Optimization of microbial poly(3-hydroxybutyrate) recover using dispersions of sodium hypochlorite solution and chloroform

Optimization was carried out for the recovery of microbiol poly(3-hydroxybutyrate) (PHB) from Alcaligenes eutrophus. This process involved the use of a dispersion made of sodium hypochlorite solution and chloroform. The dispersion enabled us to take advantage of both differential digestion by hypochlorite and solvent extraction by chloroform. The PHB recovery (%) from cell powder was maximized using a 30% hypochlorite concentration, a 90-min treatment time, and a 1:1 (v/v) chloroform-to-aqueous-phase ratio. Under these optimal conditions, the recovery was about 91% and the purity of recovered PHB was higher than 97%. The number average molecular weight, M(n) of recovered PHB was about 300,000 and the weight average molecular weight M(w) was about 1,020,000, compared to the original M(n) of 530,000 and M(w) of 1,272,000. The moderate decrease in both M(n) and M(w) might be ascribed to the shielding effect of chloroform. In addition, the relatively small decrease in M(w) probably resulted from the loss of short PHB chains which might be water soluble. The crystallinity of recovered PHB was in the range of 60 to 65%although a slightly higher crystallinity was observed when the dispersion was used. (c) 1994 John Wiley & Sons, Inc.     

Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli.

We studied recovery of poly(3-hydroxybutyric acid) (PHB) from Alcaligenes eutrophus and a recombinant Escherichia coli strain harboring the A. eutrophus poly(3-hydroxyalkanoic acid) biosynthesis genes. The amount of PHB degraded to a lower-molecular-weight compound in A. eutrophus during the recovery process was significant when sodium hypochlorite was used, but the amount degraded in the recombinant E. coli strain was negligible. However, there was no difference between the two microorganisms in the patterns of molecular weight change when PHB was recovered by using dispersions of a sodium hypochlorite solution and chloroform. To understand these findings, we examined purified PHB and lyophilized cells containing PHB by using a differential scanning calorimeter, a thermogravimetric analyzer, and nuclear magnetic resonance. The results of our analysis of lyophilized whole cells containing PHB with the differential scanning calorimeter suggested that the PHB granules in the recombinant E. coli strain were crystalline, while most of the PHB in A. eutrophus was in a mobile amorphous state. The stability of the native PHB in the recombinant E. coli strain during sodium hypochlorite treatment seemed to be due to its crystalline morphology. In addition, as determined by the thermogravimetric analyzer study, lyophilized cell powder of the recombinant E. coli strain containing PHB exhibited greater thermal stability than purified PHB obtained by chloroform extraction. The PHB preparations extracted from the two microorganisms had identical polymer properties.     

Extraction of poly-3-hydroxybutyrate using chlorinated solvents

Summary Recovery of poly-3-hydroxybutyrate (PHB) in three chlorinated solvents with or without acetone pretreatment and degradation of extracted PHB (99% pure) in hot chloroform were studied. When lyophilized Alcaligenes eutrophus biomass was used, the best results were obtained with acetone pretreatment and solvent reflux for 15 min in methylene chloride or chloroform. Recovered PHB had a 95% purity and molecular weights (M_w) of 1,050,000 and 930,000 g/mol respectively. Further heating resulted in a serious M_w, loss at reflux temperatures. Degradation of extracted PHB at 110°C in chloroform was due to random and chain-end scission, the former being predominant.     

PHB recovery by hypochlorite digestion of non-PHB biomass

Summary Hypochlorite digestion of bacterial biomass from intracellular poly--hydroxybutyrate (PHB) has not been used on a large scale since it has been reported to severely degrade PHB. In this study, to minimize degradation, the initial biomass concentration, digestion time and pH of the hypochlorite solution were optimized. Consequently, PHB of 95% purity with a weight average molecular weight (M_W) of 600,000 and a polydispersity index (PI) of 4.5 was recovered from biomass initially containing PHB with a M_W of 1,200,000 and a PI of 3.     

