Effects of environmental conditions on cell growth and poly-β-hydroxybutyrate accumulation in Alcaligenes eutrophus

Influences of the control of glucose and oxygen concentrations on cell growth and poly--hydroxybutyrate (PHB) accumulation in Alcaligenes eutrophus were studied. Glucose affects both biosynthesis and glycolysis directly and the other pathways indirectly. PHB accumulation could also be stimulated under oxygen limitation conditions, but the final PHB content within the cells was less than in the case of nitrogen limitation. When the culture was shifted from the PHB accumulation state to balanced growth conditions, PHB degradation occurred in the cells. The cell growth was inhibited by high PHB content within the cells.     

Identification of gene disruptions for increased poly‐3‐hydroxybutyrate accumulation in Synechocystis PCC 6803

Inverse metabolic engineering (IME) is a combinatorial approach for identifying genotypes associated with a particular phenotype of interest. In this study, gene disruptions that increase the biosynthesis of poly‐3‐hydroxybutyrate (PHB) in the photosynthetic bacterium Synechocystis PCC6803 were identified. A Synechocystis mutant library was constructed by homologous recombination between the Synechocystis genome and a mutagenized genomic plasmid library generated through transposon insertion. Using a fluorescence‐activated cell sorting‐based high throughput screen, high PHB accumulating mutants from the library grown in different nutrient conditions were isolated and characterized. While several mutants isolated from the screen had increased PHB accumulation, transposon insertions in only two ORFs could be linked to increased PHB production. Disruptions of sll0461, coding for gamma‐glutamyl phosphate reductase (proA), and sll0565, a hypothetical protein, resulted in increased accumulation in standard growth media and acetate supplemented media. These genetic perturbations have increased PHB accumulation in Synechocystis and serve as markers for engineering increased polymer production in higher photosynthetic organisms. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Polyhydroxybutyrate in <Emphasis Type="Italic">Rhizobium</Emphasis> and <Emphasis Type="Italic">Bradyrhizobium</Emphasis>: quantification and <Emphasis Type="Italic">phbC</Emphasis> gene expression

Polyhydroxyalkanoates (PHAs) are hydroxyalkanoate polymers that are produced and accumulate by many kinds of bacteria. These polymers act as an energy store for bacteria. Polyhydroxybutyrate (PHB) is the most studied polymer in the PHA family. These polymers have awakened interest in the environmental and industrial research areas because they are biodegradable and have thermoplastic qualities, like polypropylene. In this work, we analyzed the PHB production in Bradyrhizobium sp., Rhizobium leguminosarum bv. phaseoli, and Rhizobium huautlense cultured with two different carbon sources. We did biochemical quantification of PHB production during the three phases of growth. Moreover, these samples were used for RNA extraction and phbC gene expression analysis via real-time PCR. The bacteria showed different manner of growth, PHB accumulation and phbC gene expression when different quantity and quality of carbon sources were used. These results showed that under different growth media conditions, the growth and metabolism of different species of bacteria were influenced. These differences reflect the increase or decrease in PHB accumulation.     

PHB accumulation in Nostoc muscorum under different carbon stress situations

Use of algae for intracellular poly-β-hydroxybutyrate (PHB) accumulation for bioplastic production offers an opportunity in economic efficiency by reduced costs. The cyanobacterium Nostoc muscorum is a PHB accumulator which presents a great potential as raw material supplier because of short generation cycles. Here, we examined a range of experimental conditions including different growth conditions of phosphate-starved cells with the addition of external carbon sources. The highest, absolute PHB accumulation was measured in a phosphate-starved medium with 1% (w/w) glucose and 1% (w/w) acetate. PHB accumulated inside algae cells. After 23 days of growth in phosphate-starved medium, 1 L of culture contained up to 145.1 mg PHB. The highest PHB accumulation based on the cell dry weight was in an experiment with aeration and CO₂ addition. The intracellular level of PHB was up to 21.5% cell dry weight after 8 days.     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

Molecular mass of poly[(R )-3-hydroxybutyric acid] produced in a recombinant Escherichia coli

 Poly[(R)-3-hydroxybutyric acid] (PHB) was produced at 37 °C by a recombinant Escherichia coli harboring the Alcaligenes eutrophus biosynthesis phbCAB genes in Luria-Bertani media containing glucose at 10–30 g/l at different pH values and the time-dependent changes in the molecular mass of PHB were studied. PHB polymers accumulated within cells while glucose was present in the medium. The number-average molecular mass of PHB decreased with time during the course of PHB accumulation, and the values for PHB were markedly dependent on the cultivation conditions of the E. coli, ranging from 0.5 MDa to 20 MDa. Under specific conditions (pH 6.0), E. coli produced PHB with an extremely high molecular mass (20 MDa). It has been suggested that a chain-transfer agent is generated in E. coli cells during the accumulation of PHB. Received: 18 July 1996 / Received revision: 4 November 1996 / Accepted: 4 November 1996     

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).     

