Possibilities for controlling a PHB accumulation process using various analytical methods

Poly-beta-hydroxybutyrate (PHB) and other polyesters can be produced by various species of bacteria. Of the possible carbon sources, methane could prove to be one of the most suitable substrates for the manufacture of PHB. The methanotrophic strain Methylocystis sp. GB 25 DSM 7674 was applied in order to accumulate PHB in a rapid, non-sterile process. Cultivation was performed in two stages: a continuous growth phase (dilution rate 0.17 h(-1)) and a PHB accumulation phase under deficiency conditions of an essential nutrient (e.g. phosphorus) in batch culture. The PHB content of the biomass was as high as 51%; efficiency was the highest during the first 5 h of the product formation process. The PHB produced is of very high quality, having a high molecular mass of up to 2.5 x 10(6) Da. In order to monitor and control the process, a rapid analysis method based upon turbidimetry in the visible range (438 nm) was applied. Moreover, the PHB content of the biomass was determined using an FT-IR-spectroscopic method with ATR sampling and multivariate calibration. We achieved a value of 1.4% as the best standard error of cross validation. The nitrogen content of the PHB final product (a product quality parameter) was estimated by spectroscopic method in the visible range.     

Production of poly-b-hydroxybutyrate by Azotobacter vinelandii in a two-stage fermentation process

Poly-b-hydroxybutyrate (PHB) production in Azotobacter vinelandii UWD, a mutant that produces PHB constitutively, was suppressed by high aeration of beet molasses medium. Thus a two-stage process was designed using aeration to promote growth and suppress PHB production in the first phase, while lower aeration of raw sugar medium containing fish peptone was used to promote PHB formation in the second phase. A PHB yield of 36 g/l and productivity of > 1 g polymer l -1 .h was obtained by this approach.     

Microbial Conversion of Methane into poly-β-hydroxybutyrate (PHB): Growth and intracellular product accumulation in a type II methanotroph

The biochemical pathways of methane to poly-β-hydroxybutyrate (PHB) in type II methanotrophs have been analyzed and used to propose stoichiometric equations for cell biomass and PHB. Conditions necessary for PHB accumulation in a batch culture of Methylocystis parvus OBBP were studied. In nitrogen limited cultures PHB started accumulating in the declining growth phase and maximum rate of PHB formation occurred during the late growth and early stationary phases. Inoculum age had an effect on maximum level of intracellular PHB, which increased with inoculum age up to ca. 70% of the cell dry weight using a 70 h inoculum. It has been shown that oxygen and methane requirements are high and pose a great stress on the mass transfer in the system; this limits the obtainment of high cell concentrations. When conditions for increased mass transfer of both CH₄ and O₂ were used, 5 g/l of cells could be produced. Finally an enrichment strategy was investigated so that cells would start accumulating PHB earlier during the growth phase. Alternatives to increase cell mass concentrations are discussed.     

Optimization of polyhydroxybutyrate production from a wild type and two mutant strains of Rhodobacter sphaeroides using statistical method

Interaction studies using central composite design (CCD) gave the optimum concentrations of acetate at 4 g l(-1) and (NH4)2SO4 at 0.01 g l(-1) with an optimum temperature of 35 degrees C. Rhodobacter sphaeroides N20 gave the highest PHB (7.8 g l(-1)) and biomass (DCW) (8.2 g l(-1)) values compared to the wild type strain and the mutant strain U7. The CCD results predicted that the optimum medium for the mutant strain N20 consisted of 3.90 g l(-1) acetate, 0.01 g l(-1) (NH4)2SO4 at 33.5 degrees C (R2=0.985). Validation of this model by culturing the mutant strain in this optimum medium exhibited similar values of PHB (7.76 g l(-1)), biomass (8.32 g l(-1)) and the PHB content in the cell 93.2% of DCW. Similar amounts of PHB were also obtained in batch fermentations using a 5-l bioreactor. The effect of pH and aeration rate was also studied and the optimum values were found to be pH 7.0 with an aeration rate of 1.0 vvm. Under these optimal conditions, strain N20 produced the highest amount of PHB production (8.76 g l(-1)), PHB content (95.4% of DCW) as well as the product yield (Yp/x) (0.72). These results are the highest values ever obtained from photosynthetic bacteria reported so far.     

Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli.

Recombinant Escherichia coli XL1-Blue harboring a high-copy-number plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate synthesis genes could efficiently synthesize poly(3-hydroxybutyrate) (PHB) in a complex medium containing yeast extract and tryptone but not in a defined medium. One of the reasons for the reduced PHB production in a defined medium was thought to be severe filamentation of cells in this medium. By overexpressing an essential cell division protein, FtsZ, in recombinant E. coli producing PHB, filamentation could be suppressed and PHB could be efficiently produced in a defined medium. A high PHB concentration of 149 g/liter, with high productivity of 3.4 g of PHB/liter/h, could be obtained by the pH-stat fed-batch culture of the filamentation-suppressed recombinant E. coli in a defined medium. It was also found that insufficient oxygen supply at a dissolved oxygen concentration (DOC) of 1 to 3% of air saturation during active PHB synthesis phase did not negatively affect PHB production. By growing cells to the concentration of 110 g/liter and then controlling the DOC in the range of 1 to 3% of air saturation, a PHB concentration of 157 g/liter and PHB productivity of 3.2 g of PHB/liter/h were obtained. For the scale-up studies, fed-batch culture was carried out in a 50-liter stirred tank fermentor, in which the DOC decreased to zero when cell concentration reached 50 g/liter. However, a relatively high PHB concentration of 101 g/liter and PHB productivity of 2.8 g of PHB/liter/h could still be obtained, which demonstrated the possibility of industrial production of PHB in a defined medium by employing the filamentation-suppressed recombinant E. coli.     

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.     

The treatment of gaseous benzene by two-phase partitioning bioreactors: a high performance alternative to the use of biofilters

A 2-l (1-l working volume) two-phase partitioning bioreactor (TPPB) was used as an integrated scrubber/bioreactor in which the removal and destruction of benzene from a gas stream was achieved by the reactor's organic/aqueous liquid contents. The organic solvent used to trap benzene was n-hexadecane, and degradation of benzene was achieved in the aqueous phase using the bacterium Alcaligenes xylosoxidans Y234. A gas stream with a benzene concentration of 340 mg l(-1) at a flow rate of 0.414 l h(-1) was delivered to the system at a loading capacity of 140 g m(-3) h(-1), and an elimination capacity of 133 g m(-3 )h(-1) was achieved (the volume in this term is the total liquid volume of the TPPB). This elimination capacity is between 3 and 13 times greater than any benzene elimination achieved by biofiltration, a competing biological air treatment strategy. It was also determined that the evaluation of TPPB performance in terms of elimination capacity should include the cell mass present in the system, as this is a readily controllable quantity. A specific benzene utilization rate of 0.57 g benzene (g cells)(-1) h(-1) was experimentally determined in a bioreactor with a cell concentration that varied dynamically between 0.2 and 1 g l(-1). If it assumed that this specific benzene utilization rate (0.57 g g(-1) h(-1)) is independent of cell concentration, then a TPPB operated at high cell concentrations could potentially achieve elimination capacities several hundred times greater than those obtained with biofilters.     

