Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.     

Production of polyhydroxybutyrate by Ralstonia eutropha from protein hydrolysates

Polyhydroxybutyrate (PHB) was produced by Ralstonia eutropha DSM 11348 (formerly Alicaligenes eutrophus) in media containing 20–30 g l⁻¹ casein peptone or casamino acids as sole sources of nitrogen. In fermentations using media based on casein peptone, permanent growth up to a cell dry mass of 65 g l⁻¹ was observed. PHB accumulated in cells up to 60%–80% of dry weight. The lowest yields were found in media without any trace elements or with casamino acids added only. The residual cell dry masses were limited to 10–15 g l⁻¹ and did not contain PHB. The highest productivity amounted to 1.2 g PHB l⁻¹ h⁻¹. The mean molecular mass of the biopolymer was determined as 750 kDa. The proportion of polyhydroxyvalerate was less than 0.2% in PHB. The bioprocess was scaled up to a 300-l plant. During a fermentation time of 39 h the cells accumulated PHB to 78% w/w. The productivity was 0.98 g PHB l⁻¹ h¹. Received: 8 July 1998 / Accepted: 26 August 1998     

Enzymatic recovery and purification of polyhydroxybutyrate produced by Ralstonia eutropha

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.     

Mutation on N-terminus of polyhydroxybutyrate synthase of Ralstonia eutropha enhanced PHB accumulation

Polyhydroxyalkanoate (PHA) synthase is the central enzyme involved in the biosynthesis of PHA, a family of bacterial biodegradable polyesters. Due to its high variability, the N-terminal fragment of this enzyme was previously considered as unnecessary for a functionally active enzyme. In this study, polyhydroxybutyrate synthase from Ralstonia eutropha (PhbC(Re)) with a deletion on N-terminal 88 amino acid residues showed a significant reduced activity, as reflected by only 1.5% PHB accumulation compared with the wild type which produced 58.4% PHB of the cell dry weight. Whilst several site-specific mutagenesis results revealed the amphiphilic alpha-helix assembled by the amino acid region, D70-E88 played an important role in both maintaining the PHB synthase activity and regulating molecular weight and polydispersity of accumulated PHB homopolymer.     

Production of two phase polyhydroxyalkanoic acid granules in Ralstonia eutropha.

To synthesize layered granules consisting of selected phases of polyhydroxybutyrate (PHB) homopolymer and PH(B-co-V) copolymer, Ralstonia eutropha was grown on fructose and limited quantities (1 g/l) of valeric acid. Exhaustion of the valerate resulted in a carbon source shift and a shift in the composition of polyhydroxyalkanoate (PHA) being synthesized within the cell. The synthesis rates were 0.030 g PH(B-co-V)/l per h and 0.033 g PHB/l per h, giving a copolymer composition of 48% HV. The valerate was exhausted at approximately 12 h at a rate of 0.0894 g/l per h after which only PHB was produced through the remaining 12 h at 0.033 g PHB/l per h from the remaining fructose, which was utilized at a constant rate of 0.0861 g/l per h throughout all 24 h of the experiment. Differential scanning calorimetry (DSC) of isolated granules showed two glass transitions, confirming the presence of two distinct polymer phases within the layered granules. Transmission electron microscopic images stained with RuO4 revealed a heavily stained copolymer core within a lighter stained PHB shell, confirming the expected morphology of granule composition. Thus, biosynthesis can be exploited for the control of domain sizes in layered granules, potentially providing metabolic control over the physical properties of the resultant polymer.     

