Poly-β-hydroxybutyrate Synthesis Research Articles

The stringent response regulates the poly-β-hydroxybutyrate (PHB) synthesis in Azotobacter vinelandii.

The stringent response exerted by (p)ppGpp and RNA-polymerase binding protein DksA regulates gene expression in diverse bacterial species. To control gene expression (p)ppGpp, synthesized by enzymes RelA and SpoT, interacts with two sites within the RNA polymerase; site 1, located in the interphase between subunits β' and ω (rpoZ), and site 2 located in the secondary channel that is dependent on DksA protein. In Escherichia coli, inactivation of dksA results in a reduced sigma factor RpoS expression. In Azotobacter vinelandii the synthesis of polyhydroxybutyrate (PHB) is under RpoS regulation. In this study, we found that the inactivation of relA or dksA, but not rpoZ, resulted in a negative effect on PHB synthesis. We also found that the dksA, but not the relA mutation reduced both rpoS transcription and RpoS protein levels, implying that (p)ppGpp and DksA control PHB synthesis through different mechanisms. Interestingly, despite expressing rpoS from a constitutive promoter in the dksA mutant, PHB synthesis was not restored to wild type levels. A transcriptomic analysis in the dksA mutant, revealed downregulation of genes encoding enzymes needed for the synthesis of acetyl-CoA, the precursor substrate for PHB synthesis. Together, these data indicate that DksA is required for optimal expression of RpoS which in turn activates transcription of genes for PHB synthesis. Additionally, DksA is required for optimal transcription of genes responsible for the synthesis of precursors for PHB synthesis.

Bacteroid Development, Transcriptome, and Symbiotic Nitrogen-Fixing Comparison of Bradyrhizobium arachidis in Nodules of Peanut (Arachis hypogaea) and Medicinal Legume Sophora flavescens.

Bradyrhizobium arachidis strain CCBAU 051107 could differentiate into swollen and nonswollen bacteroids in determinate root nodules of peanut (Arachis hypogaea) and indeterminate nodules of Sophora flavescens, respectively, with different N2 fixation efficiencies. To reveal the mechanism of bacteroid differentiation and symbiosis efficiency in association with different hosts, morphologies, transcriptomes, and nitrogen fixation efficiencies of the root nodules induced by strain CCBAU 051107 on these two plants were compared. Our results indicated that the nitrogenase activity of peanut nodules was 3 times higher than that of S. flavescens nodules, demonstrating the effects of rhizobium-host interaction on symbiotic effectiveness. With transcriptome comparisons, genes involved in biological nitrogen fixation (BNF) and energy metabolism were upregulated, while those involved in DNA replication, bacterial chemotaxis, and flagellar assembly were significantly downregulated in both types of bacteroids compared with those in free-living cells. However, expression levels of genes involved in BNF, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, hydrogenase synthesis, poly-β-hydroxybutyrate (PHB) degradation, and peptidoglycan biosynthesis were significantly greater in the swollen bacteroids of peanut than those in the nonswollen bacteroids of S. flavescens, while contrasting situations were found in expression of genes involved in urea degradation, PHB synthesis, and nitrogen assimilation. Especially higher expression of ureABEF and aspB genes in bacteroids of S. flavescens might imply that the BNF product and nitrogen transport pathway were different from those in peanut. Our study revealed the first differences in bacteroid differentiation and metabolism of these two hosts and will be helpful for us to explore higher-efficiency symbiosis between rhizobia and legumes. IMPORTANCE Rhizobial differentiation into bacteroids in leguminous nodules attracts scientists to investigate its different aspects. The development of bacteroids in the nodule of the important oil crop peanut was first investigated and compared to the status in the nodule of the extremely promiscuous medicinal legume Sophora flavescens by using just a single rhizobial strain of Bradyrhizobium arachidis, CCBAU 051107. This strain differentiates into swollen bacteroids in peanut nodules and nonswollen bacteroids in S. flavescens nodules. The N2-fixing efficiency of the peanut nodules is three times higher than that of S. flavescens. By comparing the transcriptomes of their bacteroids, we found that they have similar gene expression spectra, such as nitrogen fixation and motivity, but different spectra in terms of urease activity and peptidoglycan biosynthesis. Those altered levels of gene expression might be related to their functions and differentiation in respective nodules. Our studies provided novel insight into the rhizobial differentiation and metabolic alteration in different hosts.