The effect of dissolved oxygen on PHB accumulation in activated sludge cultures

Nitrogen removal from wastewater is often limited by the availability of reducing power to perform denitrification, especially when treating wastewaters with a low carbon:nitrogen ratio. In the increasingly popular sequencing batch reactor (SBR), bacteria have the opportunity to preserve reducing power from incoming chemical oxygen demand (COD) as poly-beta-hydroxybutyrate (PHB). The current study uses laboratory experiments and mathematical modeling in an attempt to generate a better understanding of the effect of oxygen on microbial conversion of COD into PHB. Results from a laboratory SBR with acetate as the organic carbon source showed that the aerobic acetate uptake process was oxygen-dependent, producing higher uptake rates at higher dissolved oxygen (DO) supply rates. However, at the lower DO supply rates (k(L)a 6 to 16 h(-1), 0 mg L(-1) DO), a higher proportion of the substrate was preserved as PHB than at higher DO supply rates (k(L)a 30, 51 h(-1), DO >0.9 mg L(-1)). Up to 77% of the reducing equivalents available from acetate were converted to PHB under oxygen limitation (Y(PHB/Ac) 0.68 Cmol/Cmol), as opposed to only 54% under oxygen-excess conditions (Y(PHB/Ac) 0.48 Cmol/Cmol), where a higher fraction of acetate was used for biomass growth. It was calculated that, by oxygen management during the feast phase, the amount of PHB preserved (1.4 Cmmol L(-1) PHB) accounted for an additional denitrification potential of up to 18 mg L(-1) nitrate-nitrogen. The trends of the effect of oxygen (and hence ATP availability) on PHB accumulation could be reproduced by the simulation model, which was based on biochemical stoichiometry and maximum rates obtained from experiments. Simulated data showed that, at low DO concentrations, the limited availability of adenosine triphosphate (ATP) prevented significant biomass growth and most ATP was used for acetate transport into the cell. In contrast, high DO supply rates provided surplus ATP and hence higher growth rates, resulting in decreased PHB yields. The results suggest that oxygen management is crucial to conserving reducing power during the feast phase of SBR operation, as excessive aeration rates decrease the PHB yield and allow higher biomass growth.     

Application of self-cycling fermentation technique to the production of poly-beta-hydroxybutyrate

The production of poly-beta-hydroxybutyrate (PHB) by Alcaligenes eutrophus DSM 545 in a cyclone bioreactor was compared using various culture methods: batch, fed-batch, and self-cycling fermentation (SCF) with and without extended periods of nutrient deprivation. SCF is a semi-continuous method that results in a nutrient limitation for every successive generation of cells and, therefore, may have advantages for products whose formation follow secondary metabolite kinetics. Use of the SCF technique without extended nutrient deprivation produced a PHB concentration of 1.2 g L(-1) as 40% of the biomass dry weight. With nitrogen deprivation for 4 or 6 h, the concentration of PHB decreased when compared to the standard SCF technique. However, nitrogen deprivation periods of 8 h resulted in an increase in PHB concentration to 2.7 g L(-1) or 59% of the biomass dry weight. The nutrient cycling may act to repress PHB accumulation during periods of nitrogen deprivation, unless a time threshold has been reached, after which PHB accumulation occurs as in normal batch culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 815-820, 1997.     

Continuous polyhydroxybutyrate production from biogas in an innovative two-stage bioreactor configuration

Biogas biorefineries have opened up new horizons beyond heat and electricity production in the anaerobic digestion sector. Added-value products such as polyhydroxyalkanoates (PHAs), which are environmentally benign and potential candidates to replace conventional plastics, can be generated from biogas. This work investigated the potential of an innovative two-stage growth-accumulation system for the continuous production of biogas-based polyhydroxybutyrate (PHB) using Methylocystis hirsuta CSC1 as cell factory. The system comprised two turbulent bioreactors in series to enhance methane and oxygen mass transfer: a continuous stirred tank reactor (CSTR) and a bubble column bioreactor (BCB) with internal gas recirculation. The CSTR was devoted to methanotrophic growth under nitrogen balanced growth conditions and the BCB targeted PHB production under nitrogen limiting conditions. Two different operational approaches under different nitrogen loading rates and dilution rates were investigated. A balanced nitrogen loading rate along with a dilution rate (D) of 0.3 day⁻¹ resulted in the most stable operating conditions and a PHB productivity of ~53 g PHB m⁻³ day⁻¹. However, higher PHB productivities (~127 g PHB m⁻³ day⁻¹) were achieved using nitrogen excess at a D = 0.2 day⁻¹. Overall, the high PHB contents (up to 48% w/w) obtained in the CSTR under theoretically nutrient balanced conditions and the poor process stability challenged the hypothetical advantages conferred by multistage vs single-stage process configurations for long-term PHB production.     

Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli arcA</Emphasis> mutant in fed-batch microaerobic cultures

Poly(3-hydroxybutyrate) (PHB) synthesis was analyzed under microaerobic conditions in a recombinant Escherichia coli arcA mutant using glycerol as the main carbon source. The effect of several additives was assessed in a semi-synthetic medium by the 'one-factor-at-a-time' technique. Casein amino acids (CAS) concentration was an important factor influencing both growth and PHB accumulation. Three factors exerting a statistically significant influence on PHB synthesis were selected by using a Plackett-Burman screening design [glycerol, CAS, and initial cell dry weight (CDW) concentrations] and then optimized through a Box-Wilson design. Under such optimized conditions (22.02 g l(-1) glycerol, 1.78 g l(-1) CAS, and 1.83 g l(-1) inoculum) microaerobic batch cultures gave rise to 8.37 g l(-1) CDW and 3.52 g l(-1) PHB in 48 h (PHB content of 42%) in a benchtop bioreactor. Further improvements in microaerobic PHB accumulation were obtained in fed-batch cultures, in which glycerol was added to maintain its concentration above 5 g l(-1). After 60 h, CDW and PHB concentration reached 21.17 and 10.81 g l(-1), respectively, which results in a PHB content of 51%. Microaerobic fed-batch cultures allowed a 2.57-fold increase in volumetric productivity when compared with batch cultures.     

Growth of Methylosinus trichosporium OB3b on methane and poly-beta-hydroxybutyrate biosynthesis

Optimal conditions for batch cultivation of the obligate methanotroph Methylosinus trichosporium OB3b on methane without superatmospheric pressure were chosen. The yield of absolutely dry biomass after 120 h of growth reached 20 g/l. This biomass contained 30% poly-beta-hydroxybutyrate (PHB) with molecular weight 300 kDa. The growth process included the stages of biomass growth and PHB biosynthesis. The latter stage occurred under nitrogen-deficiency conditions. It was accompanied by an increase in the activity of PHB biosynthesis enzymes (beta-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase) and the main NAD(P)H producer, methylenetetrahydromethanopterin dehydrogenase. The activity of PHB depolymerase increased insignificantly.     

Growth of <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b on methane and poly-β-hydroxybutyrate biosynthesis

Optimal conditions for batch cultivation of the obligate methanotroph Methylosinus trichosporium OB3b on methane without superatmospheric pressure were chosen. The yield of absolutely dry biomass after 120 h of growth reached 20 g/l. This biomass contained 30% poly-β-hydroxybutyrate (PHB) with molecular weight 300 kDa. The growth process included the stages of biomass growth and PHB biosynthesis. The latter stage occurred under nitrogen-deficiency conditions. It was accompanied by an increase in the activity of PHB biosynthesis enzymes (β-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase) and the main NAD(P)H producer, methylenetetrahydromethanopterin dehydrogenase. The activity of PHB depolymerase increased insignificantly.     

Removal of endotoxin during purification of poly(3-hydroxybutyrate) from gram-negative bacteria.

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30 degrees C was higher than 10(4) EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.     

Fed-batch cultivation of Wautersia eutropha

Batch kinetics of polyhydroxybutyrate (PHB) synthesis in a bioreactor under controlled conditions of pH and dissolved oxygen gave a biomass of 14 g l(-1) with a PHB concentration of 6.1 g l(-1) in 60 h. The data of the batch kinetics was used to develop a mathematical model, which was then extrapolated to fed-batch by incorporating the dilution due to substrate feeding. Offline computer simulation of the fed-batch model was done to develop the nutrient feeding strategies in the fed-batch cultivation. Fed-batch strategies with constant feeding of only nitrogen and constant feeding of both nitrogen and fructose were tried. Constant feeding strategy for nitrogen and fructose gave a better PHB production rate of 0.56 g h(-1) over the value obtained in batch cultivation (PHB production rate - 0.4 g h(-1)).     

Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli.

