Biosynthesis of poly-3-hydroxybutyrate with a high molecular weight by methanotroph from methane and methanol

Poly-3-hydroxybutyrate （PHB） can be produced by various species of bacteria. Among the possible carbon sources, both methane and methanol could be a suitable substrate for the production of PHB. Methane is cheap and plentiful not only as natural gas, but also as biogas. Methanol can also maintain methanotrophic activity in some conditions. The methanotrophic strain Methylosinus trichosporium IMV3011 can accumulate PHB with methane and methanol in a brief nonsterile process. Liquid methanol （0.1%） was added to improve the oxidization of methane. The studies were carried out using shake flasks. Cultivation was performed in two stages： a continuous growth phase and a PHB accumulation phase under the conditions short of essential nutrients （ammonium, nitrate, phosphorus, copper, iron （Ⅲ）, magnesium or ethylenediamine tetraacetate （EDTA）） in batch culture. It was found that the most suitable growth time for the cell is 144 h. Then an optimized culture condition for second stage was determined, in which the PHB concentration could be much increased to 0.6 g/L. In order to increase PHB content, citric acid was added as an inhibitor of tricarboxylic acid cycle （TCA）. It was found that citric acid is favorable for the PHB accumulation, and the PHB yield was increased to 40% （w/w） from the initial yield of 12% （w/w） after nutrient deficiency cultivation. The PHB produced is of very high quality with molecular weight up to 1.5 × 10^6Da.

Preparation and characterization of biomimetic adsorbent from poly-3-hydroxybutyrate

Biomimetic adsorbent named as PHBBMA was prepared from lipophilic poly-3-hydroxybutyrate （PHB） by a modified double emulsion solvent evaporation method. PHBBMA, characterized by using scanning electron microscope and nitrogen adsorption/desorption measurements, is porous spherical particles. The characterization with the thermal gravimetric analysis and differential scanning calorimetry, 1 H nuclear magnetic resonance and Fourier transform infrared spectroscopy showed that PHBBMA preparation was a physical process without chemical reaction. The adsorption of PHBBMA for o-nitrochlorobenzene （o-NCB） was fitted better by Langmuir model than by Freundlich model, while the pseudo second-order model fitting was better than the pseudo first-order model fitting. The maximal adsorption capacity of PHBBMA for o-NCB was 57.83 mg/g at 30°C, although its specific surface area （S BET ） was only 8.45 m 2 /g. PHBBMA is a safe and environmental friendly adsorbent with high adsorption capacity because its component is innocuous and biodegradable PHB produced reusing wastes and contaminants, no byproduct can produced, and its ester and hydrocarbyl groups have strong affinity with organochlorine compounds. The further work will focus on the modification and improvement of PHBBMA in order to increase its S BET and adsorption capacity.

Construction of plant seed-specific expression vectors pSCB and pSCAB and the obtainment of transgenic Brassica napus H165 expressing poly-3-hydroxybutyrate synthetic genes

The seed-specific promoter and transit peptide were amplified and fused to the three genes phbA, phbB and phbC encoding PHB synthetic enzymes, respectively. Seed-specific expression vectors pSCB containing phbC and phbB, and pSCAB containing phbC, phbB and phbA, were constructed by introducing the genes with promoter and peptide into the binary vector pBI101. Transgenic Brassica napus H165 were obtained by Agrobacterium-mediated transformation with these vectors. They were confirmed by PCR, Southern and RT-PCR analyses.

Effect of Poly-β-hydroxybutyrate on Reproductive Performance and Larval Development of Fenneropenaeus chinensis

