Purification and Properties of an Extracellular Polyhydroxybutyrate Depolymerase from Pseudomonas mendocina DSWY0601

An extracellular polyhydroxybutyrate（PHB） depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage for the ability to form clear zones on the PHB mineral agar plates. The molecular weight of the purified PHB depolymerase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） was ap- proximately 59800 at the optimal temperature and pH value being 50 ℃ and 8.5, respectively. PHB depolymerase was stable in a temperature range of 20--50 ℃ and sensitive to pH value within a pH range of 8.0-9.5. PHB depo- lymerase degraded poly-3-hydroxybutyrate-co-4-hydroxybutyrate（P3/4HB） and poly-3-hydroxybutyrate-co-3- hydroxyvalerate（PHBV） but did not degrade poly（lactic acid）（PLA）, poly（butylene succinate）（PBS） or poly- （caprolactone）（PCL）. PHB depolymerase was sensitive to phenylmethylsulfonyl fluoride（PMSF）, H202 and SDS. The main product after enzymatic degradation of PHB was indentified as 3-hydroxbutyrate monomer（3HB） by mass spectrometric analysis, suggesting that PHB depolymerase acted as an exo-type hydrolase. Analysis ofphaZpm gene reveals that PHB depolymerase is a typical denatured short-chain-length PHA（dPHAscL, PHA=polyhydroxyalkanoate） depolymerase containing catalytic domain, linker and substrate-binding domain.

Transformation of Streptococcus zooepidemicus with Genes Responsible for Polyhydroxybutyrate Synthesis

A procedure for transformation of intact Streptococcus zooepidemicus cells by electroporation was developed through a systematic examination of the effects of various parameters, including growth conditions, electric field strengths used for electroporation, and concentrations of plasmid used for transformation. Efficiencies higher than 10 4 cfu/μg (cfu, clone forming unit) plasmid DNA were obtained for Streptococcus zooepidemicus H2004 cells. Results demonstrate that the broad host range plasmid pDL276 can be replicated in Streptococcus zooepidemicus H2004 and foreign genes responsible for polyhydroxybutyrate (PHB) synthesis inserted into the pDL276 can be successfully expressed in the transformant, in which PHB is detected using the Fourier transform infrared spectroscopy (FT IR) method.

Polyhydroxybutyrate/Hydroxyapatite Highly Porous Scaffold for Small Bone Defects Replacement in the Nonload-bearing Parts

In the present work, Polyhydroxybutyrate （PHB）/Hydroxyapatite （HA） porous composites （10%, 20%, 30 %, 40%, 50% weight HA） were obtained by sintering. PHB/20% HA optimally combines satisfactory mechanical properties with a high content of the bioactive component （HA）. Porous PHB/20% HA scaffolds have shown high mechanical properties （compressive strength of 106 MPa and Young＇s modulus of 901 MPa）. A high volume fraction of interconnected pores （〉 50 vol.%） was achieved with pore size of 50 grn - 500 gm. Biocompatibility of porous pure PHB and PHB/20%HA, as its osseointegration were assessed in vitro and after implantation in laboratory animals. PHB/20% HA （-5% ＋ 0.9%） and pure PHB ~3% ~ 1.4%） samples after 24 hours of incubation with human leucocytes showed no significant level of cytotoxicity when p = 0.648 （p-value）. In vitro massive adhesion of mouse Multipotent Mesenchymal Stromal Cells （MMSC） to the surface of both porous samples was shown. PHB/20% HA induced more intensive MMSC proliferation compared to pure PHB, which are 31% ＋ 6.1% and 20% ＋ 5.7 % respectively when p = 0.039. We observed the resorption （implant surface area was reduced by 49 %） and integration of the porous PHB/20% HA samples into surrounding tissues after 30 days of implantation. The signs of osteoclasts accumulation, neo-angigenesis and new bone formation were observed, which make PHB/20% HA promising for bone tissue engineering.