In dialysis fermentations inhibiting metabolites can be removed from cell suspensions resulting in a prolonged exponential growth phase and higher production yields. Because of successful high cell density cultivations of Escherichia coli in a laboratory dialysis reactor, a scale-up of the process was investigated. To provide sufficient membrane area for dialysis in a technical scale fermenter, an external membrane module was used, that was also applied for oxygen supply to the culture in the external loop. Cultivations with recombinant E. coli K12, with and without induction, in 2- and 300-l reactors were carried out using external modules. Cell densities exceeding 190 g l(-1), previously obtained in laboratory dialysis fermentation, were also produced with external dialysis modules. Protein concentration in a 300-l reactor was increased to the 3.8-fold of industrial fed-batch-fermentations.     

Batch cultivation of Methylosinus trichosporium OB3b: V. Characterization of poly-beta-hydroxybutyrate production under methane-dependent growth conditions

Methanotrophs have promising applications in the bioremediation of chlorinated hydrocarbons and in the production of a biopolymer, poly-beta-hydroxybutyrate (PHB). Batch bioreactor culture conditions were studied for the accumulation of PHB by methane-grown Methylosinus trichosporium OB3b, and to evaluate the effect of PHB on the bacterial capacity to degrade trichloroethylene (TCE), a common groundwater contaminant. The PHB content of the washed and lyophilized cells was measured by gas chromatography (GC), after hydrochloric acid (HCl) propanolysis. A differential GC-based assay was developed for the monomer and the polymer of beta-hydroxybutyrate utilizing 1% and 10% HCl (v/v) reaction mixtures, respectively. During bioreactor growth in a Cu-deficient modified Higgins' medium, the cells accumulated PHB upon depletion of nitrate. A biomass yield of 3.2 g dry wt/L and a PHB accumulation of approximately 10% (w/w) were reached after 140 to 160 h, without adversely affecting the propene or TCE epoxidation specific rate given by whole cells containing soluble methane monooxygenase (sMMO). The TCE biotransformation capacity ( approximately 0.25 mg TCE oxidized/mg dry cell wt) of resting cells containing approximately 10% PHB was consistently approximately 1.6-fold greater than that of cells containing only approximately 2% PHB. Higher levels (>10%) of accumulated PHB did not enhance this biotransformation capacity further. By replacing the bioreactor inlet air + CO(2) mixture with pure O(2) at approximately 85 h of batch operation, a PHB accumulation of approximately 45% was achieved after 160 h, but the whole-cell sMMO activity was markedly decreased. In contrast, cells grown in a 10 muM Cu-supplemented Higgins' nitrate minimal salts medium (particulate MMO formation) accumulated up to 50% PHB in only 120 h, coupled with a very high biomass yield of 18 g dry cell wt/L. High PHB accumulations above approximately 20% by both the -Cu and the +Cu grown cells resulted in a decreased ratio of the electronic cell count to the absorbance at 660 nm, which is commonly used to monitor bacterial growth. (c) 1996 John Wiley & Sons, Inc.     

Comparison of recombinant Escherichia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid) and morphological changes

A stable high-copy-number plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoic acid (PHA) biosynthesis genes was constructed. This plasmid was transferred to seven Escherichia coli strains (K12, B, W, XL1-Blue, JM109, DH5alpha, and HB101), which were subsequently compared for their ability to synthesize and accumulate ploy- (3-hydroxybutyric acid) (PHB). Growth of recombinant cells and PHB synthesis were investigated in detail in Luria-Bertani (LB) medium containing 20 g/L glucose. Cell growth, the rate of PHB synthesis, the extent of PHB accumulation, the amount of glucose utilized, and the amount of acetate formed varied from one strain to another. XL1-Blue (pSYL105) and B (pSYL105) synthesized PHB at the fastest rate, which was ca. 0.2 g PHB/g true cell mass-h, and produced PHB up to 6-7 g/L. The yields of cell mass, true cell mass, and PHB varied considerably among the strains. The PHB yield of XL1-Blue (pSYL105) in LB plus 20 g/L glucose was as high as 0.369 g PHB/g glucose. Strains W (pSYL105) and K12 (pSYL105) accumulated the least amount of PHB with the lowest PHB yield at the lowest synthesis rate. JM109 (pSYL105) accumulated PHB to the highest extent (85.6%) with relatively low true cell mass (0.77 g/L). Considerable filamentation of cells accumulating PHB was observed for all strains except for K12 and W, which seemed to be due either to the overexpression of the foreign PHA biosynthesis enzymes or to the accumulation of PHB. (c) 1994 John Wiley & Sons, Inc.     

