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[Study on mechanism of different PHAs during heating by FTIR].

In this paper, PHB, P(HO-co-HD), a various composition of P(HB-co-HV) copolymer and P(HB-co-HH) copolymer were studied both at normal temperature and in the process of increasing and decreasing temperature using FTIR spectroscopy technology. Results showed that FTIR spectra of PHAs with various compositions have distinguishing characteristics. Physical transformation occurred when PHAs were treated with heat denaturalization, and this physical process was reversible.

Bacteria↗

[Experimental studies on the poly-hydroxybutyrate membrane modified by gamma-radiation and mixed with calcium sulfate].

The objective of this study was to learn the property of poly-hydroxybutyrate membrane (PHBm) modified by gamma-radiation and mixture of calcium sulfate, and to explore the possibility of using modified PHBm for guided tissue regeneration (GTR). The PHB was treated by 5 KGy gamma-radiation and mixed with 1/10 calcium sulfate. The modified PHB membrane was prepared by solvent-casting techniques. The mechanical properties and molecular weight of the modified PHBm were tested. Degradability of the modified PHBm was analyzed in vitro in a buffer solution of KH2PO4-Na2HPO4. Biodegradability and biocompatibility of the modified PHBm were inspected 1, 2, 3 and 6 months after the embedding of the modified PHBm into dogs. The morphology was analyzed by scanning electron microscopy (SEM) and molecular weight was tested to evaluate the biodegradability of PHBm. Biocompatibility of the modified PHBm was observed through tissue response by light microscopy. The extension strength and the extension strain at fracture of the modified PHBm were 23.8 MPa and 1.0% respectively. The morphologic observation of the modified PHBm at different terms showed that the modified PHBm was biodegraded gradually in vitro and in vivo. The capsule surrounding the modified PHBm was mainly composed of fibrocytes and few lymphocytes. The longer the time elapsed, the thinner the capsule enveloping the modified PHBm grew. The modified PHBm possesses satisfactory mechanical properties and biocompatibility, and it is biodegradable in vitro and in vivo. The modified PHB membrane could be applied as GTR membrane.

Animals↗

Construction of recombinant Escherichia coli strains for production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate).

Plastic wastes constitute a worldwide environmental problem, and the demand for biodegradable plastics has become high. One of the most important characteristics of microbial polyesters is that they are thermoplastic with environmentally degradable properties. In this study, pUC19/PHA was cloned and transformed into three different Escherichia coli strains. Among the three strains that were successfully expressed in the production of polyhydroxyalkanoates (PHA), E. coli HMS174 had the highest yield in the production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-HV]). The cell dry weight and PHA content of recombinant HMS174 reached as high as 10.27 g/L and 43% (w/w), respectively, in fed-batch fermentor culture. The copolymer of PHA, P(HB-HV), was found in the cells, and the biopolymers accumulated were identified and analyzed by gas chromatography, proton nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. We demonstrated clearly that the E. coli host for PHA production has to be carefully selected to obtain a high yield. The results obtained indicated that a superior E. coli with high PHA production can be constructed with a desirable ratio of P(HB-HV), which has potential applications in industry and medicine.

Biodegradation, Environmental↗

[Biocompatibility of p(3HB-co-3HH) and marrow mesenchymal stem cells].

OBJECTIVE: To investigate the biocompatibility of p(3HB-co-3HH) and marrow mesenchymal stell cells (MSCs). METHODS: MSCs were inoculated to p(3HB-co-3HH), and then cultured for 2-4 weeks in vitro and embedded for 2 weeks in vivo. The growth, proliferation, morphology and phenotype properties of MSCs were observed by use of phase contrast microscope, electron microscope, HE staining and staining of type I collagen. RESULTS: p(3HB-co-3HH) had good compatibility. The inoculated MSCs could be well-distributed, attached well and obtain the phenotype of MSCs in p(3HB-co-3HH). After osteogenic inducer were added, MSCs differentiated to osteoblasts and secreted matrix. Type I collagen was stained positively by immunohistochemical techenique. CONCLUSION: The above results demonstrate that there is satisfactory biocompatibility between p(3HB-co-3HH) and MSCs.

3-Hydroxybutyric Acid↗

Prevention of postoperative pericardial adhesions by closure of the pericardium with absorbable polymer patches. An experimental study.

Pericardial adhesions after cardiac operations are a widely known phenomenon. They may severely complicate reoperations, making reentry hazardous, increasing bleeding, and prolonging the operation time. The anatomic orientation and visibility of both bypass grafts and coronary arteries are also impaired. With the aim of minimizing pericardial adhesions after cardiac operations, we studied the course of tissue regeneration after implantation of a new absorbable patch made from poly-hydroxy-butyrate. A total of 23 sheep were studied. Of these, 18 formed the test group and five served as control animals. The animals were killed at intervals of 2 to 30 months after the operation. In 14 of the 18 test animals no adhesions developed. In three animals loose adhesions were found, and in one with signs of postoperative infection there were moderate, generalized adhesions. All control sheep showed moderate adhesions; no infection was noted in this group. Light microscopy in the test group revealed a layer of mesothelium-like cells facing the epicardial side; this was already present in the early specimens. Poly-hydroxy-butyrate appeared to be slowly phagocytosed by polynucleated macrophages, which were still found occasionally in the late samples. Lymphocytes and platelets were rare. Scanning electron microscopy showed, on the epicardial side of the regenerated tissue, a mesothelium-like lining that completely covered the underlying collagen layer. The surface cell morphology grossly resembled that of native pericardium. It was concluded that in this animal model poly-hydroxy-butyrate pericardial patches decreased adhesions and preserved coronary anatomy. The findings in the control group demonstrated that pericardial surgery in the sheep was associated with adhesion formation.

