Poly-β-hydroxybutyrate Accumulation Research Articles

Promoting of abiotic stress–induced resistance using Poly-β-Hydroxybutyrate (PHB) by Rhizobium phaseoli in common bean plants.

In total, 50 Rhizobium isolates were isolated from the mature root nodules of common beans plants (Phaseolus vulgaris) grown in different nine governorates of Egypt. PHB was optimized by the identified strain using response surface methodology. A total of 11 parameters (pH, incubation period, inoculum size, temperature, agitation speed, mannitol, sucrose, yeast extract, glycine, K2HPO4, and MgSO4) were analyzed for their significant effects on PHB production by the Plackett–Burman design (PBD). Sucrose, yeast extract, glycine, and MgSO4 were the main significant factors affecting PHB accumulation. Central composite design (CCD) of the response surface methodology was used to determine the optimum levels of the selected factors. Rhizobium phaseoli reached the maximum production (4.997 g/L) at run 36 in the presence of 25 g/L of sucrose, 0.0 g/L of yeast extract, 0.87 g/L of glycine, 0.3 g/L of MgSO4, and 5% of inoculation size. In vitro experiments were carried out to test the effect of different stress conditions (pH: 6–11, temperature: 5°C–50°C, salinity: 0.01%–7%, and drought: 0%–5% w/v) on the growth of Rhizobium phaseoli. The results showed that Rhizobium phaseoli can withstand 3% –5% NaCl, high temperature of 30°C– 45°C, alkalinity at pH value of 8 – 10, and drought stress at 3% – 5% w/v polyethylene glycol with growth loss of 50% when grown on modified medium and 75% when grown on the basal one. In vivo experiments were done to study the effect of drought stress levels on the growth parameters of common bean plants. In general, all the treatments with Rhizobium phaseoli grown on the modified medium were superior to Rhizobium phaseoli grown on the basal medium. Also, they showed high tolerance of drought conditions.

Light excess stimulates Poly-beta-hydroxybutyrate yield in a mangrove-isolated strain of Synechocystis sp.

Poly-β-hydroxybutyrate (PHB) is a biodegradable biopolymer that may replace fossil-based plastics reducing its negative environmental impact. One highly sustainable strategy to produce these biopolymers is the exploitation of photosynthetic microorganisms that use sunlight and CO2 to produce biomass and subsequently, PHB. Exploring environmental biological diversity is a powerful tool to find resilient microorganisms potentially exploitable to produce bioproducts. In this work, a cyanobacterium (Synechocystis sp.) isolated from a contaminated area close to an important industrial complex was shown to produce PHB under different culture conditions. Carbon, nutrients supply and light intensity impact on biomass and PHB productivity were assessed, showing that the highest yield of PHB achieved was 241 mg L−1 (31%dcw) under high light intensity. Remarkably this condition not only stimulated PHB accumulation by 70% compared to other conditions tested but also high cellular duplication rate, maximizing the potential of this strain for PHB production.

Industrial Production of Poly-β-hydroxybutyrate from CO2: Can Cyanobacteria Meet this Challenge?

The increasing impact of plastic materials on the environment is a growing global concern. In regards to this circumstance, it is a major challenge to find new sources for the production of bioplastics. Poly-β-hydroxybutyrate (PHB) is characterized by interesting features that draw attention for research and commercial ventures. Indeed, PHB is eco-friendly, biodegradable, and biocompatible. Bacterial fermentation processes are a known route to produce PHB. However, the production of PHB through the chemoheterotrophic bacterial system is very expensive due to the high costs of the carbon source for the growth of the organism. On the contrary, the production of PHB through the photoautotrophic cyanobacterium system is considered an attractive alternative for a low-cost PHB production because of the inexpensive feedstock (CO2 and light). This paper regards the evaluation of four independent strategies to improve the PHB production by cyanobacteria: (i) the design of the medium; (ii) the genetic engineering to improve the PHB accumulation; (iii) the development of robust models as a tool to identify the bottleneck(s) of the PHB production to maximize the production; and (iv) the continuous operation mode in a photobioreactor for PHB production. The synergic effect of these strategies could address the design of the optimal PHB production process by cyanobacteria. A further limitation for the commercial production of PHB via the biotechnological route are the high costs related to the recovery of PHB granules. Therefore, a further challenge is to select a low-cost and environmentally friendly process to recover PHB from cyanobacteria.