Partnering of anammox and denitrifying bacteria benefits anammox's recovery from starvation and complete nitrogen removal

The cooperative metabolic activity of anammox and denitrifying bacteria could speed up anammox's recovery and reduce nitrate generated from the anammox reaction. In this study, a laboratory-scale model system containing a defined anammox culture AMX and a simultaneous nitrification and denitrification (SND) bacterium - Thauera sp. strain SND5 was established and investigated. Several lines of evidence revealed that strain SND5 consumed soluble microbial products (SMPs) generated by culture AMX (as high as 1.5 mg/L), stimulating anammox activity after long-term starvation. At low C/N ratios with an optimal C/N of 1, SND5 completely consumed organic carbon first at anoxic condition, storing carbon intracellularly as poly-β-hydroxybutyrate (PHB) (as high as 0.6 mg/L biomass), thereby creating a favorable environment for the growth of anammox bacteria. The anammox reaction and nitrate reduction supported by PHB catabolism could then proceed simultaneously, resulting in enhanced nitrogen removal. Cooperative interactions between anammox and denitrifying bacteria involving SMPs consumption and PHB synthesis may play a significant role in nitrogen cycling at nitrite- and carbon-limited environments.

Effects of carbon sources and operation modes on the performances of aerobic denitrification process and its microbial community shifts

Carbon source, operation mode and microbial species have great effects on the synthesis of poly-β-hydroxybutyrate (PHB) which has been identified as the key issue for aerobic denitrification process. In this study, an aerobic denitrification SBR was operated under anoxic/oxic mode and completely oxic mode with the carbon source of CH3COONa and CH3CH2CH2COONa, respectively. Total nitrogen (TN) removal efficiencies, PHB content in activated sludge, production of nitric oxide (NO) and nitrous oxide (N2O) of the process were investigated in great detail. The main results obtained from the trial were: (1) the average TN removal was in the range of 86.11%–90.05%; (2) the maximum TN removal efficiency and the maximum PHB content of the process being achieved when the carbon source of CH3CH2CH2COONa was applied under anoxic/oxic mode; (3) in case of CH3COONa as the carbon source, the concentrations of NO and N2O in the bulk liquid were ∼0.4 mg/L and ∼0.02 mg/L, respectively, while in case of CH3CH2CH2COONa, N2O of ∼0.2 mg/L and NO of ∼2.5 mg/L were recorded and the latter was decreased to ∼1.0 mg/L at the end of the cycle; (4) no obvious dominant genus in case of using CH3COONa, while Plasticicumulans sp. being the major microbial community when using CH3CH2CH2COONa. Overall, the effect of carbon source on microbial community is obvious. Nevertheless, operation mode affects the PHB synthesis, while PHB plays an important role in aerobic denitrification process for achieving a relatively high TN nitrogen removal efficiency. CH3COONa is a better carbon source for aerobic denitrification compared with CH3CH2CH2COONa.

Poly-β-hydroxybutyrate (PHB) biosynthesis, tricarboxylic acid activity and PHB content in chickpea (Cicer arietinum L.) root nodule

An experiment was conducted to assess the relationship between poly-β-hydroxybutyrate (PHB) biosynthesis and tricarboxylic acid (TCA) activity in desi and kabuli chickpea (Cicer arietinum L.) genotypes. The specific activities of enzymes of PHB metabolism viz., β-ketothiolase (PHB-A), acetoacetyl coenzyme A reductase (PHB-B) and PHB synthase (PHB-C), and those of tricarboxylic acid cycle (citrate synthase (CS) and malate dehydrogenase (MDH) under symbiosis were measured in bacteroids and compared with the PHB accumulation in the nodule and the root. The significant positive correlation was observed between shoot and nodule mass and PHB-A, PHB-B, and PHB-C activities. However, nodule and shoot weights were not significantly correlated with PHB content either in the roots or nodules. The same was true for PHB levels and citrate synthase activity. MDH activity showed a significant negative correlation with nodule PHB. A marked variation and an age dependant increase in malate dehydrogenase activity were measured. A higher capacity for malate oxidation by an increased MDH is likely alter the balance between malate decarboxylation and oxidation, resulting in a higher steady-state concentration of oxaloacetate and that may favor the utilization of acetyl-CoA in the TCA cycle rather than for the synthesis of PHB.