6-Ketoprostaglandin F1 alpha↗

Growth-associated production and characterization of poly (3-hydroxybutyric acid) of Azotobacter beijerinckii DAR-102.

Production of poly (3-hydroxybutyric acid) [P(3HB)] by Azotobacter beijerinckii DAR-102 isolated in this laboratory has been optimized under batch-culture. The accumulatad polymer attained 58% of cell dry mass during mid-stationary phase with an yield of 0.58 g/l when grown in nitrogen-free medium. The optimum concentration of glucose and fructose for P(3HB) production was 3% (w/v) and 2% (w/v) respectively while that of casamino acid and tryptose was 0.1% (w/v). Phosphate at a concentration suboptimal for growth and limitation of oxygen in the medium favoured P(3HB) accumulation. The production of P(3HB) was maximum with an inoculum dose of 4% (v/v). The accumulated polymer was isolated by direct chloroform extraction of the dry cell mass and purified by precipitation with diethyl ether. The purified polymer has been characterized in terms of its solubility properties, melting temperature, and UV-, IR- and NMR-spectroscopic analyses.

Azotobacter↗

[Transplantation of adrenocortical cells in allorat using porous PHB as cell carrier].

This experiment was designed to investigate the feasibility of transplanatation of using porous PHB as cell carrier for the transplanatation of adrenocortical cells. Adrenocortical cells from rat adrenal gland were separated and cultured in vitro. The effect of PHB on the proliferation and secretory function of adrenocortical cells were evaluated by MTT and RIA methods. Then adrenocortical cells were seeded into porous PHB. After the cells were cultured in vitro for about seven days, they were implanted into the rats having undergone bilateral adrenalectomy. The changes of blood corticosterone and aldosterone and the local histological changes in these rats were observed. Adrenocortical cells were able to grow and survive on PHB. No effect on the proliferation and secretory function of adrenocortical cells were observed. Most bilateral adrenalectomized rats bearing the transplanted adrenocortical cells within PHB (study group) survived longer than did the adrenalectomized rats in control group. The blood corticosterone level and aldosterone level of study group were higher than those of control group. It was found that PHB has no effects on the survival, proliferation and secretory function of adrenocortical cells. Adrenocortical cells within PHB can survive a period of time and can secrete corticosterone and aldosterone which can meet the needs of the adrenalectomized rats. PHB can degrade slowly in vivo. It is feasible to perform transplantation of adrenocortical cells using porous PHB as cell carrier.

Adrenal Cortex↗

Microbial polyhydroxyalkanoates (PHAs): an emerging biomaterial for tissue engineering and therapeutic applications.

Among the various biomaterials available for tissue engineering and therapeutic applications, microbial polyhydroxyalkanoates (PHAs) offer the most diverse range of thermal and mechanical properties. Of particular interest are the PHAs that contain 4-hydroxybutyrate such as poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB) and poly(4-hydroxybutyrate) [P(4HB)]. These polyesters can only be synthesized by a few types of bacteria, among which Comamonas acidovorans has the most efficient metabolic pathways to channel 4HB monomers. The resulting polyesters are bioabsorbable and are being developed as a new biomaterial for medical applications. By controlling the molar ratio of the monomers, it is possible to produce materials that are as tough and elastic as rubber.

3-Hydroxybutyric Acid↗

Optimisation of storage driven denitrification by using on-line specific oxygen uptake rate monitoring during SND in a SBR.

This study builds on previous experience of maximising the formation of COD as poly-hydroxybutyrate (PHB) and now describes a feedback technique of preserving the use of PHB for denitrification resulting in enhanced nitrogen removal rather than allowing its wasteful oxidation by oxygen. The feedback technique uses on-line SOUR monitoring for detecting the end-point of nitrification and controlling the aerobic phase length accordingly. The laboratory SBR was operated such that all organic substrate (acetate) was rapidly converted to PHB, which then served as the electron donor for nitrogen removal via simultaneous nitrification and denitrification (SND) during the aerobic phase (up to 70% SND). During SBR cycling with a fixed aeration length (240 minutes), PHB was unnecessarily oxidised after ammonium depletion, resulting in little denitrification and poor total nitrogen removal (69%). However, when the aerobic phase length was controlled via the SOUR, up to 1.8 CmM PHB (58 mg L(-1) COD) could be preserved, enabling improved total nitrogen removal (86%). The drop in the SOUR after ammonium depletion was a reproducible event that could be detected even when using raw wastewater and fresh activated sludge. The SOUR-control technique holds promise to build up PHB over a number of SBR cycles. While advanced oxygen-control is used for improved N-removal in several existing WWTPs, this study investigates the importance of oxygen control with relevance to PHB driven SND in sequencing batch reactors.

