Metabolic flux analysis of a poly-β-hydroxybutyrate producing cyanobacterium,Synechococcus sp. MA19, grown under photoautotrophic conditions

To understand the utilization property of light energy,Synechococcus sp. MA19, a poly-β-hydroxybutyrate (PHB) producer, was cultivated at the different incident light intensities of 15.3, 50.0 and 78.2 W/m² using media with and without phosphate. From the results of metabolic flux analysis, it was found that the cell yield based on ATP synthesis was estimated as 3.5×10⁻³ kg-biomass/mol-ATP in these cultures. Under the examined conditions, there were no significant differences in the efficiency of light energy conversion to chemical energies estimated as ATP synthesis and reducing potential (NADH+NADPH) formation whether the PHB synthesis took place or not. The energy converted from light to ATP was kept relatively high around the energy absorbed by the cells of 2.5–3.0×10⁶ J h⁻¹ kg⁻¹, whereas the energy of reducing potential was hardly changed in the examined range of the energy absorbed by the cells.     

Production of poly-β-hydroxybutyrate by fed-batch culture of recombinantEscherichia coli

Summary A recombinantEscherichia coli strain harboring the PHB biosynthesis genes fromAlcaligenes eutrophus was used to produce poly--hydroxybutyrate (PHB) by pH-stat fedbatch culture. Initial glucose concentration for optimal growth was found to be 20g/L from a series of flask cultures. A final PHB concentration of 88.8 g/L could be obtained after 42 hrs of cultivation.     

Production of poly-β-hydroxybutyrate on molasses by recombinant Escherichia coli

Beet molasses successfully replaced glucose as sole carbon source to produce poly--hydroxybutyrate by a recombinant Escherichia coli strain (HMS174/pTZ18u-PHB). The fermentation with molasses was cheaper than with glucose. The final dry cell weight, PHB content and PHB productivity were 39.5 g/L, 80% (w/w) and 1 g/Lh, respectively, in a 5 L stirred tank fermenter after 31.5 h fed-batch fermentation with constant pH and dissolved O2 content. © Rapid Science Ltd. 1998     

Production of poly-β-hydroxybutyrate by Alcaligenes eutrophus on different carbon sources

Poly--hydroxybutyrate was produced in shake cultures by Alcaligenes eutrophus H16 on fructose, xylose, and fumaric, itaconic, lactic and propionic acids in a three-stage process. The maximum polymer concentration of 6.9 g l^–1 (69% of cell dry matter) was obtained with 20g l^–1 of fructose with a volumetric productivity of about 0.22 g l^–1 h^–1 at 24h. Up to about 3 g l^–1 (about 50% of cell dry matter) of polymer was also produced on lactic and propionic acids as the sole carbon source during the production phase. In multivatiate optimization employing an orthogonal 2³-factorial central composite experimental design with fructose as the substrate in a single-stage process, the optimal initial fructose concentration decreased from 35 g l^–1 to 24 g l^–1 when the incubation time was increased from about 35 h to 96 h. The optimal shaking speed range was 90–113 rpm. Correspondence to: S. Linko     

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.     

Production of poly-(β-hydroxybutyrate) from saponified Vernonia galamensis oil by Alcaligenes eutrophus

Saponified vernonia oil was converted exclusively to poly(β-hydroxybutyrate) (PHB) by Alcaligenes eutrophus in a single-stage batch culture. After harvesting, centrifugation followed by lyophilization, the resulting dried cells contained up to 42.8 wt% PHB having a peak molecular mass of 381 863 Da, weight-average molecular mass of 308 390 Da, and a polydispersity of 1.1. The PHB had a melting point (T_m) range of 163–174°C with a maximum at 172°C (lit. T_m, 175°C), and heat of fusion of 18.43 cal g⁻¹. Fermentation performed under varying conditions of nitrogen limitation indicated that there was no significant effect of nitrogen concentration on the molecular mass of PHB produced from vernonia oil by A. eutrophus. Received 27 March 1998/ Accepted in revised form 17 July 1998     

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.     

Improvement in cell yield of Methylobacterium sp. by reducing the inhibition of medium components for poly-β-hydroxybutyrate production

The inhibitory effect of the concentrations of medium components on the growth of Methylobacterium sp. for poly--hydroxybutyrate production was investigated by measuring the specific growth rates for various concentrations of each medium component. When the methanol concentration was increased, the cell growth decreased and was strongly inhibited above 6% (v/v) methanol. Ammonia, calcium and iron ion did not significantly inhibit the cell growth while there were some inhibitory effects at high concentrations of sodium, potassium, and magnesium. In particular, phosphate gave most significant inhibition at concentrations higher than 75 mM. By using an automatic feeding control system of methanol, ammonia, phosphate, and minerals, their concentrations were maintained within the level necessary to reduce the inhibition of medium components. The finial dry cell weight of Methylobacterium sp. in such a system was 172 g/l at 84 h.     