Genetic engineering of Synechocystis sp. PCC6803 for poly-β-hydroxybutyrate overproduction

The biosynthesis of poly-β-hydroxybutyrate (PHB) directly from carbon dioxide is a sustainable alternative for non-renewable, petroleum-based polymer production. Synechocystis sp. PCC6803 can naturally accumulate PHB using CO2 as the sole carbon source, particularly when major nutrients such as nitrogen become limiting. Many previous studies have tried to genetically engineer PHB overproduction; mostly by increasing the expression of enzymes directly involved in its biosynthesis pathway. Here, we have instead concentrated on engineering the central carbon metabolism of Synechocystis such that (i) the PHB synthesis pathway becomes deregulated, and/or (ii) the levels of its substrate, acetyl-CoA, were increased. Seven different mutants were constructed harboring, separately or in combination, three different genetic modifications to Synechocystis' metabolic network. These were the deletions of phosphotransacetylase (Pta) and acetyl-CoA hydrolase (Ach), and the expression of a heterologous phosphoketolase (XfpK) from Bifidobacterium breve. The wild type Synechocystis and the derivative strains were compared in terms of biomass and the PHB production capability during photoautotrophic growth. This was performed in a photobioreactor exposed to a diel light/dark rhythm and using standard BG11 as the growth medium. We found that the strain that combined all three genetic modifications, i.e. xfpk overexpression in a double pta and ach deletion background, showed the highest levels of PHB production from all the strains tested here. Encouragingly, the production levels obtained: 232mgL−1, ~12% (w/w) of the dry biomass weight, and a productivity of 7.3mgL−1d−1; are to the best of our knowledge, the highest ever reported for PHB production directly from CO2.

Enrichments of methanotrophic–heterotrophic cultures with high poly-β-hydroxybutyrate (PHB) accumulation capacities

Methanotrophic–heterotrophic communities were selectively enriched from sewage sludge to obtain a mixed culture with high levels of poly-β-hydroxybutyrate (PHB) accumulation capacity from methane. Methane was used as the carbon source, N2 as sole nitrogen source, and oxygen and Cu content were varied. Copper proved essential for PHB synthesis. All cultures enriched with Cu could accumulate high content of PHB (43.2%–45.9%), while only small amounts of PHB were accumulated by cultures enriched without Cu (11.9%–17.5%). Batch assays revealed that communities grown with Cu and a higher O2 content synthesized more PHB, which had a wider optimal CH4:O2 range and produced a high PHB content (48.7%) even though in the presence of N2. In all methanotrophic–heterotrophic communities, both methanotrophic and heterotrophic populations showed the ability to accumulate PHB. Although methane was added as the sole carbon source, heterotrophs dominated with abundances between 77.2% and 85.6%. All methanotrophs detected belonged to type II genera, which formed stable communities with heterotrophs of different PHB production capacities.

Generation of poly-β-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice

It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) 14C-PHB generated from 2-14C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1–10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination.

The Role of the ncRNA RgsA in the Oxidative Stress Response and Biofilm Formation in Azotobacter vinelandii.

