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.

Miscibility, Crystallization, and Rheological Behavior of Solution Casting Poly（3-hydroxybutyrate）/poly（ethylene succinate） Blends Probed by Differential Scanning Calorimetry, Rheology, and Optical Microscope Techniques

The miscibility and crystallization of solution casting biodegradable poly（3-hydroxybuty- rate）/poly（ethylene succinate） （PHB/PES） blends was investigated by differential scanning calorimetry, rheology, and optical microscopy. The blends showed two glass transition temperatures and a depression of melting temperature of PHB with compositions in phase diagram, which indicated that the blend was partially miscible. The morphology observation supported this result. It was found that the PHB and PES can crystallize simultaneously or upon stepwise depending on the crystallization temperatures and compositions. The spherulite growth rate of PHB increased with increasing of PES content. The influence of compositions on the spherulitic growth rate for the partially miscible polymer blends was discussed.

γ-hydroxybutyrate protects the liver from warm ischemia-reperfusion injury in rat

BACKGROUND: Ischemia-reperfusion (I/R) syndrome remains an important clinical consideration in hepatic sur- gery, hemorrhagic shock, and liver transplantation, -y-hy- droxybutyrate (GHB) has been reported to exert protective effects against ischemia-reperfusion injury to various or- gans. To investigate whether GHB protects the liver from warm ischemia-reperfusion injury, we performed this study in rats. METHODS: Thirty male Wistar rats were randomly divided into a sham-operation group, a control group, and three I/R groups pretreated with GHB, GHB plus naloxone or naloxone. After 30 minutes of partial ischemia, followed by 60 minutes of reperfusion in the liver, histomorphological and enzymological changes, lipid peroxidation, apoptosis, and the plasma level of endothelin-1 were observed. RESULTS: I/R increased the serum levels of alanine ami- notransferase, aspartate aminotransferase and lactate dehy- drogenase and the plasma level of endothelin-1 significantly (P<0.01), in addition to increase of apoptotic index (AI) from 0.28%±0.25% to 17.68%±1.91%. The levels of he- patic malondialdehyde were markedly increased, whereas the activities of superoxide dismutase were markedly de- creased. GHB pretreatment prevented the liver from warm ischemia-reperfusion injury significantly, but naloxone par- tially blocked this effect. CONCLUSION: GHB may significantly protect the liver from hepatic warm ischemia-reperfusion injury via several different mechanisms.

D-β-hydroxybutyrate inhibits microglial activation in a cell activation model in vitro

Microglial activation plays an important role in a panel of neurological disorders such as multiple sclerosis(MS) and Parkinson's disease(PD),and is a key target for developing therapeutic strategies for these diseases.Ketogenic diet (KD),which is able to inhibit microglial activation in substantia nigra pars compacta of mice,has been shown effective in a mouse model of PD,possibly through increasing D-β-hydroxybutyrate(D-β-HB),a major component of ketone bodies.To verify this,we developed an in vitro model of microglia activation with a microglia line,BV-2,and investigated how D-β-HB have an effect on the LPS-stimulated BV-2 cells.We found D-β-HB is able to recover the cell viability,and inhibit the production of inflammatory mediators and cytokines such as ROS,nitrite,IL-1β,TNF-α,and IL-6,which otherwise were increased in LPS-stimulated BV-2 cells.We conclude that the LPS induced BV-2 cells activation is a valid in vitro model of microglia activation.D-β-HB is able to suppress the activation of BV-2 cells, which might account for one of the possible reasons of KD therapy on the PD model.

Weavability of β-Hydroxybutyrate and β-Hydroxyvalerate Copolymers( PHBV ) /PLA Used in Artificial Blood Vessels

The β-hydroxybutyrate and β-hydroxyvalerate copolymers( PHBV) /polylactic acid( PLA) is a new biocompatible material,which is developed through bacterial fermentation in vivo systems.The PHBV / PLA material could be used to make continuous filaments.However,features of artificial blood vessels,especially small diameter vascular grafts made of PHVB / PLA materials are not known.This research are to evaluate and improve weavability of the PHBV / PLA material, and to explore feasibility of using it in artificial blood vessels.Preliminary results showed that weavability of PHBV / PLV was not good,but its weavability could be improved by using methods of weak chemical,such as sizing.In this research,scanning electron microscope( SEM) was adopted to evaluate weavability of PHBV / PLV after sizing and observe surfaces of yarns and fabrics.Also,in order to set proper parameters in heat settings,differential scanning calorimetry( DSC) was used to identify glass transition temperature.