Production of poly-β-hydroxybutyrate by Azotobacter vinelandii UWD in media containing sugars and complex nitrogen sources

Summary Vigorously aerated batch cultures of Azotobacter vinelandii UWD formed < 1 g poly--hydroxybutyrate (PHB)/l in media containing pure sugars and 3 g PHB/l in media containing cane molasses, corn syrup or malt extract. However, > 7 g PHB/l was formed when the medium contained 5% beet molasses. Increased yields of PHB were promoted in the media containing pure or unrefined sugars by the addition of complex nitrogen sources. The greatest effect was obtained with 0.05–0.2% fish peptone (FP), proteose peptone no. 3 or yeast extract. Peptones caused a 1.6-fold increase in residual non-PHB biomass and up to a 25-fold increase in PHB content. Hence the increased PHB formation was not simply due to stimulation of culture growth. The amount of PHB per cell protein formed by UWD in media containing FP was greatest in glucose = corn syrup > malt extract > sucrose = fructose = cane molasses > maltose, as carbon sources. The addition of FP to medium containing beet molasses did not stimulate PHB yield. The peptone effect was most significant in well-aerated cultures, which were fixed nitrogen and consuming glucose at a high rate. An explanation for the peptone effect on PHB yield stimulation is proposed.     

Wastewater utilization for poly-β-hydroxybutyrate production by the cyanobacterium Aulosira fertilissima in a recirculatory aquaculture system

Intensive aquaculture releases large quantities of nutrients into aquatic bodies, which can lead to eutrophication. The objective of this study was the development of a biological recirculatory wastewater treatment system with a diazotrophic cyanobacterium, Aulosira fertilissima, and simultaneous production of valuable product in the form of poly-β-hydroxybutyrate (PHB). To investigate this possible synergy, batch scale tests were conducted under a recirculatory aquaculture system in fiber-reinforced plastic tanks enhanced by several manageable parameters (e.g., sedimentation, inoculum size, depth, turbulence, and light intensity), an adequate combination of which showed better productivity. The dissolved-oxygen level increased in the range of 3.2 to 6.9 mg liter⁻¹ during the culture period. Nutrients such as ammonia, nitrite, and phosphate decreased to as low as zero within 15 days of incubation, indicating the system''s bioremediation capability while yielding valuable cyanobacterial biomass for PHB production. Maximum PHB accumulation in A. fertilissima was found in sedimented fish pond discharge at 20-cm culture depth with stirring and an initial inoculum size of 80 mg dry cell weight (dcw) liter⁻¹. Under optimized conditions, the PHB yield was boosted to 92, 89, and 80 g m⁻², respectively for the summer, rainy, and winter seasons. Extrapolation of the result showed that a hectare of A. fertilissima cultivation in fish pond discharge would give an annual harvest of ∼17 tons dry biomass, consisting of 14 tons of PHB with material properties comparable to those of the bacterial polymer, with simultaneous treatment of 32,640 m³ water discharge.     

Rapid and simple analysis of poly-β-hydroxybutyrate content by capillary isotachophoresis

Summary We present a rapid method for the direct analysis of poly--hydroxybutyrate (PHB) content in the soil bacteria Alcaligenes eutrophus. PHB from the fresh cells was converted by sulfuric acid to the crotonic acid and measured by capillary isotachophoresis after the neutralization by CaCO₃. The method can be used for rapid and routine monitoring of the fermentation processes in samples containing 0.001 to 20 mg of PHB.     

Production and characterization of poly-β-hydroxybutyrate (PHB) polymer from Aulosira fertilissima