Isolation and characterization of a thermophilic Bacillus shackletonii K5 from a biotrickling filter for the production of polyhydroxybutyrate

Polyhydroxyalkanoates（PHAs） are aliphatic polyesters accumulated intracellularly by both Gram-negative and Gram-positive bacteria. However, compared to the PHAs of Gramnegative bacteria, few endotoxins（lipopolysaccharides, LPS）, which would be co-purified with PHAs and cause immunogenic reactions, are found in the PHAs produced by Gram-positive bacteria. A thermophilic Gram-positive bacterium K5, which exhibited good growth and polyhydroxybutyrate（PHB）-accumulating ability, has been isolated and characterized from a biotrickling filter designed for the removal of NOx from flue gas in a coal-fired power plant in China. Based on the biochemical characterization and 16 S rRNA gene sequence（Genbank accession no. JX437933）, the strain K5 has been identified as Bacillus shackletonii, which has rarely been reported in the literature, and this report is the first time that B. shackletonii has been found to accumulate PHB. The strain K5 was able to utilize glucose as carbon source to synthesize PHB at a broad range of temperatures（from 35 to 50 °C）, and the ideal temperature was 45 °C. The strain K5 could effectively yield PHB of up to 69.9% of its cell dry weight（CDW）（2.28 g/L） in flask experiments employing glucose as carbon source at 45 °C, followed by 56.8% and 52.3% of its CDW when using sodium succinate and glycerol as carbon source, respectively. For batch cultivation, the strain K5 was able to produce PHB of up to 72.6% of its cell dry weight（9.76 g/L） employing glucose as carbon source at 45 °C and pH 7.0.

Utilization of pulp and paper industrial wastewater for production of polyhydroxybutyrate by Bacillus sonorensis NAM5

Given the environmental pollution caused by petroleum-based plastics,finding alternative substitutes for sustainability has become critical.Polyhydroxybutyrate(PHB),a storage food material that is accumulated by several bacteria,is biodegrad-able,safe,environment friendly and comparable to conventional plastics.However,scale-up is an issue due to high produc-tion cost.Substrate replacement using renewable,plentiful,sustainable and low-cost carbon sources derived from industrial waste facilitates waste reduction,while also enabling the synthesis of value-added products.In this context,inexpensive pulp and paper industrial waste as carbon source was exploited for production of PHB by using previously isolated(Source:hot springs of Manikarn,Himachal Pradesh,India)thermophilic bacteria Bacillus sonorensis NAM5 under optimized conditions in a fermenter.Production was done in a fermenter under optimized conditions(72 h of incubation at 50℃temperature and 7 pH)to enhance the accumulation of PHB.The bacterial strain was able to produce 5.28±0.11 gL^(-1)after 72 h of growth without any carbon and nitrogen source supplementation to the industrial effluent.The culture accumulated 66%PHB of cell dry weight(CDW).The produced polymer was characterized through FTIR,NMR and TGA.Additionally,bacteria-treated industrial wastewater was used for phytotoxicity assay on agriculturally important crops such as wheat,maize and mung,which exhibited considerable difference in growth parameters.

FUNCTIONAL POLYHYDROXYALKANOATES SYNTHESIZED BY MICROORGANISMS

Many bacteria have been found to synthesize a family of polyesters termed polyhydroxyalkanoate, abbreviated as PHA. Some interesting physical properties of PHAs such as piezoelectricity, non-linear optical activity, biocompatibility and biodegradability offer promising applications in areas such as degradable packaging, tissue engineering and drug delivery. Over 90 PHAs with various structure variations have been reported and the number is still increasing. The mechanical property of PHAs changes from brittle to flexible to elastic, depending on the side-chainlength of PHA. Many attempts have been made to produce PHAs as biodegradable plastics using various microorganisms obtained from screening natural environments, genetic engineering and mutation. Due to the high production cost, PHAs still can not compete with the nondegradable plastics, such as polyethylene and polypropylene. Various processes have been developed using low cost raw materials for fermentation and an inorganic extraction process for PHA purification. However, a super PHA production strain may play the most critical role for any large-scale PHA production. Our recent study showed that PHA synthesis is a common phenomenon among bacteria inhabiting various locations, especially oil-contaminated soils. This is very important for finding a suitable bacterial strain for PHA production. In fact, PHA production strains capable of rapid growth and rapid PHA synthesis on cheap molasses substrate have been found on molasses contaminated soils. A combination of novel properties and lower cost will allow easier commercialization of PHA for many applications.

