use of mealworm, which is the larva of mealworm beetle (Tenebrio molitor) to recover PHA granules from Cupriavidus necator. Mealworms were shown to readily consume the freeze-dried C. necator cells and excrete the PHA granules in the form of whitish feces. Further purification using water, detergent and heat resulted in almost 100% pure PHA granules. Comparison with chloroform extraction showed no signs of reduction in the molecular weight and dispersion of the PHA molecules. Scanning electron microscopy and dynamic light scattering measurements revealed that the biologically recovered PHA granules retained their native spherical morphology. The PHA granules were subjected to a battery of tests to determine their purity and properties in comparison to the chloroform extracted PHA. This study has demonstrated the possibility of using mealworms as a biological agent to partially purify the PHA granules.

Levulinic acid or 4-ketovaleric acid is a potential renewable substrate for production of polyhydroxyalkanoates. In this work, the initial reactions of LA metabolism by Cupriavidus necator were examined in vitro. The organic acid was converted by membrane-bound crude enzymes obtained from the cells pre-grown on LA, while no LA activity was detected from cells pre-grown on acetic acid. Acetyl-CoA and propionyl-CoA were two major intermediates in the initial reactions of LA conversion. A mass balance on propionyl-CoA accounts for 84 mol% of LA added in vitro. It explains an interesting phenomenon that 3-hydroxbutyrate and 3-hydroxyvalerate are two major monomers of the biopolyester formed from LA, instead of 4-hydroxvalerate that has the similar chemical structure of LA as the precursor. A Monod model was used to describe the kinetics of LA utilization as a sole carbon source or a co-substrate of glucose and fructose. The μ(max) and K(m) of LA alone were 0.26 h⁻¹ and 0.01 g/L, respectively. The content and composition of PHA are also dependent on the culture conditions such as carbon to nitrogen ratio. The in vitro observation is supported by the high utilization rate of LA and the high molar percentage of 3HB and 3HV in the PHA derived from LA.

Escherichia coli was engineered to intracellularly manufacture streptavidin beads. Variants of streptavidin (monomeric, core and mature full length streptavidin) were C-terminally fused to PhaC, the polyester granule forming enzyme of Cupriavidus necator . All streptavidin fusion proteins mediated formation of the respective granules in E. coli and were overproduced at the granule surface. The monomeric streptavidin showed biotin binding (0.7 ng biotin/μg bead protein) only when fused as single-chain dimer. Core streptavidin and the corresponding single-chain dimer mediated a biotin binding of about 3.9 and 1.5 ng biotin/μg bead protein, respectively. However, biotin binding of about 61 ng biotin/μg bead protein with an equilibrium dissociation constant ( K D ) of about 4 × 10 −8 M was obtained when mature full length streptavidin was used. Beads displaying mature full length streptavidin were characterized in detail using ELISA, competitive ELISA and FACS. Immobilisation of biotinylated enzymes or antibodies to the beads as well as the purification of biotinylated DNA was used to demonstrate the applicability of these novel streptavidin beads. This study proposes a novel method for the cheap and efficient one-step production of versatile streptavidin beads by using engineered E. coli as cell factory.

Only the PHA synthase is required for formation of spherical intracellular PHA granules emerging at cell poles. This study aims to assign the polar targeting signal in the PHA synthase and to provide insight into molecular mechanisms of granule formation. Random in-frame insertion mutagenesis indicated dispensable and essential regions suggesting that only the N terminus (

Ralstonia eutropha H16 (Cupriavidus necator H16) is a Gram-negative, facultative chemolithoautotrophic bacterium which can use H2 and CO2 as sole energy and carbon sources in the absence of organic substrates. The biotechnological use of R. eutropha H16 on an industrial scale has already been established; however, only a small number of tools promoting inducible gene expression is available. Within this study two systems promoting inducible expression were designed on the basis of the strong j5 promoter and the Escherichia coli lacI or the Pseudomonas putida cumate regulatory elements. Both expression vectors display desired regulatory features and further increase the number of suitable inducible expression systems for the production of metabolites and proteins with R. eutropha H16.