BIOSYNTHESIS OF POLY‐3‐HYDROXYBUTYRATE (PHB) IN THE TRANSGENIC GREEN ALGA CHLAMYDOMONAS REINHARDTII1

A cell‐wall deficient strain of Chlamydomonas reinhardtii P. A Dang. CC‐849 was cotransformed with two expression vectors, p105B124 and pH105C124, containing phbB and phbC genes, respectively, from Ralstonia eutropha. The transformants were selected on Tris‐acetate‐phosphate media containing 10 μg · mL^?1 Zeomycin. Upon further screening, the transgenic algae were subcloned and maintained in culture. PCR analysis demonstrated that both phbB and phbC genes were successfully integrated into the algal nuclear genome. Poly‐3‐hydroxybutyrate (PHB) synthase activity in these transgenic algae ranged from 5.4 nmol · min^?1 · mg protein^?1 to 126 nmol · min^?1 · mg protein^?1. The amount of PHB in double transgenic algae was determined by gas chromatography–mass spectrometry (GC–MS) when comparing with PHB standard. In addition, PHB granules were observed in the cytoplasm of transgenic algal cells using TEM, which indicated that PHB was synthesized in transgenic C. reinhardtii. Hence, results clearly showed that producing PHB in C. reinhardtii was feasible. Further studies would focus on enhancing PHB production in the transgenic algae and targeting the chloroplast for PHB accumulation.     

Optimization of cultural and nutritional conditions for accumulation of poly-beta-hydroxybutyrate in Synechocystis sp. PCC 6803

Poly-beta-hydroxybutyrate (PHB) accumulation in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, was studied under various cultural and nutritional conditions. Under controlled condition, cells harvested at the stationary phase of growth depicted maximum accumulation of PHB, i.e., 4.5% (w/w of dry cells) as compared to lag (1.8%) or logarithmic (2.9%) phases of cultures. A temperature range of 28-32 degrees C and pH between 7.5 and 8.5 were preferred for PHB accumulation. Cells cultivated under regular light-dark cycles accumulated more PHB (4.5%) than those grown under continuous illumination (2.4%). Nitrogen and phosphorus starvation stimulated PHB accumulation up to the tune of 9.5 and 11% (w/w of dry cells), respectively. Synechocystis cells pre-grown in glucose (0.1%)-supplemented BG-11 medium when subjected to P-deficiency in presence of acetate (0.4%), PHB accumulation was boosted up to 29% (w/w of dry cells), the value almost 6-fold higher with respect to photoautotrophic condition. Fishpond discharges were found as suitable media for PHB accumulation in the test cyanobacterium.     

Poly(3-hydroxybutyrate) influences biofilm formation and motility in the novel Antarctic species <Emphasis Type="Italic">Pseudomonas extremaustralis</Emphasis> under cold conditions

Polyhydroxyalkanoates (PHAs) are highly reduced bacterial storage compounds that increase fitness in changing environments. It has previously shown that polyhydroxybutyrate (PHB) accumulation is essential during the growth under cold conditions. In this work, the relationship between PHB accumulation and biofilm development at low temperature was investigated. P. extremaustralis, an Antarctic strain able to accumulate PHB, and its phaC mutant, impaired in the synthesis of this polymer, were used to analyze microaerobic growth, biofilm development, EPS content and motility. PHB accumulation increased motility and survival of planktonic cells in the biofilms developed by P. extremaustralis under cold conditions. Microaerobic conditions rescued the cold growth defect of the mutant strain. The PHB accumulation capability could constitute an adaptative advantage for the colonization of new ecological niches in stressful environments.     

Influence of electron acceptor,carbon, nitrogen,and phosphorus on polyhydroxyalkanoate (PHA) production by <Emphasis Type="Italic">Brachymonas</Emphasis> sp. P12

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorus-removing bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH₄Cl), and phosphorus (KH₂PO₄) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g⁻¹ biomass, but the PHB concentration of an anoxic culture decreased (−218 mg PHB g⁻¹ biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g⁻¹ biomass h⁻¹, was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.     