Disposable Electrochemical Biosensor for Amperometric Measurement of β-hydroxybutyrate and Total Cholesterol

A new type of disposable thin-film amperometric biosensor has been developed for measurement ofβ-hydroxybutyrate and total cholesterol in serum.The biosensor consists of two plain gold electrodes mounted on a Polyethylene terephthalate (PET) substrate.The reagent solution contains bienzymes (cholesterol oxidase and cholesterol esterase for total cholesterol,β-hydroxybutyrate dehydrogenase and diaphorase forβ-hydroxybutyrate respectively) with mediator (ferricyanide) were absorption at the surface of electrodes coated by electrodepositing platinum black.The presence of the mediator lowers the applied potential and eliminates the interference from other oxidizable species enhancing the sensitivity and selectivity of the biosensor without modifying the dynamic parameters of the response.The enzymes stably retains in the matrices of platinum black film improving the performance of the mediated sensor.The linearity is observed in the concentration range from 1.0×10^(-4) to 1.0×10^(-2) mM and 1.0×10^(-4) mM to 4.99×10^(-3) mM with sensitivity of 1.958μA/mM and 2.447μA/mM respectively.The optimized biosensor exhibits excellent reproducibility and stability retaining more than 90% of its original activity over a period of one month.The simple operation of the biosensor mass-produced at low cost is expected to find clinical application and homecare.

Study on Kinetics of Dissolving Biomass Using Sodium Hypochlorite in the Recovery of Poly-β-hydroxybutyrate

The effects of sodium hypochlorite concentration in the recovery of poly-β-hydroxybutyrate (PHB) are investigated. A simple reaction equation and a mathematical model describe the reaction kinetics. Results showed that the stoichiometric coefficient increases with increasing CAO/CBO, where CAO is the concentration of the active chlorine at t = 0 and CBO is the initial concentration of the non-PHB materials.

Production of Poly(3-Hydroxybutyrate)by Fed-Batch Culture of Alcaligenes Eutrophus at Phosphorus Limitation in 50 L fermenter

Poly(3-hydroxybutyrate) (PHB) is an intracellular carbon and energy storage material accumulated by many kinds of microorganism under unfavorable growth conditions. For the production of PHB, Alcaligenes eutrophus has been widely used because it is easy to grow, and its physiological and biochemical changes during the PHB synthesis is understood in details. A very high concentration and productivity of PHB could be obtained by fed-batch culture of Alcaligenes eutrophus with phosphate limitation in 50 L fermenter.

Analysis of γ-Hydroxybutyrate in Human Urine by LC-MSIMS Method and Its Forensic Application

GHB （γ-hydroxybutyrate） is becoming popular recreational drugs. As a result of its strong sedative and amnesiac effects, GHB has been implicated in a number of DFSA cases. The natural presence of GHB in the human body and its rapid elimination after ingestion make it difficult to detect and to evaluate its roles in suspected GHB-facilitated assaults. The paper describes an analytical method for the determination of GHB in urine using LC-MS/MS. Samples were acidified by ammonium chloride solution and extracted with ethyl acetate, and then the extracts were analysed by LC-MS/MS. The limit of detection was 0.05 p.g/mL （S/N = 3）. The intra- and inter-day precision was within 10.0% at three concentrations. The methods were found to be sensitive, accurate, rapid and suitable for the forensic toxicology to test GHB in real cases.

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.

Complete Valorization of Olive Mill Wastewater through an Integrated Process for Poly-3-hydroxybutyrate Production

An economical and environmental sustainability of bioplastic production is dependent on the use of low cost and waste C-sources as raw materials. OMW （Olive Mill Wastewater） with its high organic load represents a dangerous polluting waste. Herein the authors present an integrated process for the simultaneous recovery of polyphenols, high value natural antioxidants, production of PHAs （polyhydroxyalkanotes）, thermoplastic bio-polymers, in particular of PHB （poly-3-hydroxybutyrate） starting from OMW. The combination of membrane filtration and bacterial digestion of OMW resulted in very high yields of polyphenols （3 2.5 g/L） and PHB （31.4 mg/L.h） if compared with the state of the art. These results make the technical approach described here effective for reducing the polluting effect of OMW and maximizing the valuable product yield. Moreover the process is readily suitable for an industrial scale PHB production from OMW.

Use of a Simple Fabrication Process to Produce a Biosensor： The 3-Hydroxybutyrate Dehydrogenase Case

This study was aimed to construct a biodegradable but reliable 3-β-hydroxybutymte biosensor. In this context a versatile paper based biosensor, quickly, easily and cheaply fabricated is reported. The procedure of fabrication is based on the assumption that the introduction of the enzyme in the carbon ink will allow enzyme stabilization and facilitate the study of the catalysis of enzymes and the detection of substrates. To prove this concept we use the enzyme 3-hydroxybutyrate dehydrogenase, in aqueous solution. This enzyme was chosen because it catalyzes the 3-β-hydroxybutyrate, which results from ketoacidosis. The quantification this substance in the diabetics＇ blood is very important as it can increase the reliability of the diagnosis of glycaemia. To prove the multi-use of this biosensor we not only study the redox process in steady state and during the catalytic process, but also detected and quantify the 3-β-hydroxybutyrate. Our results showed that it was possible to study the redox process that occurred during the catalysis and to confirm the amino acid residues that participate in it. It was also observed that glucose and ascorbic acid can interfere in the detection and quantification of the 3-β-hydroxybutyrate, what should be in mind when the quantification of the 3-β-hydroxybutyrate is made in blood samples.

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.

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.