Biopolymers such as polyhydroxyalkanoates (PHAs) are a class of secondary metabolites with promising importance in the field of environmental, agricultural, and biomedical sciences. To date, high-cost commercial production of PHAs is being carried out with heterotrophic bacterial species. In this study, a photoautotrophic N₂-fixing cyanobacterium, Aulosira fertilissima, has been identified as a potential source for the production of poly-β-hydroxybutyrate (PHB). An accumulation up to 66% dry cell weight (dcw) was recorded when the cyanobacterium was cultured in acetate (0.3%) + citrate (0.3%)-supplemented medium against 6% control. Aulosira culture supplemented with 0.5% citrate under P deficiency followed by 5?days of dark incubation also depicted a PHB accumulation of 51% (dcw). PHB content of A. fertilissima reached up to 77% (dcw) under P deficiency with 0.5% acetate supplementation. Optimization of process parameters by response surface methodology resulted into polymer accumulation up to 85% (dcw) at 0.26% citrate, 0.28% acetate, and 5.58?mg?L^?1 K₂HPO₄ for an incubation period of 5?days. In the A. fertilissima cultures pre-grown in fructose (1.0%)-supplemented BG 11 medium, when subjected to the optimized condition, the PHB pool boosted up to 1.59?g?L^?1, a value ～50-fold higher than the control. A. fertilissima is the first cyanobacterium where PHB accumulation reached up to 85% (dcw) by manipulating the nutrient status of the culture medium. The polymer extracted from A. fertilissima exhibited comparable material properties with the commercial polymer. As compared with heterotrophic bacteria, carbon requirement in A. fertilissima for PHB production is lower by one order magnitude; thus, low-cost PHB production can be envisaged.     

Transport of poly-β-hydroxybutyrate in human plasma

Poly-β-hydroxybutyrate (PHB) is an amphiphilic lipid that has been found to be a ubiquitous component of the cellular membranes of bacteria, plants and animals. The distribution of PHB in human plasma was investigated using chemical and immunological methods. PHB concentrations proved highly variable; in a random group of 24 blood donors, total plasma PHB ranged from 0.60 to 18.2 mg/l, with a mean of 3.5 mg/l. In plasma separated by density gradient ultracentrifugation, lipoproteins carried 20–30% of total plasma PHB; 6–14% in the very low density lipoproteins (VLDL), 8–16% in the low density lipoproteins (LDL), and < 3% in the high density lipoproteins (HDL). The majority of plasma PHB (70–80%) was found in protein fractions of density > 1.22 g/ml. Western blot analysis of the high density fractions with anti-PHB F(ab')₂ identified albumin as the major PHB-binding protein. The affinity of albumin for PHB was confirmed by in vitro studies which demonstrated transfer of ¹⁴C-PHB from chloroform into aqueous solutions of human and bovine serum albumins. PHB was less tightly bound to LDL than to other plasma components; the polymer could be isolated from LDL by extraction with chloroform, or by digestion with alkaline hypochlorite, but it could not similarly be recovered from VLDL or albumin. PHB in the LDL correlated positively with total plasma cholesterol and LDL cholesterol, and negatively with HDL cholesterol. The wide concentration range of PHB in plasma, its presence in VLDL and LDL and absence in HDL, coupled with its physical properties, suggest it may have important physiological effects.     

Production of poly-β-hydroxybutyrate from methanol: characterization of a new isolate of Methylobacterium extorquens

Summary Poly--hydroxybutyric acid (PHB) and similar bacterial polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of methanol, one of the cheapest noble substrates available, may help to reduce the cost of producing such bioplastics. As a first step, a culture collection of 118 putative methylotrophic microorganisms was obtained from various soil samples without any laboratory enrichment step to favour culture diversity. The most promising culture was selected based on rapidity of growth and PHB accumulation and later identified as Methylobacterium extorquens. This isolate was obtained from soml contaminated regularly with used oil products for some 40 years. Concentrations of methanol greater than 8 g/l affected growth significantly and the methanol concentration was optimal at 1.7 g/l. PHB concentrations averaged 25–30% (w/v) of dry weight under non-optimized conditions. Controlling methanol concentration, using an open-loop configuration, led to biomass levels of 9–10 g/l containing 30–33% PHB while preventing methanol accumulation. The new isolate was also able to produce the co-polymer PHB/poly--hydroxyvalerate (PHV) using the mixture methanol + valerate. The PHV-to-PHB ratio was about 0.2 at the end of the fermentation. An average molecular mass varying between 2 and 3 × 10⁵ Da was obtained for three PHB samples using two different measurement methods.Publication number NRCC No. 33672 Offprint requests to: D. Groleau     

Neural network designs for poly- β-hydroxybutyrate production optimization under simulated industrial conditions

Improvement of the fermentation efficiency of poly--hydroxybutyrate (PHB) may make it competitive with chemically synthesized petroleum-based polymers. One step toward this is optimization of fluid dispersion and the feed rates to a fed-batch bioreactor. In a recent study using a fermentation model, dispersion corresponding to a Peclet number of 20 was shown to maximize the productivity of PHB. Here further improvement has been investigated using neural optimization. A comparison of seven neural topologies has shown that while feed-forward and radial basis neural networks are computationally efficient, recurrent networks generate higher concentrations of PHB. All networks enhanced the productivity by 16–93% over model-based optimization.     