Azotobacter vinelandii is a soil bacterium that forms desiccation-resistant cysts, and the exopolysaccharide alginate is essential for this process. A. vinelandii also produces alginate under vegetative growth conditions, and this production has biotechnological significance. Poly-β-hydroxybutyrate (PHB) is another polymer synthetized by A. vinelandii that is of biotechnological interest. The GacS/A two-component signal transduction system plays an important role in regulating alginate production, PHB synthesis, and encystment. GacS/A in turn controls other important regulators such as RpoS and the ncRNAs that belong to the Rsm family. In A. vinelandii, RpoS is necessary for resisting oxidative stress as a result of its control over the expression of the catalase Kat1. In this work, we characterized a new ncRNA in A. vinelandii that is homologous to the P16/RsgA reported in Pseudomonas. We found that the expression of rgsA is regulated by GacA and RpoS and that it was essential for oxidative stress resistance. However, the activity of the catalase Kat1 is unaffected in rgsA mutants. Unlike those reported in Pseudomonas, RgsA in A. vinelandii regulates biofilm formation but not polymer synthesis or the encystment process.

Rhizobial Diversity and Nodulation Characteristics of the Extremely Promiscuous Legume Sophora flavescens.

In present study, we report our extensive survey on the diversity and biogeography of rhizobia associated with Sophora flavescens, a sophocarpidine (matrine)-containing medicinal legume. We additionally investigated the cross nodulation, infection pattern, light and electron microscopies of root nodule sections of S. flavescens infected by various rhizobia. Seventeen genospecies of rhizobia belonging to five genera with seven types of symbiotic nodC genes were found to nodulate S. flavescens in natural soils. In the cross-nodulation tests, most representative rhizobia in class α-Proteobacteria, whose host plants belong to different cross-nodulation groups, form effective indeterminate nodules, while representative rhizobia in class β-Proteobacteria form ineffective nodules on S. flavescens. Highly host-specific biovars of Rhizobium leguminosarum (bv. trifolii and bv. viciae) and Rhizobium etli bv. phaseoli could establish symbioses with S. flavescens, providing further evidence that S. flavescens is an extremely promiscuous legume and it does not have strict selectivity on either the symbiotic genes or the species-determining housekeeping genes of rhizobia. Root-hair infection is found as the pattern that rhizobia have gained entry into the curled root hairs. Electron microscopies of ultra-thin sections of S. flavescens root nodules formed by different rhizobia show that the bacteroids are regular or irregular rod shape and nonswollen types. Some bacteroids contain poly-β-hydroxybutyrate (PHB), while others do not, indicating the synthesis of PHB in bacteroids is rhizobia-dependent. The extremely promiscuous symbiosis between S. flavescens and different rhizobia provide us a basis for future studies aimed at understanding the molecular interactions of rhizobia and legumes.

A Study on Production of Poly-β-Hydroxybutyrate Bioplastic from Sago Starch by Indigenous Amylolytic Bacteria

Bacillus sp. PSA10 and Bacillus sp. PPK5 were two indigenous strain amylolytic bacteria from SoutheastSulawesi that have ability to produce bioplastic poly-β-hydroxybutyrate (PHB) from sago starch. The study wasattempted to determine the mechanism of PHB production by bacteria amylolytic was grown on sago starchcontainingmedia. Two amylolytic bacteria i.e. Bacillus sp. PSA10 and Bacillus sp. PPK5 was grown for 168 hin a mineral salts medium with sago starch as carbon source. Growth of amylolytic bacteria was monitoredby cell dry weight. Extraction of PHB was done by N-hexane acetone-diethyl ether method and PHB contentwas quantifi ed with UV spectrophotometer at 235 nm. Glucose level was determined by using kit of glucoseGOD 10” and was quantifi ed with spectrophotometer at 500 nm. Sago starch concentration was determinedby phenol method using specthrophotometer at 490 nm. The result of the study showed that Bacillus sp.PSA10 was produced PHB up to 66,81 % (g PHB/g cell dry weight) at 48 h and Bacillus sp. PPK5 up to 24,83% (g PHB/g cell dry weight) at 84 h. Bacillus sp. PSA10 has ability to converse sago starch to be PHB directlywithout glucose accumulation in the media, whereas Bacillus sp. PPK5 have to accumulate glucose as productof sago starch hydrolysis to produce of PHB. PHB synthesis by Bacillus sp. PHB production on sago starchof the Bacillus sp. PSA10 was found to be growth-associated whereas Bacillus sp. PPK5 was found to be nongrowth-associated. Therefore, two indigenous amylolytic bacteria were having of difference in biosynthesismechanism of PHB in sago starch medium and their characteristics of PHB synthesis should be consideredin developing cultivation methods for the effi cient production of PHB. Keywords : Production, PHB, Amylolytic bacteria, Sago starch.