In the present study, Fenneropenaeus chinensis individuals were assigned into six groups, and fed with a diet supplemented with 0 （Control）, 0.5% （Group Eos）, 1.0% （Group E1.0）, 2.0% （Group E2.0）, 3.0% （Group E3.0） or 5.0% （Group E5.0） of poly-13-hydroxybutyrate （PHB） during their growth and spawning periods, to explore the effect of PHB on the reproductive performance of parents and the development of larvae. The experi- ment was lasted for 192 d to measure the mortality rate, relative percentage of survival（RPS）, weight gain rate（WGR）, specific growth rate （SGR） and cycle of gonadal development of parental shrimps, number of eggs, number of nauplii, stages of larval development and metamorphosis. The results indicated that PHB had an obvious effect on the fecundity of parental shrimps and growth performance of larva. Compared with control group, the mortality rate of broodstock in experimental groups decreased initially and increased subsequently with the increase of PHB concentration, while RPS showed an opposite pattern. The mortality rate of group E2.0 was the lowest among all groups, showing significant difference from that of other groups（P 〈 0.05） except group E1.0 （P 〉 0.05）. WGR of all experimental groups was higher than that of the control group, among which, group E2.0 and group E5.0 had significantly higher WGR than the control （P〈0.05）. However, there was no significant difference in the cycle of gonadal development between the experimental groups and the control group （P 〉 0.05）. The numbers of eggs and nauplii in the experimental groups were higher than those of the control group. And the numbers of eggs and nauplii in group E2.0 were significantly higher than those of the control （P 〈 0.05）, bud showed no significant difference from those of other groups （P 〉 0.05）. However, there was no significant difference in the duration of larval development and metamorphosis among all the groups. In summary, PHB is capable of improving the reproductive performance of parents and the survival and development of larva in F. chinensis, and 2.0% is the optimal concentration of PHB supplemented to the diet.

Effect of Poly-β-Hydroxybutyrate on the Activity of Nonspecific Immunity Related Enzymes in Fenne rope naeus chinensis

[Objective] This study was carried out to explore the effect of poly-β-hydroxybutyrate（PHB） on the performance of the nonspecific immune system in Fenneropenaeus chinensis. [Method] F. chinensis individuals were assigned into six groups, and each group was fed with a diet containing 0（Control）, 0.5%（Group E0.5）, 1.0%（Group E1.0）, 2.5%（Group E2.5）, 5.0%（Group E5.0） or 10.0% PHB（Group E10.0）. The mortality rate and relative percent of survival（RPS） of each group were calculated after 6 weeks. Meanwhile, the total antioxidant capacity（T-AOC）, the activity of acid phosphatase（ACP）, peroxidase（POD） and catalase（CAT）, and the content of malondialdehyde（MDA） in hepatopancreas and serum were measured,and their correlation with PHB concentration was analyzed. [Result] The RPS in PHB treated shrimps increased initially and decreased subsequently with increasing PHB concentration. RPS of Group E1.0 was the highest, showing significant difference from that of other groups（ P 〈0.05）.With the increase in PHB concentration, the activity of immunity related enzymes changed in a similar pattern with RPS： increasing at first and decreasing subsequently. In addition, the activity of the enzymes was elevated in the 2nd and 3rd weeks of PHB administration. Among them, T-AOC in serum of groups E1.0 and E2.5, T-AOC in hepatopancreas of Group E1.0, ACP activity in serum of groups E1.0 and E2.5, ACP activity in hepatopancreas of Group E1.0, CAT activity in groups E0.5, E1.0 and E2.5, CAT activity in hepatopancreas of groups E0.5, E1.0 and E10.0, POD activity in both serum and hepatopancreas of groups E0.5, E1.0 and E2.5, SOD activity in both serum and hepatopancreas of Group E1.0, MDA content in serum of Group E1.0 and MDA content in hepatopancreas of groups E0.5 and E1.0 showed significant difference from those of other groups（P〈0.05）. [Conclusion] PHB can improve the immunity of F. chinensis, 1.0% in feed has the best effect, and the total enzyme activity reaches the highest level in the 2nd and 3rd weeks of PHB administration.

Production of Poly-b-hydroxybutyrate by Activated Sludge in Sequencing Batch Reactor under Aerobic Conditions

In order to improve poly-β-hydroxybutyrate（PHB） production in activated sludge, the anaerobic/aerobic alternative operating sequencing batch reactor（SBR） process was applied in this paper to accumulate PHB. Effects of nutritional conditions and carbon concentration on PHB accumulation were studied. Results indicated that PHB accumulation reached the highest level and accounted for 11.2 % under anaerobic condition for phosphate limitation and 20.84 % under aerobic condition for nitrogen and phosphate limitation of mixed liquor suspended solid（MLSS）, respectively. In addition, 4 g/L was proved to be the optimum carbon concentration in both anaerobic and aerobic experiments, and the PHB accumulation reached 17.1 %（anaerobic, phosphorus limitation） and 60.4 %（aerobic, nitrogen and phosphorus limitation） of MLSS, respectively. PHB could be successfully extracted with sodium hypochlorite and chloroform method from the activated sludge. In addition, the infrared spectrum showed that the PHB sample extracted was of high purity.