Production and Characterization of Bioplastic by Polyhydroxybutyrate Accumulating Erythrobacter aquimaris Isolated from Mangrove Rhizosphere.

The synthesis of bioplastic from marine microbes has a great attendance in the realm of biotechnological applications for sustainable eco-management. This study aims to isolate novel strains of poly-β-hydroxybutyrate (PHB)-producing bacteria from the mangrove rhizosphere, Red Sea, Saudi Arabia, and to characterize the extracted polymer. The efficient marine bacterial isolates were identified by the phylogenetic analysis of the 16S rRNA genes as Tamlana crocina, Bacillus aquimaris, Erythrobacter aquimaris, and Halomonas halophila. The optimization of PHB accumulation by E. aquimaris was achieved at 120 h, pH 8.0, 35 °C, and 2% NaCl, using glucose and peptone as the best carbon and nitrogen sources at a C:N ratio of 9.2:1. The characterization of the extracted biopolymer by Fourier-transform infrared spectroscopy (FTIR), Nuclear magnetic resonance (NMR), and Gas chromatography-mass spectrometry (GC-MS) proves the presence of hydroxyl, methyl, methylene, methine, and ester carbonyl groups, as well as derivative products of butanoic acid, that confirmed the structure of the polymer as PHB. This is the first report on E. aquimaris as a PHB producer, which promoted the hypothesis that marine rhizospheric bacteria were a new area of research for the production of biopolymers of commercial value.

Outer membrane protein I is associated with poly-β-hydroxybutyrate granules and is necessary for optimal polymer accumulation in Azotobacter vinelandii on solid medium.

Azotobacter vinelandii is a soil bacterium that is able to synthesize poly-β-hydroxybutyrate (PHB), a polymer used to produce biodegradable plastic. PHB is stored in the cytoplasm as granules surrounded by several proteins such as the major phasin PhbP, PHB synthase and PHB depolymerase, among others. Many studies have reported the presence of membrane proteins on PHB granules due to contamination during the polymer extraction procedures. Previously, the outer membrane protein I (OprI) was detected on the polymer granules in A. vinelandii. In this study, by using random transposon mutagenesis, we identified that a mutation in the oprI gene diminished PHB accumulation in A. vinelandii on solid medium. Electron microscopy confirmed the low polymer production by the oprI mutant. Analysis of PHB granules by Tricine-SDS-PAGE revealed that the absence of OprI affected the protein profile of the granules, suggesting that OprI could have a structural role in A. vinelandii. Thus, some membrane proteins on PHB granules may not be artefacts as previously described.

Plant Tissue Localization and Morphological Conversion of Azospirillum brasilense upon Initial Interaction with Allium cepa L.

The genus Azospirillum is recognized as plant growth-promoting bacteria that exert beneficial effects on the host plant and is morphologically converted into cyst-like cells (i.e., c-form) in association with poly-β-hydroxybutyrate (PHB) accumulation in the cells under stress conditions. We constructed Azospirillum brasilense, labeled with reporter genes (gus/gfp, mCherry) and examined the plant tissue localization along with a morphological conversion into the c-form upon its initial interaction with onion seedlings (Allium cepa L.). The PHB granules in the A. brasilense cells were easily detected under fluorescence as “black holes”, rendering it possible to monitor the morphological conversion from vegetative to the c-form cells. The results showed that the A. brasilense cells on the surface of the roots and bulbs (underground stem) began converting at three days following inoculation and that the cell conversion was significantly advanced with time along with the cell population increase. The endophytic infection of A. brasilense into the bulb tissues was also confirmed, although these likely constituted vegetative cells. Moreover, the morphological conversion into the c-form was induced under nitrogen-restricted conditions. Analysis of the biochemical properties of the A. brasilense cells during cell conversion revealed that the acetylene reduction activity correlated positively with the PHB accumulation in the cells converting into the c-form under nitrogen-restricted conditions.