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters considered as alternatives to petroleum-based plastics. Ralstonia eutropha is a model organism for PHA production. Utilizing industrially rendered waste animal fats as inexpensive carbon feedstocks for PHA production is demonstrated here. An emulsification strategy, without any mechanical or chemical pre-treatment, was developed to increase the bioavailability of solid, poorly-consumable fats. Wild type R. eutropha strain H16 produced 79-82% (w/w) polyhydroxybutyrate (PHB) per cell dry weight (CDW) when cultivated on various fats. A productivity of 0.3g PHB/(L × h) with a total PHB production of 24 g/L was achieved using tallow as carbon source. Using a recombinant strain of R. eutropha that produces poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)], 49-72% (w/w) of PHA per CDW with a HHx content of 16-27 mol% were produced in shaking flask experiments. The recombinant strain was grown on waste animal fat of the lowest quality available at lab fermenter scale, resulting in 45 g/L CDW with 60% (w/w) PHA per CDW and a productivity of 0.4 g PHA/(L × h). The final HHx content of the polymer was 19 mol%. The use of low quality waste animal fats as an inexpensive carbon feedstock exhibits a high potential to accelerate the commercialization of PHAs.

Different recombinant R-3-hydroxybutyryl-CoA (3-HB) synthesis pathways strongly influenced the rate and accumulation of the biopolymer poly[(R)-3-hydroxybutyrate] (PHB) in Saccharomyces cerevisiae. It has been previously shown that expression of the Cupriavidus necator PHB synthase gene leads to PHB accumulation in S. cerevisiae [Leaf, T., Peterson, M., Stoup, S., Somers, D., Srienc, F., 1996. Saccharomyces cerevisiae expressing bacterial polyhydroxybutyrate synthase produces poly-3-hydroxybutyrate. Microbiology 142, 1169-1180]. This finding indicates that native S. cerevisiae expresses genes capable of synthesizing the correct stereochemical substrate for the synthase enzyme. The effects of variations of 3-HB precursor pathways on PHB accumulation were investigated by expressing combinations of C. necator PHB pathway genes. When only the PHB synthase gene was expressed, the cells accumulated biopolymer to approximately 0.2% of their cell dry weight. When the PHB synthase and reductase gene were co-expressed, the PHB levels increased approximately 18 fold to about 3.5% of the cell dry weight. When the beta-ketothiolase, reductase and synthase genes were all expressed, the strain accumulated PHB to approximately 9% of the cell dry weight which is 45 fold higher than in the strain with only the synthase gene. Fluorescent microscopic analysis revealed significant cell-to-cell heterogeneity in biopolymer accumulation. While the population average for the strain expressing three PHB genes was approximately 9% of the cell dry weight, some cells accumulated PHB in excess of 50% of their cell volume. Other cells accumulated no biopolymer. In addition, the recombinant strain was shown to co-produce ethanol and PHB under anaerobic conditions. These results demonstrate that the technologically important organism S. cerevisiae is capable of accumulating PHB aerobically and anaerobically at levels similar to some bacterial systems. The easily assayed PHB system also creates a convenient means of probing in vivo the presence of intracellular metabolites which could be useful for studying the intermediary metabolism of S. cerevisiae.

Polyhydroxybutyrate (PHB) is the most studied among a wide variety of polyhydroxyalkanoates, bacterial biodegradable polymers known as potential substitutes for conventional plastics. This work aimed at evaluating the use of enzymes to recover and purify the PHB produced by Ralstonia eutropha DSM545. Screening experiments allowed the selection of trypsin, bromelain and lysozyme among six enzymes, based on their efficiency in lysing cells of a non-PHB producing R. eutropha strain. Then, process conditions for high efficiency in PHB purification from the DSM545 cells were searched for the enzymes previously selected. The best result was achieved with 2.0% of bromelain (enzyme mass per biomass), equivalent to 14.1 U ml(-1), at 50 degrees C and pH 9.0, resulting in 88.8% PHB purity. Aiming at improving the process efficiency and reducing the enzyme cost, experiments were carried out with pancreatin, leading to 90.0% polymer purity and an enzyme cost three times lower than the one obtained with bromelain. The molecular mass analysis of PHB showed no polymer degradation. Therefore, this work demonstrates the potential of using enzymes in order to recover and purify PHB and bacterial biopolymers in general.