Poly-ß-hydroxybutyrate accumulation by Rhodobacter sphaeroides phase variants

The Rand M variants of a purple photosynthetic bacterium Rhodobacter sphaeroides 2R grown on a medium with acetate accumulate poly- β-hydroxybutyrate (PHB). Accumulation of this polymer occurs in the cells grown either anaerobically on the light or aerobically in the dark. On the medium with C/N imbalance (C/N = 4), PHB content during the stationary growth phase under aerobic conditions in the dark was 40 and 70% of the dry biomass of the R and M variant, respectively. The Rba. sphaeroides M variant is therefore a promising culture for large-scale PHB production. Investigation of activity of the TCA cycle enzymes revealed that decreased activity of citrate synthase, the key enzyme for acetate involvement in the reactions of the tricarboxylic acids cycle, was primarily responsible for enhanced PHB synthesis by Rba. sphaeroides. Moreover, the Rba. sphaeroides M variant grown under aerobic conditions in the dark exhibited considerably lower activity of NADH oxidase, which participates in the oxidation of reduced NADH produced in the TCA cycle during acetate oxidation. The combination of these two factors increases the possibilities for acetate assimilation via an alternative mechanism of PHB synthesis.

Experimental Investigation of Poly-β-Hydroxybutyrate Production by Azohydromonas lata: Kinetics and Artificial Neural Network Modeling

Batch culture of Azohydromonas lata was investigated for the production of intracellular poly-β-hydroxybutyrate (PHB). In order to determine the C:N value of the culture media for maximizing the microbial productivity of PHB, different concentrations of glucose and ammonium chloride were used as carbon and nitrogen sources, respectively. The optimal temperature and shaking rate was obtained at 30°C and 180 rpm, respectively. The maximum intracellular PHB concentration obtained was 5.09 g/l, which was 20% (w/w) of the cell dry weight (CDW) after 72 h. Also, the synthesis of PHB was growth associated with the C:N ratio of 153.71. The maximum calculated Yp/s was 0.212 (gr/gr) and the specific production rate value after 12 h was 0.264 g/l/h, with 40 and 50 g/l of glucose concentrations, respectively, with 0.5 g/l ammonium chloride kept constant. The chemical composition of the resulting PHB was analyzed by Fourier transform infrared spectroscopy and proton nuclear magnetic resonance spectroscopy. The Leudeking–Piret model was used for kinetic analysis of the PHB production, the statistical analysis of which was modeled by response surface methodology. An artificial neural network technique was applied for modeling of the microbial production of PHB by A. lata as a function of the glucose concentration and CDW, where the minimum mean square error of the model was 0.0012 and 0.0038 for glucose concentrations of 50 g/l and 40 g/l, respectively, when 0.5 g/l ammonium chloride was kept constant.

The Synthesis of Poly-β-Hydroxybutyrate by Moderately Halophilic Bacteria <i>Halomonas venusta</i>

In this study, poly-β-hydroxybutyrate (PHB) was synthesized by moderately halophilic bacteria Halomonas venusta and identified by 1H-NMR. The effects of different carbon sources, salt concentration, initial carbon nitrogen quality ratio, initial phosphate concentration were studied on the synthesis of PHB. The optimum conditions of the PHB synthesis were detemined. Glucose was as carbon source with the concentration of 80 g/L and salt concentration, initial carbon nitrogen ratio, initial phosphate concentration was 3%, 15 and 1.9 g/L respectively. Under the above conditions, a two-phase synthesis system of PHB was constructed. The PHB synthesis amount and yield of cell dry weight was reached up to 1.5 g/L and 69.19 wt.%, respectively. The efficient synthesis of PHB was realized.