Poly-β-hydroxybutyrate Production by Methylosinus trichosporium OB3b at Different Gas-phase Conditions.

BackgroundThe utilization of methane for production of Poly-β-hydroxybutyrate (PHB) not only cuts the emissions of greenhouse gases but also greatly reduces PHB production cost.ObjectivesThe aim of this study was to determine the effects of gas-phase conditions on PHB production by Methylosinus trichosporium OB3b.Materials and MethodsBacterial cultivation and PHB production were conducted in a series of sealed serum bottles. Nitrogen-free mineral salts medium was used to induce PHB production in the presence or absence of N2 in the headspace.ResultsIn the absence of N2, the highest PHB content (i.e., 52.9% of the dry cell weight with a PHB concentration of 814.3 mg.L-1) was obtained at a ratio of CH4:O2=2:1. Further study at different O2 concentrations with a fixed CH4 partial pressure in absence of N2 showed that PHB accumulation by methanotroph could be tolerated high oxygen partial pressure and its respond to the variation of the oxygen concentration depends on the methane partial pressure. In presence of N2, with headspace gas replenished only when oxygen was almost depleted, the degradation of intracellular PHB has appeared. In the regimen of updating headspace gas at the point when the PHB content began to decrease, the highest PHB content (i.e., 55.5% of the dry cell weight with 901.8 mg.L-1 PHB concentration and 12.5 mg.L-1.h-1PHB productivity) was obtained at 0.2 atm O2 and PHB accumulation was depressed with an oxygen concentration greater than 0.3 atm.ConclusionsThe methanotroph responses differentially to the increase in the oxygen partial pressure with regard to PHB accumulation either in the presence or in the absence of N2.

Physiological characterization of poly-β-hydroxybutyrate accumulation in the moderately thermophilic hydrogen-oxidizing bacterium Hydrogenophilus thermoluteolus TH-1

Hydrogenophilus thermoluteolus strain TH-1 is a thermophilic hydrogen-oxidizing microorganism that has the highest growth rate among autotrophs. Genomic analysis revealed that this strain comprises the complete gene set for poly-β-hydroxybutyrate (PHB) synthesis, i.e., three copies of acetyl-CoA acetyltransferase and polyhydroxyalkanoate synthase and one copy of acetoacetyl-CoA reductase and 3-hydroxyacyl-CoA dehydrogenase/3-hydroxybutyryl-CoA epimerase. An investigation on PHB accumulation in strain TH-1 demonstrated that PHB accumulation was induced by nitrogen limitation under autotrophic as well as heterotrophic conditions. This strain accumulated up to 430.4±14.3mgL-1 PHB during a 3-h incubation under nitrogen-limited heterotrophic conditions. The highest PHB accumulation rates under autotrophic and heterotrophic conditions were 38.6% (w/w) of the dry cells after a 6-h induction and 53.8% after 3h, respectively. Although PHB granules started to accumulate after 15min of nitrogen limitation under heterotrophic conditions, a drastic decrease of PHB was observed after 9h of induction.

Poly-β-hydroxybutyrate (PHB)-accumulating Halomonas improves the survival, growth, robustness and modifies the gut microbial composition of Litopenaeus vannamei postlarvae

