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.

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.

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.

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.

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.

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.

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.

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.

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.

Kinetic analysis of anaerobic phosphorus release during biological phosphorus removal process

Enhanced biological phosphorus removal(EBPR)is a commonly used and sustainable method for phosphorus removal from wastewater.Poly-β-hydroxybutyrate(PHB),polyphosphate,and glycogen are three kinds of intracellular storage polymers in phosphorus accumulation organisms.The variation of these polymers under different conditions has an apparent influence on anaerobic phosphorus release,which is very important for controlling the performance of EBPR.To obtain the mechanism and kinetic character of anaerobic phosphorus release,a series of batch experiments were performed using the excessively aerated sludge from the aerobic unit of the biological phosphorus removal system in this study.The results showed that the volatile suspended solid(VSS)had an increasing trend,while the mixed liquid suspended sludge(MLSS)and ashes were reduced during the anaerobic phosphorus release process.The interruption of anaerobic HAc-uptake and phosphorus-release occurs when the glycogen in the phosphorus-accumulating-organisms is exhausted.Under the condition of lower initial HAc-COD,HAc became the limiting factor after some time for anaerobic HAc uptake.Under the condition of higher initial HAc-COD,HAc uptake was stopped because of the depletion of glyco-gen in the microorganisms.The mean ratio ofΔ_(ρP)/Δ_(ρPHB),Δ_(ρ)GLY/ΔρPHB,Δ_(ρP)/ΔCOD,andΔ_(ρPHB)/ΔCOD was 0.48,0.50,0.44,and 0.92,respectively,which was nearly the same as the theoretical value.The calibrated kinetic parameters of the HAc-uptake and phosphorus-release model were evaluated as follows:QHAc,max was 164 mg/(g·h),QP,max was 69.9 mg/(g·h),Kgly was 0.005,and KCOD was 3 mg/L.An apparently linear correlation was observed between the ratio ofΔ_(ρP)/ΔCOD and pH of the solution,and the equation between them was obtained in this study.Enhanced biological phosphorus removal(EBPR)is a commonly used and sustainable method for phosphorus removal from wastewater.Poly-β-hydroxybutyrate(PHB),polyphosphate,and glycogen are three kinds of intracellular storage polymers in phosphorus accumulation organisms.The variation of these polymers under different conditions has an apparent influence on anaerobic phosphorus release,which is very important for controlling the performance of EBPR.To obtain the mechanism and kinetic character of anaerobic phosphorus release,a series of batch experiments were performed using the excessively aerated sludge from the aerobic unit of the biological phosphorus removal system in this study.