Screening and comparative assay of poly-hydroxyalkanoates produced by bacteria isolated from the Gavkhooni Wetland in Iran and evaluation of poly-β-hydroxybutyrate production by halotolerant bacterium Oceanimonas sp. GK1

The screening of bacteria isolated from soil, water and sediment samples of the Gavkhooni Wetlandin Iranled to the isolation of about 161 moderately halophilic and halotolerant bacteria which were able to accumulate intracel- lular lipid inclusions under conditions of nitrogen limitation. Primary studies showed that most of the isolates were able to produce these inclusions but that only a Gram-negative, rod- shaped halotolerant bacterium, designated as strain GK1(IBRC-M10197), showed high-capacity production in a wide range of culture conditions. Gas chromatography and Fourier transform infrared spectroscopy studies revealed that the inclusions were poly-β-hydroxybutyrate (PHB). Pheno- typic characterization and phylogenetic analysis based on 16S rRNA gene sequence comparison showed that this strain is a member of the genus Oceanimonas. The genome sequence study of this bacterium revealed the presence of genes in- volved in the PHB synthesis pathway, which was supported by results of the transmission electron microscopy analysis. The isolate was capable of producing PHBunder a widerange of physicochemical conditions, including pH 6-9, tempera- tures ranging from 20 to 40 °C and various salt concentrations and carbon/nitrogen sources. Under the optimum condition, cultivation in shake flasks (200 rpm) led to a PHB accumula- tion of about 75 wt% at 35 °C, pH 8 and 5 % (w/v) NaCl in a medium supplemented with 5 % sucrose as the carbon source and 0.5 % peptone as the nitrogen source.

Posttranscriptional regulation of PhbR, the transcriptional activator of polyhydroxybutyrate synthesis, by iron and the sRNA ArrF in Azotobacter vinelandii

Azotobacter vinelandii is a Gram-negative bacterium able to synthesize poly-β-hydroxybutyrate (PHB), a biodegradable plastic of industrial interest. The phbBAC operon encodes the enzymes of PHB synthesis and is activated by the transcriptional regulator PhbR and the sigma factor RpoS. Iron limitation has been previously reported to increase PHB accumulation in A. vinelandii; however, the mechanism by which iron controls PHB synthesis is unknown. Under iron starvation in Escherichia coli, the RyhB sRNA modulates the translation of genes involved in iron homeostasis. ArrF is the RyhB analogue in A. vinelandii and similarly increases in quantity during Fe(2+) depletion. In this study, we evaluate the effect of iron and ArrF on PHB accumulation, and on phbR and phbBAC expression in A. vinelandii strain UW136. Using transcriptional and translational fusions of phbR and phbB with gusA reporter gene, we found that iron limitation increased the expression of phbBAC at the transcriptional level and posttranscriptionally increased the expression of phbR. We also found that the ArrF sRNA is a positive regulator of phbR expression at the posttranscriptional level. Collectively, these data suggest that iron limitation increases the translation of phbR through ArrF.

High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process

A mixed fermentation strategy based on exponentially fed-batch cultures (EFBC) and nutrient pulses with sucrose and yeast extract was developed to achieve a high concentration of PHB by Azotobacter vinelandii OPNA, which carries a mutation on the regulatory systems PTSNtr and RsmA-RsmZ/Y, that negatively regulate the synthesis of PHB. Culture of the OPNA strain in shake flaks containing PY-sucrose medium significantly improved growth and PHB production with respect to the results obtained from the cultures with the parental strain (OP). When the OPNA strain was cultured in a batch fermentation keeping constant the DOT at 4%, the maximal growth rate (0.16h−1) and PHB yield (0.30gPHBgSuc−1) were reached. Later, in EFBC, the OPNA strain increased three fold the biomass and 2.2 fold the PHB concentration in relation to the values obtained from the batch cultures. Finally, using a strategy of exponential feeding coupled with nutrient pulses (with sucrose and yeast extract) the production of PHB increased 7-fold to reach a maximal PHB concentration of 27.3±3.2gL−1 at 60h of fermentation. Overall, the use of the mutant of A. vinelandii OPNA, impaired in the PHB regulatory systems, in combination with a mixed fermentation strategy could be a feasible strategy to optimize the PHB production at industrial level.