Mechanism of the polymerization reaction initiated and catalyzed by the polyhydroxybutyrate synthase of Ralstonia eutropha

Polyhydroxybutyrate (PHB) synthases (polymerases) catalyze the polymerization of the coenzyme A thioester of 3-hydroxybutyrate to PHB. The Ralstonia eutropha PHB synthase purified from recombinant E. coli cells exists in aqueous solution in both monomeric (single subunit) and homodimeric (two subunits) forms in equilibrium. Several lines of evidence suggest that the homodimer is the active form of the synthase. The initial mechanistic model for the polymerization reaction proposed that two different thiol groups form the catalytic site. The cysteine at 319 has been shown to provide one thiol group that is involved in the covalent catalysis, but a second thiol group on the same protein molecule has not yet been identified. It is suggested that cysteines at 319 from each of the two molecules of a homodimer synthase provide two identical thiol groups to jointly form a single catalytic site. To verify this model using the strategy of in vitro reconstitution, heterodimers composed of the wild-type subunit and of the C(319) mutated subunit were constructed and the activities at various ratios of the wild-type subunit to the mutated subunit were measured. The experimental results indicate that the homodimer is the active form of the enzyme, that the heterodimer containing the mutated subunit has no activity, and that a single cysteine is not sufficient for catalysis. Two identical thiol groups from C(319) residues on each subunit of the homodimer are required to form the catalytic site for the initiation and propagation reactions. We further demonstrate that a dimer synthase that has initiated the polymerization reaction (primed synthase) is significantly more stable against dissociation than the unprimed (unreacted) dimer synthase. These two properties explain the nature of lag phenomenon during the in vitro polymerization reaction catalyzed by this enzyme     

The production of polyhydroxybutyrate by Methylobacterium rhodesianum and Ralstonia eutropha in media containing glycerol and casein hydrolysates

Methylobacterium rhodesianum and Ralstonia eutropha were cultivated to produce polyhydroxybutyrate (PHB) using media which contained glycerol and casein hydrolysates as C/N-substrates. In these media the pH had not to be regulated during the fermentations. The first strain accumulated an average of 39% PHB during 92 h of cultivation in flasks and 50% PHB during 45 h of cultivation in fermenters. The second one yielded an average of 47% PHB during 67 h of cultivation using casein peptone and 65% PHB during 45 h of cultivation using Casamino acids in the medium. Calculated N-balances showed that about 65% of the supplied nitrogen was used for growth of non-PHB cell dry mass. The conversion of glycerol to PHB was 17% (w/w).     

New insight into the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate

Phasins are proteins that are proposed to play important roles in polyhydroxyalkanoate synthesis and granule formation. Here the phasin PhaP of Ralstonia eutropha has been analyzed with regard to its role in the synthesis of polyhydroxybutyrate (PHB). Purified recombinant PhaP, antibodies against PhaP, and an R. eutropha phaP deletion strain have been generated for this analysis. Studies with the phaP deletion strain show that PhaP must accumulate to high levels in order to play its normal role in PHB synthesis and that the accumulation of PhaP to low levels is functionally equivalent to the absence of PhaP. PhaP positively affects PHB synthesis under growth conditions which promote production of PHB to low, intermediate, or high levels. The levels of PhaP generally parallel levels of PHB in cells. The results are consistent with models whereby PhaP promotes PHB synthesis by regulating the surface/volume ratio of PHB granules or by interacting with polyhydroxyalkanoate synthase and indicate that PhaP plays an important role in PHB synthesis from the early stages in PHB production and across a range of growth conditions.     

Monitoring the in situ crystallization of native biopolyester granules in Ralstonia eutropha via infrared spectroscopy

Poly(3-hydroxybutyrate) (PHB), a representative polyhydroxyalkanoate (PHA), is a naturally occurring biopolyester stored as tiny, intracellular granules in microbial cells. In vivo, native PHB granules are amorphous, stabilized by a monolayer membrane and intra-granule water. When subjected to varying environmental conditions, the native granules may become partially crystalline. The in situ crystallinity of native PHB granules in Ralstonia eutropha cells suspended in aqueous solution was monitored with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). No sample preparation was required for measurement. A major measurement error could be caused by the evaporation of water. Therefore, the infrared absorption spectra should be taken after the initial settlement of cells, but before excessive dehydration. Background interference caused by water and non-PHB biomass was constant throughout the time course of measurement, regardless of granule crystallinity. The wavenumber 1184 cm⁻¹ was found to be most sensitive to the in situ crystallinity of native PHB granules.     