Diminazene aceturate modified nanocomposite for improved efficacy in acute trypanosome infection

Objective: To investigate the improved antitrypanocidal activity and toxicity of diminazene aceturate modified Nano drug in experimental rats. Methods: Aqueous leaf extract of Hyptis suaveolens was used to reduce gold tetrachloride to its nanoparticle size and this was characterized and formulates with naturally synthesized polyhydroxybutyrateas a Nano carrier. A total of thirty (30) albino rats were group into 6 (A-F) of 5 rats each & infected intraperitoneally with 0.2 mL of the inoculum containing about 1×103 Trypanosoma brucei brucei parasites per 0.2 mL of blood. Groups A and B were treated with 3 and 6 minutes released orange PHB, Groups C and D were treated with 15 and 30 minutes released mango PHB formulated tablet while Groups E and F were negative (untreated) and standard drug (Dininazene aceturare) respectively. Results: The free drug and modified orange synthesized polyhydroxy butyrate shows antitrypanocidal activities by reducing the replicating rate of the parasite as compared to infect untreated. While the modified- mango synthesized shows increasing order of replication. There were significant increases in all the haematological parameter evaluated in the infected treated groups compared to infect untreated. But no significant difference (P<0.05) observed in the Catalase activity in the serum and liver of all the groups whereas, the modified orange synthesized shows significant decrease in other enzymes activities evaluated when compared with the free drug, mango synthesized and the infected untreated groups. Conclusion: Orange synthesized modified diminazene aceturate show efficacy as free drug with limited toxicity that can enhance the therapeutic.

Influence of the wastewater composition on denitrification and biological P-removal in the S-DN-P process: (c) Dissolved and undissolved substrates

The denitdfication and P-removal in the sorption-denitrification-P-removal （S-DN-P） process were carded out under various wastewater compositions. It is noted that P-removal largely depends on the wastewater composition as well to the quantity of the substrates present in wastewater fraction. Three different wastewater fractions are obtained as： raw wastewater, dissolved wastewater （obtained with filtration using 0.45 μm filter）, and undissolved wastewater （i.e., infiltrate obtained by above filtration）. The ratio of P-release/CODtotal-consumption clearly inferred that undissolved wastewater possess very low value i.e., 0.0008 followed by raw wastewater 0.008 and dissolved wastewater 0.03. When this ratio was nearby 0.01, enhanced P-removal was observed. Moreover, the ratio of P-uptake to NO3^--N decomposition for raw wastewater was two times for dissolved wastewater. Interestingly, it was observed that the P-removal and denitrification depend not only on the dissolved substrates but also the undissolved substrates present in the wastewater. The result of the P-removal obtained with this S-DN-P process did not show a big difference of 36%, 34% and 30%, respectively, for raw, dissolved and undissolved wastewater.

Development of sustainable thermal insulation based on bio-polyester filled with date pits

Date palm pit(DPP)-filled poly(-hydroxybutyrate)(PHB)composites were prepared,evaluated,and characterized to determine their thermal insulation ability.Thermal conductivity values ranged between 0.086 and 0.100 W/(m·K).At a maximum filler concentration(50%(w)),the specific heat capacity and thermal diffusivity were 1183 J/(kg·K)and 0.0689 mm^(2)/s,respec-tively.The DPP increased the thermal stability,and the highest compressive strength obtained was 80 MPa at 30%filler content.The PHB-DPP composites exhibited promising water absorption(less than 6%)and tensile strength(6-14 MPa).Date-pit-based PHB composites could be used in sustainable building engineering and cleaner production.

Mechanical Properties of Polyhydroxyalkanoate Bioceramic Nanocomposites

New composites prepared containing nanoscale hydroxyapatite or monetite uniformly distributed in a polyhydroxyalkanoate (polyhydroxybutyrate or polyhydroxybutyrate-hydroxyvalerate) matrix with mass fractions of 1%5% were then injected into dumb-like specimens. The results show that the tensile strength and the tensile modulus of the composites are improved. The microstructures of the composites were observed using transmission electron microscopy (TEM).

Biosynthesis of Polyhydroxyalkanoates

Many bacteria are able to synthesize polyhydroxyalkanoates, abbreviated as PHA, as carbon and energy storage compounds. PHA with over 90 different monomer structures has been reported and the number keeps increasing. The piezoelectricity, non linear optical activity, biocompatibility and biodegradability of PHA have offered promising applications in areas such as tissue engineering, drug delivery, smart materials and degradable packaging. Various microorganisms obtained from nature, genetic engineering and mutations have been used for microbial production of PHA as biodegradable plastics. In fact, PHA synthesizing bacteria can be easily found in various locations, especially oil contaminated soils. Many bacterial strains possessing the ability to synthesize PHA with various monomers have been isolated from oil contaminated soils. In molasses contaminated soil, 40% of the isolated bacteria were capable of growing rapidly and synthesizing PHA at the same time. It seems that nature has provided endless numbers of novel PHA synthesizing strains.