SYNTHESIS OF NOVEL BLOCK COPOLYMERS OF POLY(3-HYDROXYBUTYRIC ACID)WITH POLY(ETHYLENE GLYCOL)THROUGH ANIONIC POLYMERISATION

A novel kind of copolymer with ABA-type block structure was synthesized by anionic ring-opening polymerization of beta-butyrolactone (beta-BL) in the presence of a PEG-based dicarboxylates as macroinitiators which were prepared by the esterification of aliphatic cyclic anhydride and poly(ethylene glycol) (PEG) oligomers (M-n = 2000, 4000 and 6000) and conversion of potassium dicarboxylates. The resultant copolymers as well as the intermediates were characterized by IR, H-1-NMR and GPC.

Poly-β-hydroxybutyrate alleviated diarrhea and colitis via Lactobacillus johnsonii biofilm-mediated maturation of sulfomucin

Maintainance of sulfomucin is a key end point in the treatment of diarrhea and inflammatory bowel disease(IBD).However,the mechanisms underlying the microbial sense to sulfomucin are poorly understood,and to date,there are no therapies targeting the secretion and maturation of sulfomucin in IBD.Herein,we biosynthesized poly-β-hydroxybutyrate(PHB)and found that PHB could alleviate inflammation caused by diarrhea and colitis by enhancing the differentiation of sulfomucin.Microbiota transplantation and clearance together demonstrate that PHB promoting sulfomucin is mediated by Lactobacillus johnsonii(L.johnsonii).Further studies revealed that PHB provides a favorable niche for L.johnsonii biofilm formation to resist disturbance and support its growth.L.johnsonii-biofilm alleviates colitis by regulating fucose residues to promote goblet cell differentiation and subsequent sulfomucin maturation.Importantly,PHB alleviates colitis by enhancing sulfomucin secretion and maturation in a L.johnsonii-dependent manner.PHB represents a class of guardians,acting as a safe probiotic-biofilm delivery system that significantly promotes probiotic proliferation.Altogether,this study adds weight to the possible role of probiotics and functional materials in the treatment of intestinal inflammation.The application of PHB and biofilm self-coating L.johnsonii carries high translational potential and may be of clinical relevance.

Effect of poly(3-hydroxyalkanoates) as natural polymers on mesenchymal stem cells

Mesenchymal stem cells(MSCs)are stromal multipotent stem cells that can differentiate into multiple cell types,including fibroblasts,osteoblasts,chondrocytes,adipocytes,and myoblasts,thus allowing them to contribute to the regeneration of various tissues,especially bone tissue.MSCs are now considered one of the most promising cell types in the field of tissue engineering.Traditional petri dish-based culture of MSCs generate heterogeneity,which leads to inconsistent efficacy of MSC applications.Biodegradable and biocompatible polymers,poly(3-hydroxyalkanoates)(PHAs),are actively used for the manufacture of scaffolds that serve as carriers for MSC growth.The growth and differentiation of MSCs grown on PHA scaffolds depend on the physicochemical properties of the polymers,the 3D and surface microstructure of the scaffolds,and the biological activity of PHAs,which was discovered in a series of investigations.The mechanisms of the biological activity of PHAs in relation to MSCs remain insufficiently studied.We suggest that this effect on MSCs could be associated with the natural properties of bacteria-derived PHAs,especially the most widespread representative poly(3-hydroxybutyrate)(PHB).This biopolymer is present in the bacteria of mammalian microbiota,whereas endogenous poly(3-hydroxybutyrate)is found in mammalian tissues.The possible association of PHA effects on MSCs with various biological functions of poly(3-hydroxybutyrate)in bacteria and eukaryotes,including in humans,is discussed in this paper.