Recovery of poly(3-hydroxybutyrate) from coagulated Ralstonia eutropha using a chemical digestion method

For economic recovery of poly(3-hydroxybutyrate) (PHB) from culture broths of Ralstonia eutropha containing PHB, Al-based and Fe-based coagulants were used in the pretreatment step. The coagulated cells were then separated by centrifugation, and PHB was extracted by chemical digestion with a sodium hypochlorite/chloroform dispersion solution. The practical upper limits of dosage were found to be 1, 500 mg-Al/L and 1,000 mg-Fe/L, respectively, for Al- and Fe-based coagulants. When the harvested cells were treated with a 50% sodium hypochlorite/chloroform dispersion solution, PHB recovery and purity were 90-94% and 98-99%, respectively. The influence of the use of coagulants on the PHB recovery process was found to be insignificant. Despite the residual Al and Fe in the recovered PHB (less than 450 mg-Al/kg-PHB and 750 mg-Fe/kg-PHB, respectively), no detectable amounts of Al and Fe were leached from films made of the recovered PHB under acidic conditions. The use of Fe-based coagulants is less recommended because the Fe impurity can cause an unwanted colorization problem in the final product.     

The effects of sodium bisulfite in extraction of PHB by hypochlorite

Summary Molecular weight of PHB becomes low when using a hypochlorite extraction method for PHB separation. This disadvantage was overcome by adding sodium bisulfite which is an anti-oxidant. The molecular weight drop was decreased from 30 % to 14 % by adding sodium bisulfite. It was also found that raising the pH to 12 and the addition of a surfactant resulted in the improvement of the PHB purity.     

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     

SEC-MALS characterization of microbial polyhydroxyalkanoates

Characterization of poly-3-hydroxybutyric acid (PHB) and poly-3-hydroxybutyric-co-valeric acid (PHBV, 13% valerate) in chloroform was performed using size exclusion chromatography coupled to a multi-angle light scattering detector (SEC-MALS). Absolute molar mass averages, molar mass distribution, and the radius of gyration were determined. Three sample preparation methods were examined: dissolution in chloroform (1) at room temperature, (2) at 60 degrees C, and (3) after thermal pretreatment of samples (annealing at 180 degrees C with subsequent quenching in liquid nitrogen). Dissolution at 60 degrees C and dissolution of thermally pretreated samples gave molecularly dissolved PHB and PHBV. At 60 degrees C using acid free chloroform, there was no indication of degradation for up to 120 min dissolution time, whereas thermal degradation of polymers did take place during annealing at 180 degrees C. The degradation rate constants for number and weight average degree of polymerization at 180 degrees C were slightly higher for PHB (5.19 x 10(-5) min(-1), 4.95 x 10(-5) min(-1)) than for PHBV (4.99 x 10(-5) min(-1), 4.54 x 10(-5) min(-1)). The dependence of the radii of gyration on molar mass showed that both polymers form random coils in chloroform. The relationship between the absolute molar masses and relative SEC results was determined. DSC and NMR characterization also gave evidence of the progress of degradation.     

Pilot-scale extraction of PHB from recombinant E. coli by homogenization and centrifugation

A new method of poly--hydroxybutyrate (PHB) extraction from recombinant E. coli is proposed, using homogenization and centrifugation coupled with sodium hypochlorite treatment. The size of PHB granules and cell debris in homogenates was characterised as a function of the number of homogenization passes. Simulation was used to develop the PHB and cell debris fractionation system, enabling numerical examination of the effects of repeated homogenization and centrifuge-feedrate variation. The simulation provided a good prediction of experimental performance. Sodium hypochlorite treatment was necessary to optimise PHB fractionation. A PHB recovery of 80% at a purity of 96.5% was obtained with the final optimised process. Protein and DNA contained in the resultant product were negligible. The developed process holds promise for significantly reducing the recovery cost associated with PHB manufacture.This revised version was published online in October 2005 with corrections to the Cover Date.     

Size analysis of poly(3-hydroxybutyric acid) granules produced in recombinant Escherichia coli

Summary Size distributions of PHB granules synthesized in recombinant Escherichia coli are determined by photosedimentation. Mean granule Stokes diameters are in the range 1.13–1.25 m, which is larger than reported values for wild type microorganisms. Treatment with 1.5% hypochlorite and mild centrifugation did not affect granule size distribution. Treatment with 10% hypochlorite led to a significant reduction in mean diameter and total PHB.     