The influence of nutritional and environmental conditions on the accumulation of poly-beta-hydroxybutyrate in Bacillus mycoides RLJ B-017

AIMS: To optimize the nutritional and environmental conditions for growth of and poly-beta-hydroxybutyrate (PHB) accumulation in Bacillus mycoides RLJ B-017. METHODS AND RESULTS: An isolate, identified as B. mycoides, was grown on different sources of carbon and nitrogen. Among these, sucrose, beef extract and di-ammonium sulphate were found to be the most suitable for growth and PHB accumulation. The overall maximum value of PHB (%) in cells, PHB yield (Yp/s) and productivities (Qp and qp) were 69.4 +/- 0.4% dry cell weight (DCW), 0.21 gp gS(-1), 0.104 +/- 0.012 gp l(-1) h(-1) and 0.03 gp gx(-1) h(-1), respectively when grown in a medium containing 20 gs l(-1) sucrose, supplemented with di-ammonium sulphate. The addition of beef extract increased the value of PHB (%) in cells, PHB yield and productivities by 17.58 +/- 0,3, 23.8, 19.23 +/- 0.3 and 13.8 +/- 0.2% , respectively. The overall maximum values of PHB (% DCW), PHB yield and productivities were obtained at pH 7.0 +/- 0 .1, temperature 30 +/- 0.5 degrees C, agitation 650 rev min(-1) and oxygen transfer rate 3.8 mmol O(2) l(-1) h(-1). CONCLUSIONS: Sucrose, glucose and fructose were found to be more suitable for cell growth and PHB accumulation, but sucrose was less expensive than glucose. Among the nitrogen sources, beef extract and di-ammonium sulphate promoted PHB synthesis. The accumulation of PHB was observed to be growth associated. SIGNIFICANCE AND IMPACT OF THE STUDY: Gram-positive bacteria have not been reported to accumulate large amounts of polyhydroxyalkanoate and hence have not been considered as potent candidates for industrial production. A number of Bacillus spp. have been reported to accumulate 9-44.5% DCW PHB. By comparison, Bacillus RLJ B-017 contained 69.4 +/- 0.4% DCW PHB. Therefore, this strain has been considered as a potent organism for industrial interest. A relatively high yield of PHB was obtained in this wild strain and PHB synthesis was independent of nutrient limitation. The conditions for the higher PHB yield and productivity will be optimized in the next phase using fed-batch culture.     

High level production of supra molecular weight poly (3-hydroxybutyrate) by metabolically engineered<Emphasis Type="Italic">Escherichia coli</Emphasis>

The supra molecular weight poly ([R]-3-hydroxybutyrate) (PHB), having a molecular weight greater than 2 million Da, has recently been found to possess improved mechanical properties compared with the normal molecular weight PHB, which has a molecular weight of less than 1 million Da. However, applications for this PHB have been hampered due to the difficulty of its production. Reported here, is the development of a new metabolically engineeredEscherichia coli strain and its fermentation for high level production of supra molecular weight PHB. RecombinantE. coli strains, harboring plasmids of different copy numbers containing theAlcaligenes latus PHB biosynthesis genes, were cultured and the molecular weights of the accumulated PHB were compared. When the recombinantE. coli XL 1-Blue, harboring a medium-copy-number pJC2 containing theA. latus PHB biosynthesis genes, was cultivated by fed-batch culture at pH 6.0, supra molecular weight PHB could be produced at up to 89.8 g/L with a productivity of 2.07 g PHB/L-h. The molecular weight of PHB obtained under these conditions was as high as 22 MDa, exceeding by an order of magnitude the molecular weight of PHB typically produced inRalstonia eutropha or recombinantE. coli