The PHB-accumulating Halomonas strain was isolated from local solar saltworks and was cultured in CM culture medium with addition of 30 g L−1 glucose to favor the PHB accumulation in the cells. The effect of Halomonas-PHB on the survival, growth, osmotic stress tolerance, resistance against Vibrio anguillarum challenge and the gut microbial composition of Litopenaeus vannamei postlarvae were investigated. The postlarvae PL6 were fed Artemia nauplii enriched with Halomonas-PHB (HM.PHB), Halomonas without PHB accumulation (HM), commercial PHB particles (par.PHB) and non-enriched Artemia nauplii (Control), respectively, for two weeks. The results revealed that both Halomonas-PHB and particle PHB could improve the survival, growth, and enhance the robustness of the shrimp PLs under both challenge and non-challenged conditions. High throughput sequencing on gut microbiota indicated that Halomonas-PHB not only suppressed the propagation of Vibrio, but also diverged to gut microbiota towards to the probiotic bacteria propagation (i.e. Bacillus and Lactobacillus) in the gut. In conclusion, the Halomonas-PHB is more efficient than particle PHB in L. vannamei larviculture and thus can be applied as feed additive in shrimp culture.

Predicting the accumulation of storage compounds by Rhodococcus jostii RHA1 in the feast-famine growth cycles using genome-scale flux balance analysis.

Feast-famine cycles in biological wastewater resource recovery systems select for bacterial species that accumulate intracellular storage compounds such as poly-β-hydroxybutyrate (PHB), glycogen, and triacylglycerols (TAG). These species survive better the famine phase and resume rapid substrate uptake at the beginning of the feast phase faster than microorganisms unable to accumulate storage. However, ecophysiological conditions favouring the accumulation of either storage compounds remain to be clarified, and predictive capabilities need to be developed to eventually rationally design reactors producing these compounds. Using a genome-scale metabolic modelling approach, the storage metabolism of Rhodococcus jostii RHA1 was investigated for steady-state feast-famine cycles on glucose and acetate as the sole carbon sources. R. jostii RHA1 is capable of accumulating the three storage compounds (PHB, TAG, and glycogen) simultaneously. According to the experimental observations, when glucose was the substrate, feast phase chemical oxygen demand (COD) accumulation was similar for the three storage compounds; when acetate was the substrate, however, PHB accumulation was 3 times higher than TAG accumulation and essentially no glycogen was accumulated. These results were simulated using the genome-scale metabolic model of R. jostii RHA1 (iMT1174) by means of flux balance analysis (FBA) to determine the objective functions capable of predicting these behaviours. Maximization of the growth rate was set as the main objective function, while minimization of total reaction fluxes and minimization of metabolic adjustment (environmental MOMA) were considered as the sub-objective functions. The environmental MOMA sub-objective performed better than the minimization of total reaction fluxes sub-objective function at predicting the mixture of storage compounds accumulated. Additional experiments with 13C-labelled bicarbonate (HCO3−) found that the fluxes through the central metabolism reactions during the feast phases were similar to the ones during the famine phases on acetate due to similarity in the carbon sources in the feast and famine phases (i.e., acetate and poly-β-hydroxybutyrate, respectively); this suggests that the environmental MOMA sub-objective function could be used to analyze successive environmental conditions such as the feast and famine cycles while the metabolically similar carbon sources are taken up by microorganisms.

Poly-β-hydroxybutyrate (PHB) production by Synechocystis PCC6803 from CO2: Model development

The biosynthesis of poly-β-hydroxybutyrate (PHB) by bioconversion of CO2 is a sustainable alternative to the non-renewable, petroleum-based polymer production. Indeed, the PHB production by conversion of CO2 contributes to the reduction of the greenhouse-gas concentration in the atmosphere. A kinetic dynamic model of PHB production by autotrophic cultures of Synechocystis PCC6803 was proposed and developed by means of the biochemical networks simulator COPASI.Two classes of cells were assumed to be present in the broth: growing cells, PHB producing cells. The model included the two classes of cells and their nitrogen and phosphate internal quota. The dynamics of the cell growth and PHB production were described taking into account: cellular growth rate; lysis rate; nitrate and phosphate utilization rate; PHB production rate. The assessment of the kinetic parameters and of the yields (model calibration) was carried out by the regression of experimental data. Tests were carried out in photobioreactors under dynamic light system (light/dark cycle) using media characterized by initial nitrate concentration ranging between 0 and 1.5g/L. The developed model was validated with respect to independent experimental set.The proposed model successfully reproduced the experimental data (cell concentration, nitrogen and phosphate concentration and PHB content): the square correlation coefficient of the investigated variable concentrations ranged between 0.81 and 0.99. Parameter sensitivity analysis was carried out to assess the role of the selected parameters on cell growth and PHB accumulation. The dynamics of cellular growth were not significantly affected by a ±20% variation of maximum specific growth rate, of velocity of conversion to PHB producing cells, and of maximum uptake rate of nitrate. The PHB accumulation dynamics were particularly sensitive to the variation of the value of the investigated parameters.The proposed model may support the design and the optimization of a PHB production process by means of autotrophic cultures.