Functional Metabolomics Reveals that Astragalus Polysaccharides Improve Lipids Metabolism through Microbial Metabolite 2-Hydroxybutyric Acid in Obese Mice

Polysaccharides are widely present in herbs with multiple activities,especially immunity regulation and metabolic benefits for metabolic disorders.However,the underlying mechanisms are not well under-stood.Functional metabolomics is increasingly used to investigate systemic effects on the host by iden-tifying metabolites with particular functions.This study explores the mechanisms underlying the metabolic benefits of Astragalus polysaccharides(APS)by adopting a functional metabolomics strategy.The effects of APS were determined in eight-week high-fat diet(HFD)-fed obese mice.Then,gas chromatography–time-of-flight mass spectrometry(GC–TOFMS)-based untargeted metabolomics was performed for an analysis of serum and liver tissues,and liquid chromatography–tandem mass spectrom-etry(LC–MS/MS)-based targeted metabolomics was performed.The potential functions of the metabo-lites were tested with in vitro and in vivo models of metabolic disorders.Our results first confirmed the metabolic benefits of APS in obese mice.Then,metabolomics analysis revealed that APS supplemen-tation reversed the HFD-induced metabolic changes,and identified 2-hydroxybutyric acid(2-HB)as a potential functional metabolite for APS activity that was significantly decreased by a HFD and reversed by APS.Further study indicated that 2-HB inhibited oleic acid(OA)-induced triglyceride(TG)accumula-tion.It was also found to stimulate the expression of proteins in lipid degradation in hepatocytes and TG lipolysis in 3T3-L1 cells.Moreover,it was found to reduce serum TG and regulate the proteins involved in lipid degradation in high-fat and high-sucrose(HFHS)-fed mice.In conclusion,our study demonstrates that the metabolic benefits of APS are at least partially due to 2-HB generation,which modulated lipid metabolism both in vitro and in vivo.Our results also highlight that functional metabolomics is practical for investigating the mechanism underlying the systemic benefits of plant polysaccharides.

Vancomycin pretreatment attenuates acetaminophen-induced liver injury through 2-hydroxybutyric acid

Liver injury caused by acetaminophen(AP)overdose is a leading public health problem.Although APinduced liver injury is well recognized as the formation of N-acetyl-p-benzoquinone(NAPQI),a toxic metabolite of AP,resulting in cell damage,emerging evidence indicates that AP-induced liver injury is also associated with gut microbiota.However,the gut microbiota-involved mechanism remains largely unknown.In our study,we found that vancomycin(Vac)pretreatment(100 mg/kg,twice a day for 4 days)attenuated AP-induced liver injury,altered the composition of gut microbiota,and changed serum metabolic profile.Moreover,we identified Vac pretreatment elevated cecum and serum 2-hydroxybutyric acid(2-HB),which ameliorated AP-induced cell damage and liver injury in mice by reducing AP bioavailability and elevating GSH levels.Our current results revealed the novel role of 2-HB in protecting AP-induced liver injury and add new evidence for gut microbiota in affecting AP toxicity.

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.

Emerging roles of GPR109A in regulation of neuroinflammation in neurological diseases and pain

Neuroinflammation plays a critical role in the pathological process of multiple neurological disorders and pathological pain conditions.GPR109A,a Gi protein-coupled receptor,has emerged as an important therapeutic target for controlling inflammation in various tissues and organs.In this review,we summarized current data about the role of GPR109A in neuroinflammation.Specifically,we focused on the pharmacological features of GPR109A and signaling pathways used by GPR109A to ameliorate neuroinflammation and symptoms in Alzheimer’s disease,Parkinson’s disease,multiple sclerosis,stroke,and pathological pain conditions.

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.

Halomonas-PHB protects gnotobiotic Artemia against Vibrio and m odifies Artemia gut microbiota in xenic culture conditions

The prokaryotic cell storage compound ploy-β-hydroxybutyrate(PHB)has been considered as prebiotics that can be applied in aquaculture.In this paper,the dietary effect of a PHB-accumulating Halomonas strain(HM·PHB)identified from our previous work were studied in Artemia under gnotobiotic and xenic culture conditions,in comparison of Halomonas without PHB accumulation(HM)and microalgae Isochrysis(ISO)feeding.Under gnotobiotic condition,both HM·PHB and HM served as sole food supporting Artemia survival.Although both HM·PHB and HM feeding had no significant difference on Artemia survival percentage(P>0.05),HM·PHB significantly improved their resistance against Vibrio anguillarum challenge(P<0.05).Mass Artemia culture were further performed in xenic condition.Compared to ISO,HM·PHB feeding protected Artemia against V.anguillarum challenge(P<0.05),and HM·PHB and HM feeding resulted in increased T-AOC,pepsin,T-SOD and CAT activities(P<0.05).High throughput sequencing analysis showed that HM·PHB and HM feeding resulted in a lower Artemia gut microbial diversity(P<0.05),and modified the gut microbial community by remarkably reducing the Vibrio proportion.The outcome of the paper confirmed the beneficial effect of Halomonas-PHB in Artemia culture,which supports the use of Halomonas-PHB in the production of bio-secured live feed Artemia.