The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.

E-lysis of Ralstonia eutropha H16, which belongs to the beta-subclass, was undertaken to verify whether transmembrane tunnel formation is possible in bacteria which do not belong to the enterobacteriaceae. For this purpose, a new gene E expression plasmid, pKG12, with two origins of replication, oriV and oriT, from plasmid pRP4, chloramphenicol and kanamycin resistance genes and a casette composed of lambda cI857 and lambda pR gene E was constructed. Temperature upshift of R. eutropha H16 (pKG12) from 28 to 45 degrees C during exponential growth resulted in lysis of the strain with features characteristic of E-mediated lysis of Escherichia coli. The cytoplasmic contents released can easily be separated from the still intact envelope fraction by centrifugation or filtration. As R. eutropha H16 represents an important industrial organism, E-mediated lysis could facilitate procedures for the recovery of intracellular mediators or products like polyhydroxyalkanoates.

The poly(β-hydroxybutyrate) (PHB) biosynthetic genes of Ralstonia eutropha that are organized in a single operon ( phaCAB ) have been cloned in Escherichia coli, where the expression of the genes in the wild-type pha operon from plasmid pTZ18U-PHB leads to the formation of 50–80% PHB/celldry mass when the cells are grown in Luria–Bertani medium supplemented with 1% glucose (w/v). In combination with the phaCAB genes, expression of cloned lysis gene E of bacteriophage PhiX174 from plasmid pSH2 has been used to release PHB granules produced in E. coli . It was shown that small PHB granules in a semiliquid stage are squeezed out of the cells through the E-lysis tunnel structure which is characterized by a small opening in the envelope with borders of fused inner and outer membranes. All envelope components remain intact after E-lysis and can be removed from the mixture of released PHB granules by density gradient centrifugation. In addition, a modified E-lysis procedure is described which enables the release of PHB from cell pellets in pure water or low ionic strength buffer. PHB granules in aqueous solution can be aggregated by divalent cations. Addition of glassmilk speeds up the agglomeration of PHB granules and binding to glass beads can either be used for collection or further purification of PHB in aqueous solutions.

The fatty acid (FA) composition of the bacterial membrane of Cupriavidus necator DSM 545 was assessed during the time course of two-stage fed-batch cultivations for the production of short-chain polyhydroxyalkanoates (PHA). Changes in the relative proportion of straight, methyl and cyclopropyl saturated, unsaturated, hydroxy substituted and polyunsaturated FA were observed, depending on the C sources and cultivation conditions used to favor the synthesis of poly(3-hydroxybutyrate) (P(3HB)), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) or poly(3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate) (P(3HB-4HB-3HV)), under N limiting conditions. The relative percentage of each FA class was studied using glucose or waste glycerol (GRP), as main C source for P(3HB) production. The FA profile was also assessed when GRP was used together with i) γ-butyrolactone (GBL) (precursor of 4HB monomers) for P(3HB-4HB) synthesis and ii) GBL and propionic acid (PA) (3HV precursor) to yield P(3HB-4HB-3HV). The effect of GBL and PA utilization as PHA monomer precursors on the FA profile of the cell membrane was studied under two different dissolved oxygen concentrations (DOC).

A combined two-cell reaction system containing Gluconobacter oxydans and Ralstonia eutropha was evaluated with regard to asymmetric ketone reduction using H(2) as the reductant. Whole cells permeabilized by EDTA/toluene were used, and synthesis was performed in a biphasic aqueous/organic reaction medium. The two-cell system was compared with a system in which G. oxydans alone was used for both ketone reduction and cofactor regeneration, using an alcohol as co-substrate. The two-cell system exhibited almost twice the initial reaction rate of the single-cell system, a higher yield (75% vs. 48%) but slightly lower enantiomeric purity (93% vs. 98%) of the product (S)-2-octanol. The permeabilized R. eutropha cells are worth evaluating for byproduct-free NADH regeneration in combination with other whole cell catalysts.