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.

Photosynthetic poly-\u03b2-hydroxybutyrate accumulation in unicellular cyanobacterium Synechocystis sp. PCC 6714

Poly-β-hydroxybutyrate (PHB) production from CO2 has the potential to reduce the production cost of this biodegradable polyesters, and also to make the material more sustainable compared to utilization of sugar feedstocks. In this study the unicellular cyanobacterium, Synechocystis sp. PCC 6714 has been identified as an unexplored potential organism for production of PHB. Synechocystis sp. PCC 6714 was studied under various cultivation conditions and nutritional limitations. Combined effects of nitrogen and phosphorus deficiency led to highest PHB accumulation under photoautotrophic conditions. Multivariate experimental design and quantitative bioprocess development methodologies were used to identify the key cultivation parameters for PHB accumulation. Biomass growth and PHB accumulation were studied under controlled defined conditions in a lab-scale photobioreactor. Specific growth rates were fourfold higher in photobioreactor experiments when cultivation conditions were controlled. After 14 days of cultivation in nitrogen and phosphorus, limited media intracellular PHB levels reached up to 16.4% from CO2. The highest volumetric production rate of PHB was 59 ± 6 mg L−1 day−1. Scanning electron microscopy of isolated PHB granules of Synechocystis sp. PCC 6714 cultivated under nitrogen and phosphorus limitations showed an average diameter of 0.7 µm. The results of this study might contribute towards a better understanding of photoautotrophic PHB production from cyanobacteria.

Production of poly-β-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.

Maximizing the accumulation of Poly-β-hydroxybutyrate (PHB) in low-carbon urban sewage

This study investigated the influence of different factors in maximizing poly-β-hydroxybutyrate (PHB) accumulation in low-carbon urban sewage. The effects of chemical oxygen demand and total phosphorus (TP) concentrations on PHB accumulation were analyzed using an anaerobic/oxygen sequencing batch reactor. Both the anaerobic phase and the aeration phase were studied. The results showed that maximum PHB accumulations increased as the carbon source increased, growing from 5.45 to 27.51 mg/g, when the carbon source concentration increased from 140 to 400 mg/L, at the end of anaerobic phase. Maximum PHB accumulation decreased from 15.89 to 13.88 mg/g when exposed to influent carbon–phosphorus C/P, suggesting that carbon source is more influential and beneficial for PHB accumulation than TP. With an increase in pre-aeration time from 10 to 50 min, PHB accumulation rose from 21.12 to 31.96 mg/g; this is higher than 15.54 mg/g PHB accumulation without pre-aeration. When the pre-aeration time was fixed at 15 min, but with different pre-aeration fluxes ranging from 20 to 50 L/h, PHB accumulation increased from 23.79 to 30.09 mg/g. These results demonstrate that pre-aeration treatment facilitates PHB accumulation.

The effect of anaerobic-aerobic and feast-famine cultivation pattern on bacterial diversity during poly-β-hydroxybutyrate production from domestic sewage sludge.