Ethanethiol degradation by Ralstonia eutropha

In the present study, a pure culture of Ralstonia eutropha was used to degrade gaseous ethanethiol. Ethane thiol at various initial concentrations ranging from 115 to 320 mg/m³ was degraded almost completely within 120 ～ 168 h, while at higher concentrations up to 452 mg/m³, removal efficiency declined. It was likely that ethanethiol was used as the source of energy by R. eutropha, since no clear increase in the biomass concentration was observed. Kinetic data of ethanethiol bidegradation could be fitted using the Monod model. The kinetic parameters were q _m = 0.23 (mg ethanethiol/g biomass/h), and K _s = 1.379 (mg/L). The mineralization pathway of ethanethiol through sulphate, as the detected product, and the energy production were discussed in some detail.     

Kinetic parameters of a culture of the hydrogen-oxidizing Ralstonia eutropha,grown under the regimen of biosynthesis of polyhydroxybutyrate

Kinetic parameters of a culture of the hydrogen-oxidizing bacterium Ralstonia eutropha, grown on a gas substrate under the conditions favoring autotrophic biosynthesis of polyhydroxybutyrate, were studied. The following parameters, making it possible to control and optimize the process in industrial situations, were determined: specific rate of substrate consumption, physical properties of culture medium, and coefficients of heat emission and mass transfer.     

In vivo blending of medium chain length polyhydroxy-alkanoates and polyhydroxybutyrate using recombinant Pseudomonas putida harboring phbCAB operon of Ralstonia eutropha

The in vivo blending of medium chain length polyhydroxyalkanoates (mcl-PHA) and polyhydroxybutyrate (PHB) was carried out using recombinant Pseudomonas putida after transforming the phbCAB operon of Ralstonia eutropha. The most suitable carbon sources for the production of mcl-PHA and PHB blends were identified to be octanoate and gluconate. The molar fractions of 3-hydroxyoctanoate and 3-hydroxybutyrate in the polymer blends were effectively modulated by controlling the mixing ratio of octanoate and gluconate, thereby producing a composition ranging from 95% mcl-PHA to 78% PHB.     

Production and purification of self-assembling peptides in Ralstonia eutropha

Self-assembling peptides have emerged as an attractive scaffold material for tissue engineering, yet the expense associated with solid phase chemical synthesis has limited their broad use. In addition, the fidelity of chemical synthesis constrains the length of polypeptides that can be produced homogeneously by this method. Template-derived biosynthesis by recombinant DNA technology may overcome both of these problems. However, recovery of polypeptides from recombinant protein expression systems typically involves multi-step purification schemes. In this study, we report an integrated approach to recombinantly produce and purify self-assembling peptides from the recently developed expression host Ralstonia eutropha. The purification is based on the specific affinity of carbohydrate binding modules (CBMs) to cellulose. In a first step, we identified CBMs that express well in R. eutropha by assembling a fusion library of green fluorescent protein (GFP) and CBMs and determining the fluorescence of cell-free extracts. Three GFP::CBM fusions were found to express at levels similar to GFP alone, of which two CBMs were able to mediate cellulose binding of the GFP::CBM fusion. These two CBMs were then fused to multiple repeats of the self-assembling peptide RAD16-I::E (N-RADARADARADARADAE-C). The fusion protein CBM::E::(RAD16-I::E)4 was expressed in R. eutropha and purified using the CBM's affinity for cellulose. Subsequent proteolytic cleavage with endoproteinase GluC liberated RAD16-I::E peptide monomers with similar properties to the chemically synthesized counterpart RAD16-I.     