Control of molecular weight of poly-β-hydroxybutyric acid produced in fet-batch culture of Protomonas extorquens

Summary To control molecular weight of poly--hydroxybutyric acid (PHB) produced in a fedbatch culture of Protomonas extorquens, the effects of cultural temperature, pH, molar ratio of methanol and ammonia, and concentration of methanol in the medium on polymerization were inverstigated. Change of methanol concentration affected average molecular weight of PHB. When the cultivation was carried out at 0.05 g/l of methanol, average molecular weight of PHB reached above 8×10⁵. On the other hand, in the case of 32 g/l of methanol average molecular weight of PHB was less than 0.5×10⁵. Although every sample had a wide molelcular weight distribution, it became possible to control voluntarily average molecular weight of PHB.     

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.     

Microbial synthesis of polyhydroxybutyrate from glycerol: Gluconeogenesis, molecular weight and material properties of biopolyester

Glycerol is considered as an ideal feedstock for producing bioplastics via bacterial fermentation due to its ubiquity, low price, and high degree of reduction substrate. In this work, we study the yield and cause of limitation in poly(3‐hydroxybutyrate) (PHB) production from glycerol. Compared to glucose‐based PHB production, PHB produced by Cupriavidus necator grown on glycerol has a low productivity (0.92 g PHB/L/h) with a comparably low maximum specific growth rate of 0.11 h^?1. We found that C. necator can synthesize glucose from glycerol and that the lithotrophical utilization of glycerol (non‐fermentative substrate) or gluconeogenesis is an essential metabolic pathway for biosynthesis of cellular components. Here, we show that gluconeogenesis affects the reduction of cell mass, the productivity of biopolymer product, and the molecular chain size of intracellular PHB synthesized from glycerol by C. necator. We use NMR spectroscopy to show that the isolated PHB is capped by glycerol. We then characterized the physical properties of the isolated glycerol‐based PHB with differential scanning calorimetry and tensile tests. We found that although the final molecular weight of the glycerol‐based PHB is lower than those of glucose‐based and commercial PHB, the thermal and mechanical properties of the biopolymers are similar. Biotechnol. Bioeng. 2012; 109: 2808–2818. © 2012 Wiley Periodicals, Inc.     

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.     

Biodegradability studies of polyhydroxyalkanoate (PHA) film produced by a marine bacteria using Jatropha biodiesel byproduct as a substrate

Polyhydroxyalkanoates are water-insoluble, hydrophobic polymers and can be degraded by microorganisms that produce extracellular PHA depolymerase. The present work was aimed to evaluate the degradability of Polyhydroxyalkanoate film produced by Halomonas hydrothermalis using Jatropha biodiesel byproduct as a substrate. PHB films were subjected to degradation in soil and compared with the synthetic polymer (acrylate) and blend prepared using the synthetic polymer (acrylate) and PHB. After 50 days, 60% of weight loss in PHB film and after 180 days 10% of blended film was degraded while no degradation was found in the synthetic film. Scanning electron microscopy and confocal microscopy revealed that after 50 days the PHB film and the blended film became more porous after degradation while synthetic film was not porous. The degradative process was biologically mediated which was evident by the control in which the PHB films were kept in sterile soil and the films showed inherent integrity over time. The TGA and DSC analysis shows that the melting temperatures were changed after degradation indicating physical changes in the polymer during degradation.     

Effect of pH on the molecular weight of poly-3-hydroxybutyric acid produced by Alcaligenes sp.

Summary The molecular weight of a polymer is an important parameter characterizing the physical properties of the polymer. The effect of pH on the molecular weight of poly-3-hydroxybutyric acid (PHB) produced by Alcaligenes sp. K-912 were investigated. PHB having higher molecular weight was obtained when cultivated under high pH conditions compared to the results cultivated under low pH conditions. The polydispersity index was almost the same as 1.6. But the cell mass was decreased as the pH became higher. The molecular weight was mainly determined in the early cultivation period and molecular weight difference could be explained by calculating the number of polymer chains.     