Poly-β-hydroxybutyrate/ectoine co-production by ectoine-excreting strain Halomonas salina

The ectoine-excreting bacterial strain of Halomonas salina was employed in the co-production of poly-β-hydroxybutyrate (PHB) and ectoine (Ect) during a fermentation process (PHB/Ect co-production). An efficient PHB/Ect co-production process was carried out at low NaCl concentration (30gL−1). It was established using 1H Nuclear Magnetic Resonance spectroscopy that H. salina produces PHB. The effects of the NaCl concentration, the initial C/N ratio, the phosphate concentration and mixed carbon sources were investigated with respect to PHB/Ect co-production. The PHB/Ect co-production system comprised growing and non-growing cell phases and was developed with NaCl concentration of 30gL−1. The optimal conditions for PHB/Ect co-production by the ectoine-excreting strain of H. salina were 30gL−1 NaCl, with an initial C/N ratio of 15, an initial phosphate concentration of 12gL−1 and mixed carbon sources of 55gL−1 glucose and 25gL−1 monosodium glutamate. Using a PHB/Ect co-production system with growing and non-growing cell phases prevents the inhibition of PHB synthesis by high concentration of NaCl and significantly reduces ectoine degradation. PHB and ectoine concentrations as high as 35.3gL−1 and 8.6gL−1, respectively, were achieved. The efficient co-production of PHB and ectoine at a low NaCl concentration has been realised.

Study on Synthesis of PHB by Moderate Halophile and Aqueous Extraction of PHB

PHB (Poly-β-hydroxybutyrate) has been widely in the areas of industrial packaging, pharmacy and agriculture. Currently, its production cost, especially in extraction and purification are high. A moderate halophilic bacterium was used for PHB synthesis and an aqueous extraction method based on osmotic downshock was investigated in this paper to further improve the extraction efficiency. PHB synthesised by moderate halophile with certain NaCl concentration can be released with cell lysis by osmotic downshock. Meanwhile the high temperature, ultrasonic and SDS (Sodium dodecyl sulfate) can enhance the release. After 48 h of fermentation, the production of PHB by Halomonas salina was 6.5 g/L. The extraction rate of PHB was 24.6% with osmotic downshock and 35.4% with additional ultrasonic assistance at 65 °C. A further addition of 0.5% SDS made the extraction rate increase to 75.4%. Thereinto, the addition of 6% NaCl had significantly improved the aqueous extraction rate to 92.3%.

In silico genomics analysis of weed Lantana camara and its proposed application in synthesis of polyhydroxybutyrate

Poly (3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers The most common PHA is poly-β-hydroxybutyrate (PHB), and this polymer can accumulate up to 90% of the cellular dry weight of some bacteria. We report using Lantana camara, a weed for the production of PHB to provide a cheap and abundant source of this polymer. Maturase K gene (m atK) of chloroplast is highly conserved in plant, which is involved in intron splicing. Based on a computational gene sequence analysis, we propose that the L. Camara, a common weed, can be an inexpensive and abundant source for biological production of PHB. Our simulations focus on the maturase K ( matK) gene – length of 1331 base pairs (bp) – of the choloroplasts, which affects the intron splicing. Our computational approach considers a reconstructed chloroplast genome of L. Camara, the trnL–trnF intergenic spacer (IGS), and trnH–psbA IGS in the chloroplast genome. The Blastn result indicates that the Verbenaceae genus Lantana displays 86% sequence identity with that of Solanaceae genus Nicotiana. Phylogenetic tree was constructed to identify an ideal comparison of L. camara matK gene and other plant species used for the insertion of phbA, phbB, and phbC enzymes, which regulate PHB synthesis. The former can be a good candidate for bioplastic biosynthesis in plant of family Verbanaceae. Our computational results propose that the chloroplast transformation may be useful in biological production of PHB.