Purification of polyhydroxybutyrate synthase from its native organism, Ralstonia eutropha: implications for the initiation and elongation of polymer formation in vivo

Class I polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha catalyzes the formation of PHB from (R)-3-hydroxybutyryl-CoA, ultimately resulting in the formation of insoluble granules. Previous mechanistic studies of R. eutropha PhaC, purified from Escherichia coli (PhaC(Ec)), demonstrated that the polymer elongation rate is much faster than the initiation rate. In an effort to identify a factor(s) from the native organism that might prime the synthase and increase the rate of polymer initiation, an N-terminally Strep2-tagged phaC (Strep2-PhaC(Re)) was constructed and integrated into the R. eutropha genome in place of wild-type phaC. Strep2-PhaC(Re) was expressed and purified by affinity chromatography from R. eutropha grown in nutrient-rich TSB medium for 4 h (peak production PHB, 15% cell dry weight) and 24 h (PHB, 2% cell dry weight). Analysis of the purified PhaC by size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography revealed that it unexpectedly copurified with the phasin protein, PhaP1, and with soluble PHB (M(w) = 350 kDa) in a "high-molecular weight" (HMW) complex and in monomeric/dimeric (M/D) forms with no associated PhaP1 or PHB. Assays for monitoring the formation of PHB in the HMW complex showed no lag phase in CoA release, in contrast to M/D forms of PhaC(Re) (and PhaC(Ec)), suggesting that PhaC in the HMW fraction has been isolated in a PHB-primed form. The presence of primed and nonprimed PhaC suggests that the elongation rate for PHB formation is also faster than the initiation rate in vivo. A modified micelle model for granule genesis is proposed to accommodate the reported observations.     

Quantifying the surface characteristics and flocculability of Ralstonia eutropha

The microbial surface and flocculability were qualitatively characterized through the combination of the surface thermodynamic and the extended DLVO approaches, with Ralstonia eutropha, a polyhydroxybutyrate-producing bacterium, as an example. The negativity of the ζ potential of R. eutropha decreased from the initial −19.5 to −11 mV in its cultivation with the consumption of glucose. The total interfacial free energy (ΔG _adh) was changed from −80 to 28.5 mJ m⁻² in its entire growth process. This suggests that the bacterial surface changed from hydrophobic into hydrophilic, resulting in an alteration of its surface characteristics and flocculability in its different growth phases. As a result, the stability ratio of suspensions increased with the increasing cultivation time, indicating that the cell particles became more repulsive with each other and led to a more stable suspension of R. eutropha in its cultivation. The obtained information in this work might be useful for better understanding the surface characteristics and the flocculability and even manipulating its flocculability in the microbial growth process.     

Metabolite profiles of polyhydroxyalkanoate-producing Ralstonia eutropha H16

This study describes metabolite profiles of Ralstonia eutropha H16 focusing on biosynthesis of polyhydroxyalkanoates (PHAs), bacterial polyesters attracted as biodegradable bio-based plastics. As CoA-thioesters are important intermediates in PHA biosynthesis, four kinds of acyl-CoAs with medium chain length were prepared and used to establish analytical conditions for capillary electrophoresis-electron spray ionization-tandem mass spectrometry (CE–ESI-MS/MS). Metabolites were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose and PHA production phase on octanoate, and subjected to stable isotope dilution-based comparative quantification by multiple reaction monitoring using CE–ESI-MS/MS and ¹³C-labeled metabolites prepared by extraction from R. eutropha mutant grown on U-¹³C₆-glucose. This procedure allowed to quantify relative changes of 94 ionic metabolites including CoA-thioesters. Hexose-phosphates except for glucose 1-phosphate were decreased in the PHA production phase than in the growth phase, suggesting reduced flux of sugar degradation after the cell growth. Several intermediates in TCA cycle and gluconeogenesis were increased in the PHA production phase on octanoate. Interestingly, ribulose 1,5-bisphosphate were detected in all the samples examined, raising possibilities of CO₂ fixation by Calvin–Benson–Bassham cycle in this bacterium even under heterotrophic growth conditions. Turnover of acyl moieties through β-oxidation was suggested to be active on fructose, as CoA-thioesters of C₆ and C₈ were detected in the fructose-grown cells. In addition, major metabolic pools in R. eutropha cells were estimated from the signal intensities. The results of the present study provided new insights into global metabolisms in PHA-producing R. eutropha.