Structures and growth mechanisms of poly-(3-hydroxybutyrate) (PHB) crystallized from solution and thin melt film

The spherulitic structures and morphologies of poly-(3-hydroxybutyrate) (PHB) crystallized from a so- lution and a thin melt film were investigated in this study. The formation mechanisms of banded spherulites under different crystallization conditions are proposed. It was found that the formation of banded spherulites was caused by the rhythmic crystal growth of the spherulites and lamellar twisting growth for the polymer crystallization from a thin melt film and a solution, respectively.

Effects of PSMA and experimental conditions on the morphologies of BaCO_3 whiskers

BaCO3 whiskers exhibiting different morphologies were fabricated by a simple precipitation reaction of barium ch/oride with sodium carbonate in the absence and presence of poly-（styrene-alt-maleic acid） （PSMA） as a crystal growth modifier at room temperature. The as-prepared products were characterized by scanning electron microscopy （SEM）, transmission electron microscopy （TEM）, and X-ray diffraction （XRD）. The influences of experimental parameters on the size and morphology of BaCO3 whiskers were investigated and discussed. It was found that the as-prepared BaCO3 whiskers are single crystals with diameter ranging from 100 to 300 run, and grow along the crystallographic a-axis or [100] direction. BaCO3 whiskers with different morphologies, such as branching and dendritic structure, can be obtained depending on the experimental conditions. With increasing PSMA concentration, the diameter of BaCO3 whiskers decreases.

Syntheses of amphiphilic biodegradable copolymers of poly(ethyl ethylene phosphate) and poly(3-hydroxybutyrate) for drug delivery

Biodegradable and amphiphilic triblock copolymers poly(ethyl ethylene phosphate)-poly(3-hydroxybutyrate)-poly(ethyl ethylene phosphate) (PEEP-b-PHB-b-PEEP) have been successfully synthesized through ring-opening polymerization. The structures are confirmed by gel permeation chromatography and NMR analyses. Crystallization investigated by X-ray diffraction reveals that the block copolymer with higher content of poly(ethyl ethylene phosphate) (PEEP) is more amorphous, showing decreased crystallizability. The obtained copolymers self-assemble into biodegradable nanoparticles with a coreshell micellar structure in aqueous solution, verified by the probe-based fluorescence measurements and transmission electronic microscopy (TEM) observation. The hydrophobic poly(3-hydroxybutyrate) (PHB) block serves as the core of the micelles and the micelles are stabilized by the hydrophilic PEEP block. The size and size distribution are related to the compositions of the copolymers. Paclitaxel (PTX) has been encapsulated into the micelles as a model drug and a sustained drug release from the micelles is observed. MTT assay also demonstrates that the block copolymers are biocompatible, rendering these copolymers attractive for drug delivery.

Construction of a sustainable 3-hydroxybutyrate-producing probiotic Escherichia coli for treatment of colitis

Colitis is a common disease of the colon that is very difficult to treat.Probiotic bacteria could be an effective treatment.The probiotic Escherichia coli Nissle 1917(EcN)was engineered to synthesize the ketone body(R)-3-hydroxybutyrate(3HB)for sustainable production in the gut lumen of mice suffering from colitis.Components of heterologous 3HB synthesis routes were constructed,expressed,optimized,and inserted into the EcN genome,combined with deletions in competitive branch pathways.The genome-engineered EcN produced the highest 3HB level of 0.6 g/L under microaerobic conditions.The live therapeutic was found to colonize the mouse gastrointestinal tract over 14 days,elevating gut 3HB and short-chain-length fatty acid(SCFA)levels 8.7-and 3.1-fold compared to those of wild-type EcN,respectively.The sustainable presence of 3HB in mouse guts promoted the growth of probiotic bacteria,especially Akkermansia spp.,to over 31%from the initial 2%of all the microbiome.As a result,the engineered EcN termed EcNL4 ameliorated colitis induced via dextran sulfate sodium(DSS)in mice.Compared to wild-type EcN or oral administration of 3HB,oral EcNL4 uptake demonstrated better effects on mouse weights,colon lengths,occult blood levels,gut tissue myeloperoxidase activity and proinflammatory cytokine concentrations.Thus,a promising live bacterium was developed to improve colonic microenvironments and further treat colitis.This proof-of-concept design can be employed to treat other diseases of the colon.