Production of β-galactosidase from thermophilic fungus Rhizomucor sp

A thermostable β-galactosidase was produced extracellularly by a thermophilic Rhizomucor sp, with maximum enzyme activity (0.21 U mg⁻¹) after 4 days under submerged fermentation condition (SmF). Solid state fermentation (SSF) resulted in a nine-fold increase in enzyme activity (2.04 U mg⁻¹). The temperature range for production of the enzyme was 38–55°C with maximum activity at 45°C. The optimum pH and temperature for the partially purified enzyme was 4.5 and 60°C, respectively. The enzyme retained its original activity on incubation at 60°C up to 1 h. Divalent cations like Co²⁺, Mn²⁺, Fe²⁺ and Zn²⁺ had strong inhibitory effects on the enzyme activity. The K _m and V _max for p-nitrophenyl-β- _D-galactopyranoside and o-nitrophenyl-β - _D-galactopyranoside were 0.39 mM, 0.785 mM and 232.1 mmol min⁻¹ mg⁻¹ respectively. The K _m and V _max for the natural substrate lactose were 66.66 μM and 0.20 μ mol min⁻¹ mg⁻¹. Received 10 March 1997/ Accepted in revised form 17 July 1997     

Some properties of Photosystem II preparations from the cyanobacterium Synechococcus sp. — presence of an l-amino acid oxidase in Photosystem II complexes from Synechococcus sp.

A highly active O₂-evolving Photosystem II complex has been purified from the cyanobacterium Synechococcus sp., and this complex has been compared with the Photosystem II complex previously isolated from this cyanobacterium (Ohno, T., Satoh, K. and Katoh, S. (1986) Biochim. Biophys. Acta 852, 1–8). Further treatment of the O₂-evolving complex with the detergent sodium taurodesoxycholate resulted in a complex which consisted mainly of the 47 and 40 kDa peptides and which had lost the O₂-evolving activity, but which could still reduce 2,6-dichlorophenolindophenol with 1,5-diphenylcarbazide. Previously, we have shown that a flavoprotein of 49 kDa which has an l-amino acid oxidase activity under certain conditions, is a component of highly active Photosystem II preparations from the cyanobacterium Anacystis nidulans (Pistorius, E.K. and Gau, A.E. (1986) FEBS Lett. 206, 243–248). Based on immunological studies with the antiserum raised against the l-amino acid oxidase protein from A. nidulans, we show that a protein which cross-reacts with this antiserum is present in the highly purified Photosystem II preparations from Synechococcus sp. Moreover, an l-amino acid oxidase activity could also be detected in Photosystem II preparations from Synechococcus sp. The enzyme preferentially oxidizes basic l-amino acids as l-arginine, l-ornithine, 2,3-diamino propionic acid and l-citrulline. In contrast to the enzyme from A. nidulansl-lysine is not oxidized. The here shown presence of an l-amino acid oxidase protein in Photosystem II preparations from Synechococcus sp. is an additional support of our hypothesis that a flavoprotein is a functional component of the water-oxidizing enzyme complex.     

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.     

Pathway of anaerobic poly-β-hydroxybutyrate degradation byIlyobacter delafieldii

Ilyobacter delafieldii produced an extracellular poly--hydroxybutyrate (PHB) depolymerase when grown on PHB; activity was not detected in cultures grown on 3-hydroxybutyrate, crotonate, pyruvate or lactate. PHB depolymerase activity was largely associated with the PHB granules (supplied as growth substrate), and only 16% was detected free in the culture supernatant. Monomeric 3-hydroxybutyrate was detectable as a product of depolymerase activity. The monomer was fermented to acetate, butyrate and H₂. After activation by coenzyme A transfer from acetyl-CoA or butyryl-CoA, the resultant 3-hydroxybutyryl-CoA was oxidized to acetoacetyl-CoA (producing NADH), followed by thiolytic cleavage to yield acetyl-CoA which was further metabolized to acetyl-phosphate, then to acetate with concomitant ATP production. The reducing equivalents (NADH) could be disposed of by the evolution of H₂, or by a reductive pathway in which 3-hydroxybutyryl-CoA was dehydrated to crotonyl-CoA and reduced to butyryl-CoA. In cocultures ofI. delafieldii withDesulfovibrio vulgaris on PHB, the H₂ partial pressure was much lower than in the pure cultures, and sulfide was produced. Thus interspecies hydrogen transfer caused a shift to increased acetate and H₂ production at the expense of butyrate.