Lipopolysaccharides free P[3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)] production was achieved using recombinant Corynebacterium glutamicum harboring polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha. Cells grown on glucose with feeding of propionate as a precursor of 3HV unit accumulated 8–47 wt% of P(3HB-co-3HV). The 3HV fraction in the copolymer was varied from 0 to 28 mol% depending on the propionate concentrations. © 2010 Published by Elsevier B.V.

Polymers based on olefins have wide commercial applicability. However, they are made from non-renewable resources and are characterised by difficulty in disposal where recycle and re-use is not feasible. Poly-beta-hydroxybutyric acid (PHB) provides one example of a polymer made from renewable resources. Before motivating its widespread use, the advantages of a renewable polymer must be weighed against the environmental aspects of its production. Previous studies relating the environmental impacts of petroleum-based and bio-plastics have centred on the impact categories of global warming and fossil fuel depletion. Cradle-to-grave studies report equivalent or reduced global warming impacts, in comparison to equivalent polyolefin processes. This stems from a perceived CO(2) neutral status of the renewable resource. Indeed, no previous work has reported the results of a life cycle assessment (LCA) giving the environmental impacts in all major categories. This study investigates a cradle-to-gate LCA of PHB production taking into account net CO(2) generation and all major impact categories. It compares the findings with similar studies of polypropylene (PP) and polyethylene (PE). It is found that, in all of the life cycle categories, PHB is superior to PP. Energy requirements are slightly lower than previously observed and significantly lower than those for polyolefin production. PE impacts are lower than PHB values in acidification and eutrophication.

Poly-3-hydroxybutyrate (PHB) is mainly accumulated by Ralstonia eutropha under unbalanced growth conditions, which limits its production in batch or fed-batch modes. The continuous production of PHB was investigated in a two-stage continuous culture system. The first-stage produced cell mass giving the maximal cell dry weight of 27.1 g l(-1) at 0.21 h(-1) of dilution rate. High specific cell growth rate results in the decrease of PHB synthesis under glucose-limited and nitrogen-rich conditions in the first-stage. The second-stage produced PHB giving the maximal PHB concentration of 47.6 g l(-1) at 0.14 h(-1) of dilution rate. Specific PHB synthetic rate reached highest value at low dilution rate under nitrogen-limited condition in the second-stage, and decreased with the increase of ammonium concentration in the culture. In the continuous culture system, the maximal PHB productivity could reach 1.43 g l(-1) h(-1) at a dilution rate of 0.12 h(-1), but with relatively low PHB content of 47.6%. Maximal yield of PHB on glucose could reach 0.36 g g(-1) glucose at 0.075 h(-1) of dilution rate with relatively high PHB productivity of 1.23 g l(-1) h(-1) and PHB content of 72.1%, respectively.

The broader usage of poly-beta-hydroxybutyrate (PHB), for instance as bulk plastics, calls for cheap raw materials and greater overall process efficiency. The bacterial synthesis is generally induced and promoted by the limitation of growth via nitrogen, oxygen or phosphate depletion with the simultaneous excess and higher concentration of the carbon substrate. Consequently, toxic substrates have been considered unsuitable for PHB synthesis. Nevertheless, a single-stage continuous process for producing PHB from toxic substrates using microorganisms was developed and is reported here. The maximum heat flux during continuous growth and the maximum yield of PHB versus the substrate consumption rate were found to coincide. This suggests the possibility of controlling the conversion of a growth-inhibiting substrate into PHB and maximizing the process efficiency. The observed correlation occurred irrespective of the substrates investigated (phenol or sodium benzoate), the PHB-producing strain (Ralstonia eutropha JMP 134 or Variovorax paradoxus JMP 116), or the type of limitation imposed. The maximum PHB yields obtained comprised up to 50% of cell dry mass.