Valorization of poly-β-hydroxybutyrate(PHB)-based bioplastic waste in anaerobic digesters of food waste for bioenergy generation:reactor performance,microbial community analysis,and bioplastic biodegradation

This study aims to investigate the significance and biodegradation pathways of PHB-based bioplastic in anaerobic digesters treating food waste,where the reactor performance of changed methane generation,bioplastic biodegradation efficiency,and bioinformatic analysis of functional microbes were emphasized.The results showed that PHB-based plastic film could be partially biodegraded in the food waste digester,and a bioaugmentation use of Alcaligenes Faecalis(AF)and Bacillus Megaterium(BM)was beneficial to largely accelerate the degradation process through a beneficial shift of both the functional bacterial and archaeal species.Microbial community analysis indicated that the major bacterial species belonged to genera Candidatus_Cloacimonas,Rikenellaceae,and Defluviitoga,while the dominant methanogenic archaeal species belonged to genera Methanomassiliicoccus,Methanosarcina,and Methanosaeta.Bioplastic biodegradation analysis suggested that the optimal fractions of AF and BM for PHB-based plastic degradation were 50% AF and 75% BM,respectively,which deserves further optimization and scale-up validation.The finding of this study would contribute to the combined management of PHB-based bioplastic with food waste for clean energy recovery and a greener environment.

碳硫比和硝酸根投加量对DS-EBPR工艺的影响研究

为获得适宜的硫循环协同反硝化生物除磷工艺(DS-EBPR)运行条件,采用批次实验研究了进水m(C)/m(S)和NO_3^--N投加量对DS-EBPR的影响,同时对工艺代谢机理进行初步研究。结果表明,m(C)/m(S)为150/200时有最大吸磷速度2.4 mg/(g·h),而PHA的储存和消耗速度却最低,同时poly-S的储存和消耗速度却最大。DS-EBPR释磷段和吸磷段的反应进行能量平衡分析,说明poly-S在DS-EBPR工艺运行中可作为潜在的能量来源。poly-S在吸磷段可调节NO_3^--N的反硝化作用,可减少NO_3^--N对下一周期释磷段的影响,以达到良好的释磷效果。DS-EBPR工艺的反应机理明显不同于传统的EBPR工艺。

功能化氧化石墨/聚羟基丁酸酯纳米复合材料的制备与性能研究

采用溶液插层技术制备功能化氧化石墨/聚羟基丁酸酯纳米复合材料(MGO/PHB),利用红外光谱分析(FTIR)和原子力显微镜(AFM)对MGO的表面结构进行表征,并用扫描电子显微镜(SEM)、X射线衍射(XRD)、示差扫描量热仪(DSC)及拉伸试验分别对MGO/PHB的微观形貌、结晶性能及力学性能进行分析测试。结果表明:MGO的加入加速了PHB基体的结晶并导致结晶度增大;氧化石墨经表面功能化处理后,改善了MGO片在PHB基体中的均匀分散以及其与PHB基体的界面相互作用;随着MGO质量分数的增加,复合材料的断裂强度先降低后增大,而与纯PHB相比,复合材料都表现出更高的断裂模量值,但复合材料的力学性能变化与MGO添加量的阀值有一定关系。

Supercritical fluid-assisted fabrication of C-doped Co_(3)O_(4) nanoparticles based on polymer-coated metal salt nanoreactors for efficient enzyme-mimicking and glucose sensor properties

Nanomaterials doped with non-metallic C have attracted tremendous attention as potential nano-artificial enzymes due to their ability to change the energy band structure to improve their intrinsic properties.Herein,we report a green,facile,efficient,fast strategy to access high-performance nanozymes via supercritical CO_(2)fluid technology-fabricated polymer nanoreactor of poly-(methyl vinyl ether-co-maleic anhydride)(PVM/MA)coated Co(NO_(3))_(2)into C-doped Co_(3)O_(4)(C-Co_(3)O_(4))nanozyme by a onestep calcination process.Converting PVM/MA to C doping into Co_(3)O_(4)shortens the entire lattice constant of the crystal structure,and the overall valence band energy level below the Fermi level shifts toward the lower energy direction.The as-prepared CCo_(3)O_(4)demonstrated significant peroxidase-like catalytic activity,significantly greater than the undoped Co_(3)O_(4)nanoparticle nanozyme.The following density functional theory(DFT)calculations revealed that the doped nano-enzyme catalytic site displayed a unique electronic structure,altering the material surface with more electrons to fill the anti-bond of the two molecular orbitals,significantly improving the peroxidase-like enzyme catalytic and glucose sensor performance.The resultant enzymatic glucose sensing in a linear range of 0.1–0.6 mM with a detection limit of 3.86μM is in line with standard Michaelis–Menten theory.Collectively,this work demonstrates that converting polymers into nanozymes of C-doped form by supercritical CO_(2)fluid technology in a step is an effective strategy for constructing high-performance glucose sensor nanozymes.This cost-effective,reliable,precise system offers the potential for rapid analyte detection,facilitating its application in a variety of fields.