Production of poly-β-hydroxybutyrate by Alcaligenes eutrophus transformants harbouring cloned phbCAB genes

Summary Three transformants of Alcaligenes eutrophus harbouring the recombinant plasmids containing phbCAB, phbAB, and phbC genes, were cultivated to investigate the effect of cloned genes on cell growth and poly--hydroxybutyrate accumulation. Both in the nutrient-rich and minimal media, the increased PHB accumulation in the transformants was observed compared to the parent strain, and this was the result of the increased enzyme activities in the transformants. Low carbon concentration and high C/N molar ratio favored higher PHB accumulations in the transformants. The transformant harbouring the phbC gene showed the highest PHB accumulation, which indicated that PHB synthase was the most critical enzyme for PHB biosynthesis in the transformant.     

Zobellella denitrificans strain MW1, a newly isolated bacterium suitable for poly(3-hydroxybutyrate) production from glycerol

Aims: To search for new bacteria for efficient production of polyhydroxyalkanoates (PHAs) from glycerol. Methods and Results: Samples were taken from different environments in Germany and Egypt, and bacteria capable of growing in mineral salts medium with glycerol as sole carbon source were enriched. From a wastewater sediment sample in Egypt, a Gram‐negative bacterium (strain MW1) was isolated that exhibited good growth and that accumulated considerable amounts of polyhydroxybutyrate (PHB) from glycerol and also from other carbon sources. The 16S rRNA gene sequence of this isolate exhibited 98·5% and 96·2% similarity to Zobellella denitrificans strain ZD1 and to Zobellella taiwanensis strain ZT1 respectively. The isolate was therefore affiliated as strain MW1 of Z. denitrificans. Strain MW1 grows optimally on glycerol at 41°C and pH 7·3 and accumulated PHB up to 80·4% (w/w) of cell dry weight. PHB accumulation was growth‐associated. Although it was not an absolute requirement, 20 g l^?1 sodium chloride enhanced both growth (5 g cell dry weight per litre) and PHB content (87%, w/w). Zobellella denitrificans strain MW1 is also capable to accumulate the poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) copolymer if sodium propionate was used as cosubstrate in addition to glycerol. Conclusions: A new PHB‐accumulating strain was isolated and identified. This strain is able to utilize glycerol for growth and PHB accumulation to high content especially in the presence of NaCl that will enable the utilization of waste glycerol from biodiesel industry. Significance and Impact of the Study: This study is the first report on accumulation of PHA in a member of the new genus Zobellella. Furthermore, utilization of glycerol as the sole carbon source for fast growth and PHB biosynthesis, growth in the presence of NaCl and high PHB contents of the cells will make this newly isolated bacterium a potent candidate for industrial production of PHB from crude glycerol occurring as byproduct during biodiesel production.     

Polysaccharide That May Serve as a Carbon and Energy Storage Compound for Sporulation in Bacillus cereus

An intracellular, glucose-containing polysaccharide accumulates in Bacillus cereus early in sporulation and is degraded at the time of spore maturation. This pattern of accumulation and degradation occurred when growth was limited by glucose or a component of yeast extract. These data suggest that the polysaccharide may be serving as a carbon and energy storage compound for sporulation. A somewhat similar pattern of accumulation and degradation of poly-beta-hydroxybutyric acid (PHB) was shown earlier by Kominek and Halvorson (1965) to occur in Bacillus cereus. When cells were grown in a medium buffered strongly at pH 7.4, however, very little accumulation of PHB occurred. We have found that polysaccharide accumulates in cells grown in both the strong and weakly buffered media. Perhaps polysaccharide is the major carbon and energy storage compound when cells are grown under conditions preventing significant accumulation of PHB. The lack of polysaccharide accumulation during the exponential phase of growth may be an indication that the needed biosynthetic enzymes are controlled by catabolite repression during growth. The polysaccharide was purified and found to consist of glucose. The iodine absorption spectrum suggests a degree of branching between that of glycogen and amylopectin.     

Role of poly-β-hydroxybutyric acid in cyst formation byAzotobacter

Several parameters associated with the growth ofAzotobacter vinelandii in liquid culture were examined in order to investigate the relationship between the accumulation and degradation of poly-β-hydroxybutyric acid (PHB), the development of viscous capsular components, and cyst formation. The amount of intracellular PHB, which increased markedly during the log phase of growth, reached a maximum during the early stationary phase and subsequently declined. During polymer degradation there was a concurrent increase in the extent of encystment in the cultures supplemented with CaCO₃. An increase was noted in the viscosity of culture supernatants during polymer degradation when CaCO₃ was deleted from the medium and the culture pH was controlled by the periodic addition of 0.1m KOH. The extent of encystment and the amount of PHB accumulated were directly proportional to the substrate concentration. The PHB was selectively labeled by the addition of sodium acetate-2-¹⁴C to late log-phase cells. During polymer utilization in either encysting or nonencysting cultures 20% of the label was evolved as CO₂. In the nonencysting cultures, 45% of the radioactivity was distributed between residual PHB and other cellular components, and 35% was in the supernatant polysaccharide-like material. Intact cysts retained 80% of the label. Experiments with ruptured cysts indicated that about 35% of the radioactivity was present in the intine material.     