Aerobiosis↗

The presence of ammonium facilitates nitrite reduction under PHB driven simultaneous nitrification and denitrification.

For economic and efficient nitrogen removal from wastewater treatment plants via simultaneous nitrification and denitrification the nitrification process should stop at the level of nitrite such that nitrite rather than nitrate becomes the substrate for denitrification. This study aims to contribute to the understanding of the conditions that are necessary to improve nitrite reduction over nitrite oxidation. Laboratory sequencing batch reactors (SBRs) were operated with synthetic wastewater containing acetate as COD and ammonium as the nitrogen source. Computer controlled operation of the reactors allowed reproducible simultaneous nitrification and denitrification (SND). The oxygen supply was kept precisely at a low level of 0.5 mgL(-1) and bacterial PHB was the only electron donor available for denitrification. During SND little nitrite or nitrate accumulated (< 20% total N), indicating that the reducing processes were almost as fast as the production of nitrite and nitrate from nitrification. Nitrite spiking tests were performed to investigate the fate of nitrite under different oxidation (0.1-1.5 mgL(-1) of dissolved oxygen) and reduction conditions. High levels of reducing power were provided by allowing the cells to build up to 2.5 mM of PHB. Nitrite added was preferentially oxidised to nitrate rather than reduced even when dissolved oxygen was low and reducing power (PHB) was excessively high. However, the presence of ammonium enabled significant reduction of nitrite under low oxygen conditions. This is consistent with previous observations in SBR where aerobic nitrite and nitrate reduction occurred only as long as ammonium was present. As soon as ammonium was depleted, the rate of denitrification decreased significantly. The significance of the observed strongly stimulating effect of ammonium on nitrite reduction under SND conditions is discussed and potential consequences for SBR operation are suggested.

Acetates↗

[Effect of carbon and nitrogen sources on the accumulation of poly-beta-hydroxybutyrate by purple non-sulfur photosynthetic bacteria].

The effect of carbon and nitrogen sources on the accumulation of Poly-beta-Hydroxybutyrate (PHB) by purple non-sulfur photosynthetic bacteria (PNSB)was investigated. The results showed that ammonia nitrogen and organic nitrogen could benefit the PNSB accumulating PHB; under certain ratio of carbon and nitrogen low carbon concentration can accumulate more PHB, but high carbon concentration is better for PNSB accumulating PHB with the growth of PNSB. As to different substrates, butyrate is the best for accumulating of PHB, but acetate is better and propionate is the poorest. Mixed substrate with butyrate as the main content are better than single substrate; carbon dioxide have certain effect on the PNSB accumulating PHB, and high carbon dioxide concentration can improve PHB content when using acetate or butyrate as substrate.

Bacteria↗

[The synthesis of hydroxybutyrate and hydroxyvalerate copolymers by the bacterium Ralstonia eutropha].

The paper deals with the study of the synthesis of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) copolymers by the bacterium Ralstonia eutropha B-5786 grown under different carbon nutrition conditions (growth on carbon dioxide, fructose, and CO2-valerate and fructose-valerate mixtures). The parameters to be analyzed included the yield of biomass, the yield, synthesis rate, and composition of copolymers, the activity of the key enzymes of polyhydroxyalkanoate (PHA) synthesis (beta-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase), the maximum tolerable concentration of valerate to the bacterium, and the conditions that govern the incorporation of hydroxyvalerate to copolymers. This allowed the relationship between cultivation conditions and the proportion of monomers in the copolymers to be deduced. We were able to synthesize a range of 3HB/3HV copolymers and found that the thermal characteristics and the degree of crystallinity of these copolymers depend on the molar fraction of 3HV.

Acetyl-CoA C-Acyltransferase↗

Microbial synthesis and characterization of physiochemical properties of polyhydroxyalkanoates (PHAs) produced by bacteria isolated from activated sludge obtained from the municipal wastewater works in Hong Kong.

The first objective of this study was the measurement of physical properties of P(3HB-co-3HV) copolymers with different (hydroxybutyrate) HB to (hydroxyvalerate) HV ratios produced by Bacillus cereus (TRY2) isolated from activated sludge. The 3HV PHBV copolymers were 0.05, 22.6, 39.2, 54.1, and 69.1 mol%, respectively. The second objective was to study possible waste water treatment and production of PHAs at the same time by B. cereus (TRY2) and Pseudomonas spp. (TOB17) (both were isolated from activated sludge), recombinant Bacillus DH5alpha, and a combination of the above three bacteria. The results were satisfactory; the maximum COD and TOC of the sewage sludge reduced were 53.5% and 67.5%, respectively.

Bacillus cereus↗