Cloning and sequencing of phbA gene of poly-β-hydroxybuty rate synthesis and its expression analysis

The gene of the first key enzyme of poly-β-hydroxybutyrate synthesis, 3-ketothiolase, has been amplified and cloned from chromosomal DNA of Alcaligenes eutrophus H16 by PCR. DNA sequencing results show that phbA cloned in pBluescriptSK^+ has an identical sequence with that reported previously except for one base pair. The plant constitutive expression vector has been constructed and tobacco has been transformed in order to examine the phbA gene function and the efficiency of ctp gene product. SDS-polyacrylamide gel electrophoresis result shows that the ctp gene product could direct foreign protein into plastid efficiently and phbA gene could be translated into corresponding protein with correct size. The enzyme activity analysis of 3-ketothiolase shows that the enzyme could catalyze acetyl-CoA to form acetoacetylCoA.

Comparative evaluation of physico-chemical characteristics of biopolyesters P（3HB） and P（3HB-co-3HV） produced by endophytic Bacillus cereus RCL 02

BACKGROUND： Bacteria endogenously residing within the plant tissues have attracted significant attention for production of biopolyester, polyhydroxyalkanoates （PHAs）. Bacillus cereus RCL 02 （MCC 3436）, a leaf endophyte of oleaginous plant Ricinus communis L. accumulates 81% poly（3-hydroxybutyrate） [P（3HB）] of its cell dry biomass when grown in mineral salts （MS） medium. METHODS： The copolymer production efficiency of B. cereus RCL 02 was evaluated in valeric acid supplemented MS medium under biphasic cultivation condition. The copolymer so produced has been compared with the P（3HB） isolated from RCL 02 in terms of thermal, mechanical and chemical properties. RESULTS： Valeric acid supplementation as co-substrate in the medium has led to the production of copolymer of 3- hydroxybutyrate （3HB） and 3-hydroxyvalerate （3HV） [P（3HB-co-3HV）] with 14.6 mol% 3HV. The identity of the polymers has been confirmed by X-ray diffraction （XRD） analysis, Fourier transform infrared （FTIR） and nuclear magnetic resonance （NMR） spectroscopic studies. Thermogravimetric analysis （TGA） revealed that P（3HB） and P（3HB-co-3HV） films degraded at 278.66℃ and 273.49℃, respectively. The P（3HB-co-3HV） showed lower melting temperature （165.03℃） compared to P （3HB） （170.74℃） according to differential scanning calorimetry （DSC）. Incorporation of 3HV monomers decreased the tensile strength （21.52 MPa）, tensile modulus （0.93 GPa）, storage modulus （E＇） （0.99 GPa） and increased % elongation at break （12.2%） of the copolyester. However, P（3HB） showed better barrier properties with lower water vapor transmission rate （WVTR） of 0.55 g-mil/100 in2/24 h. CONCLUSION： These findings emphasized exploration of endophytic bacterial strain （RCL 02） to produce biodegradable polyesters which might have significant potential for industrial application.

Expressing PHB synthetic genes through chloroplast genetic engineering

Chloroplast integration and expression vector containing expression cassettes for phbB, phbA, phbC and aadA genes was constructed and bombarded into the tobacco chloroplast genome. Transplastomic plants were analyzed with PCR and Southern blot. Their homoplastomy was also judged. Northern dot and RT-PCR analysis were employed to investigate transgene expression at transcriptional level. The results indicate that the chloroplast transformation system is compatible for poly-3-hydroxybutyrate (PHB) production.