Effect of anaerobic promoters on the microaerobic production of polyhydroxybutyrate (PHB) in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from Ralstonia eutropha for the micro- or anaerobic PHB production in recombinant Escherichia coli. Among the promoters, the one for alcohol dehydrogenase (P _adhE ) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring P _adhE and phbCAB achieved a 48% PHB accumulation in the cell dry weight after 48 h of static culture compared with only 30% PHB production under its native promoter. Sixty-seven percent PHB was produced in the dry weight (CDW) of an acetate pathway deleted (Δpta deletion) E. coli JW2294 harboring the vector pWCY09. In a batch process conducted in a 5.5-l NBS fermentor containing 3 l glucose LB medium, E. coli JW2294 (pWCY09) grew to 7.8 g/l CDW containing 64% PHB after 24 h of microaerobic incubation. In addition, molecular weight of PHB was observed to be much higher under microaerobic culture conditions. The high activity of P _adhE appeared to be the reason for improved micro- or anaerobic cell growth and PHB production while high molecular weight contributed to the static culture condition.     

Formation and utilisation of carbon reserves by Rhizobium

The accumulation and utilisation of poly-β-hydroxybutyrate (PHB) and extracellular polysaccharide (ECPS) have been studied in Rhizobium NZP 2037 and it was found that PHB synthesis was slow during growth whereas ECPS formation was not affected. PHB synthesis only was dependent on the pH of the medium. PHB and ECPS were both used as carbon sources in the absence of other exogenous carbon. ECPS when given to starved cells as sole carbon source in the absence of nitrogen was used for PHB synthesis by the organism. In the presence of nitrogen ECPS was found to support growth. The extracellular breakdown of ECPS was demonstrated and was accompanied by the release of its constituent glucose. This was favoured by low pH.     

Growth-associated production of poly-β-hydroxybutyrate from glucose or alcoholic distillery wastewater by Actinobacillus sp. EL-9

Summary The characteristics of poly--hydroxybutyrate (PHB) production from glucose or alcoholic distillery wastewater by isolated Actinobacillus sp. EL-9 were investigated. PHB production was not dependent on nutrients limitation in Actinobacillus sp. EL-9. The PHB accumulation of Actibobacillus sp. EL-9 followed a growth-associated type where the cell growth and PHB accumulation were carried out simultaneously. The Actinobacillus sp. EL-9 was shown to synthesize and accumulate PHB from alcoholic distillery wastewater during growth. The best growth and PHB production were obtained with enzyme-hydrolyzed alcoholic distillery wastewater.     

Poly(β-hydroxybutyric acid) thermoplastic production by Alcaligenes latus: Behavior of fed-batch cultures

Fed-batch culture of Alcaligenes latus, ATCC 29713, was investigated for producing the intracellular bioplastic poly(β–hydroxybutyric acid), PHB. Constant rate feeding, exponentially increasing feeding rate, and pH-stat fed batch methods were evaluated. pH-stat fed batch culture reduced or delayed accumulation of the substrate in the broth and led to significantly enhanced PHB productivity relative to the other modes of feeding. Presence of excessive substrate appeared to inhibit PHB synthesis, but not the production of cells. In fed-batch culture, the maximum specific growth rate (0.265?h^?1) greatly exceeded the value (0.075?h^?1) previously observed in batch culture of the same strain. Similarly, the maximum PHB production rate (up to 1.15?g?·?l^?1?·?h^?1) was nearly 8-fold greater than values observed in batch operations. Fed-batch operation was clearly superior to batch fermentation for producing PHB. A low growth rate was not a prerequisite for PHB accumulation, but a reduced or delayed accumulation of substrate appeared to enhance PHB accumulation. Under the best conditions, PHB constituted up to 63% of dry cell mass after 12?h of culture. The average biomass yield coefficient on sucrose was about 0.35, or a little less than in batch fermentations. The highest PHB concentrations attained were about 18?g?·?l^?1.     

Overexpression of NAD kinase in recombinant <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> harboring the <Emphasis Type="BoldItalic">phbCAB</Emphasis> operon improves poly(3-hydroxybutyrate) production

NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16–35 wt.% compared with the controls; meanwhile, NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with 7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield Y _PHB/glucose showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain, a 76% increase for the Y _PHB/glucose. These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.