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.

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.

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.

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.

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.

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.