Nature’s fight against plastic pollution:Algae for plastic biodegradation and bioplastics production

The increased global demand for plastic materials has led to severe plastic waste pollution,particularly to the marine environment.This critical issue affects both sea life and human beings since microplastics can enter the food chain and cause several health impacts.Plastic recycling,chemical treatments,incineration and landfill are apparently not the optimum solutions for reducing plastic pollution.Hence,this review presents two newly identified environmentally friendly approaches,plastic biodegradation and bioplastic production using algae,to solve the increased global plastic waste.Algae,particularly microalgae,can degrade the plastic materials through the toxins systems or enzymes synthesized by microalgae itself while using the plastic polymers as carbon sources.Utilizing algae for plastic biodegradation has been critically reviewed in this paper to demonstrate the mechanism and how microplastics affect the algae.On the other hand,algae-derived bioplastics have identical properties and characteristics as petroleum-based plastics,while remarkably being biodegradable in nature.This review provides new insights into different methods of producing algae-based bioplastics(e.g.,blending with other materials and genetic engineering),followed by the discussion on the challenges and further research direction to increase their commercial feasibility.

Potential one-step strategy for PET degradation and PHB biosynthesis through co-cultivation of two engineered microorganisms

The management and recycling of plastic waste is a challenging global issue.Polyethylene terephthalate(PET),one of the most widely used synthetic plastics,can be hydrolyzed by a series of enzymes.However,upcycling the resulting monomers is also a problem.In this study,we designed a co-cultivation system,in which PET degradation was coupled with polyhydroxybutyrate(PHB)production.First,PETase from Ideonalla sakaiensis was expressed in Yarrowia lipolytica Po1f with a signal peptide from lipase.The engineered PETase-producing Y.lipolytica was confirmed to hydrolyze bis(2-hydroxyethyl)terephthalate(BHET)and PET powder into the monomers terephthalate(TPA)and ethylene glycol(EG).Simultaneously,a TPA-degrading Pseudomonas stutzeri strain isolated from PET waste was transformed with a recombinant plasmid containing the phb CAB operon from Ralstonia eutropha,which encodes enzymes for the biosynthesis of PHB.The two co-cultivated engineered microbes could directly hydrolyze BHET to produce the bioplastic PHB in one fermentation step.During this process,5.16 g/L BHET was hydrolyzed in 12 h,and 3.66 wt%PHB(3.54 g/L cell dry weight)accumulated in 54 h.A total of 0.31g/L TPA was produced from the hydrolyzation of PET in 228 h.Although PHB could not be synthesized directly from PET because of the low hydrolyzing efficiency of PETase,this study provides a new strategy for the biodegradation and upcycling of PET waste by artificial microflora.

Formation of Polyhydroxyalkanoate Blends by Pseudomonas pseudoalcaligenes M1-2 from Various Carbon Sources

Pseudomonas pseudoalcaligenes strain M1\|2 isolated from oil\|contaminated soil collected from an oilfield in northern China was found to be able to synthesize a blend of polyhydroxyalkanoates (PHAs) containing monomers of 3\|hydroxybutyrate (C4), 3\|hydroxyvalerate (C5), 3\|hydroxyheptanoate (C7), 3\|hydroxyoctanoate (C8), 3\|hydroxynonanoate (C9), 3\|hydroxydecanoate (C10) and 3\|hydroxydodecanoate (C12) from various carbon sources. The hydroxyalkanoate (HA) monomer composition varied both quantitatively and qualitatively, depending on the carbon sources used. The presence of octanoate in substrates of myristic acid or tridecanoate promoted the synthesis of HB monomer in the blend. Concentration of octanoate was also found to significantly affect the PHB content in the blend. A PHA biosynthesis pathway in Pseudomonas pseudoalcaligens M1\|2 was proposed.

Effect of Surface Modification on Microbiol Polyhydroxyalkanoate Films on Biocompatibility

The purpose of this study was to investigate in vitro biocompatibility of a new type of polymer, polyhydroxybutyrate co hexanoate (PHBHHx). The hydrophilicity and biocompatibility were studied with two kinds of enzymes, amylase BAN480L and lipase Novozym388. The degree of hydrophilicity was observed using contact angle measurements. In vitro biocompatibility evaluations were carried out by direct incubation of mouse fibroblast cell line L929 on the polyhydroxyalkanoate (PHA) films. The samples treated with BAN480L showed that the PHA biocompatibility increased while the hydrophilicity decreased. Relative to untreated samples, the number of cells on the Novozym388 modified PHBHHx significant decrease as the hydrophilicity also decreased. The results indicated that other surface characteristics besides hydrophilicity influence the biocompatibility of PHBHHx films.