An extracellular poly (3-hydroxybutyrate) depolymerase from Penicillium sp. DS9713a-01

Summary Penicillium sp. DS9713a-01 was obtained by ultraviolet (u.v.) light mutagenesis from the Penicillium sp. DS9713a which can degrade poly (3-hydroxybutyrate) (PHB). The enzymatic activity of DS9713a-01 was 97% higher than that of the wild-type strain. The DS9713a-01 mutant could completely degrade PHB films in 5 days; however, the wild-type strain achieved only 61% at the same time. The extracellular PHB depolymerase was purified from the culture medium containing PHB as the sole carbon source by filtration, ammonium sulfate precipitation and chromatography on Sepharose CL-6B. The molecular weight of the PHB depolymerase was about 15.1kDa determined by SDS-polyacrylamide gel electrophoresis. The optimum activity of the PHB depolymerase was observed at pH 8.6 and 50 °C. The enzyme was stable at temperatures below 37 °C and in the pH range from 8.0 to 9.2. The activity of PHB depolymerase could be activated or inhibited by some metal ions. The apparent K _m value was 0.164 mg ml⁻¹. Mass spectrometric analysis of the water-soluble products after enzymatic degradation revealed that the primary product was the monomer, 3-hydroxybutyric acid.     

Effect of ethanol and hydrogen peroxide on poly(3-hydroxybutyrate) biosynthetic pathway in <Emphasis Type="Italic">Cupriavidus necator</Emphasis> H16

Exposition of Cupriavidus necator to ethanol or hydrogen peroxide at the beginning of the stationary phase increases poly(3-hydroxybutyrate) (PHB) yields about 30%. Hydrogen peroxide enhances activity of pentose phosphate pathway that probably consequently increases intracellular ratio NADPH/NADP⁺. This effect leads to stimulation of the flux of acetyl-CoA into PHB biosynthetic pathway and to an increase of enzymatic activities of β-ketothiolase and acetoacetyl-CoA reductase while activity of PHB synthase remains uninfluenced. During ethanol metabolisation, in which alcohol dehydrogenase is involved, acetyl-CoA and reduced coenzymes NAD(P)H are formed. These metabolites could again slightly inhibit TCA cycle while flux of acetyl-CoA into PHB biosynthetic pathway is likely to be supported. As a consequence of TCA cycle inhibition also less free CoA is formed. Similarly with hydrogen peroxide, activities of β-ketothiolase and acetoacetyl-CoA reductase are increased which results in over-production of PHB. Molecular weight of PHB produced under stress conditions was significantly higher as compared to control cultivation. Particular molecular weight values were dependent on stress factor concentrations. This could indicate some interconnection among activities of β-ketothiolase, acetoacetyl-CoA reductase and PHB molecular weight control in vivo.     

羟基脂肪酸聚酯制备与热性质研究

不同的碳源条件下,真养产碱杆菌可在胞内积累聚羟丁酸(PHB)或含羟基戊酸单体(HV)比例不等的聚羟丁戊酸共聚物(PHBv)。利用次氯酸钠,氯仿混合液体系提取上述羟基脂肪酸聚酯(PHA),提取率为85％,纯度达97．O％。以差示扫描热分析法对PHB和PHBV材料进行热性质研究,发现材料中的HV组分逐渐增加．材料的熔点Tm,熔化焓Hm逐渐下降。热分解峰值逐渐向低温区偏移。但含HV为6lmol％的PHBV材料有关热性质出现回升现象。     

Studies on intracellular degradation of polyhydroxyalkanoic acid-polyethylene glycol copolymer accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 accumulates poly(3-hydroxybutyric acid) [PHB] when grown in glucose containing nitrogen-free Stockdale medium. The same medium supplemented with valerate alone and valerate plus polyethylene glycol (PEG) leads to the accumulation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBV] and PEG containing PHBV-PEG polymers, respectively. The intracellular degradation of these polymers as studied in carbon-free Stockdale medium showed a rapid degradation of PHB followed by PHBV, while it was least in case of PHBV-PEG. The rate of such degradation was 44.16, 26.4 and 17.0 mg h(-1)l(-1) for PHB, PHBV and PHBV-PEG, respectively. During the course of such of PHBV and PHBV-PEG degradation the 3HB mol% of polymers decreased significantly with increase of 3HV mol fraction, the EG mol% in PHBV-PEG, however, remained constant. After 50h of degradation the decrease in intrinsic viscosity and molecular mass of PHBV-PEG were 37.5 and 43.6%, respectively. These values appeared low compared to PHB and PHBV. Moreover, the increasing EG content of polymer retarded their extent of degradation. Presence of PEG, particularly of low molecular weight PEG was inhibitory to intracellular PHA depolymerise (i-PHA depolymerase) activity and the relative substrate specificity of the i-PHA depolymerase of MAL-201 appeared to be PHB > PHBV > PHBV-PEG.