The main objective of this work was to investigate the influence of different oxygen supply patterns on poly-β-hydroxybutyrate (PHB) yield and bacterial community diversity. The anaerobic-aerobic (A/O) sequencing batch reactors (SBR1) and feast-famine (F/F) SBR2 were used to cultivate activated sludge to produce PHB. The mixed microbial communities were collected and analyzed after 3months cultivation. The PHB maximum yield was 64wt% in SBR1 and 53wt% in SBR2. Pyrosequencing analysis 16S rRNA gene of two microbial communities indicated there were nine and four bacterial phyla in SBR1 and SBR2, respectively. Specifically, Proteobacteria (36.4% of the total bacterial community), Actinobacteria (19.7%), Acidobacteria (14.1%), Firmicutes (4.4%), Bacteroidetes (1.7%), Cyanobacteria/Chloroplast (1.5%), TM7 (0.8%), Gemmatimonadetes (0.2%), and Nitrospirae (0.1%) were present in SBR1. Proteobacteria (94.2%), Bacteroidetes (2.9%), Firmicutes (1.9%), and Actinobacteria (0.7%) were present in SBR2. Our results indicated the SBR1 fermentation system was more stable than that of SBR2 for PHB accumulation.

Impact of Various Stress Conditions on Poly-β-Hydroxybutyrate (PHB) Accumulation in Aulosira fertilissima CCC 444

Background: Poly-β-hydroxybutyrate (PHB) from cyanobacteria is considered to be a potentially viable source for degradable plastic. Yet, to date, only a modest level of research has been reported in this area. PHB has variety of properties such as natural origin, biocompatibility, biodegradability, piezoelectricity, streospecificity, thermoplasticity and optical activity, which make it suitable for various applications in health industry. Methods: This article elucidates comprehensive information and knowledge with regard to several stress conditions (e.g., salt stress, heat and chilling stresses, heavy metal supplementation, limitation of gas exchange and nutrient deficiency) to enhance PHB production from a diazotrophic cyanobacterium, Aulosira fertilissima CCC 444. Results: The results showed that cultures reached a maximum biomass concentration of 631 mg l-1, regardless of initial inoculum levels. However, with an initial inoculum of 80 mg dry cell weight (dcw) l-1, maximum PHB accumulation was achieved under regular light-dark cycles. A. fertilissima supplemented with heavy metals showed enhancement in PHB accumulation up to 18.7% of dcw in 1.0 mg l-1 Ni-supplemented medium for 7 days, followed by 17.6% (dcw) in 1.5 mg l-1 Cu-supplemented medium. NaCl stress, heat and chilling stresses and deficiencies of potassium, calcium, and magnesium had negative effects on PHB accumulation. Interestingly, supplementation of 0.5% acetate at the initiation of gas exchange limitation boosted the accumulation of PHB to 48.7% (dcw) on 14 days of incubation, a value 7-fold higher compared to the control. Conclusion: Gas exchange limitation with carbon supplementation found to be an appropriate condition for enhanced PHB accumulation in cyanobacteria. Thus, using cyanobacteria for PHB production is one of the most promising ecofriendly ways as these are oxygen evolving photoautotrophic organisms; require minimal inorganic nutrients for growth and due to aquatic in nature, cyanobacteria do not compete for arable land for their cultivation. Currently, intensive research is essential in this area for ingenious utilization of cyanobacterial biomass to produce environmentally affordable bioplastic. Keywords: Aulosira fertilissima, gas exchange limitation, heavy metals, poly-β-hydroxybutyrate.

Production, optimization and characterization of polyhydroxybutyrate, a biodegradable plastic by Bacillus spp.

Poly-β-hydroxybutyrate (PHB) is the intracellular lipid reserve accumulated by many bacteria. The most potent terrestrial bacterium Bacillus cereus SE-1 showed more PHB accumulating cells (22.1 and 40% after 48 and 72 h) than that of the marine Bacillus sp. CS-605 (5 and 33% after 48 and 72 h). Both the isolates harbored phbB gene and the characteristics C=O peak was observed in the extracted PHB by Fourier transformed infrared spectroscopy analysis. Maltose was found to be the most suitable carbon source for the accumulation of PHB in B. cereus SE-1. The extracted PHB sample from B. cereus SE-1 was blended with a thermoplastic starch (TS) and an increased thermoplasticity and decreased crystallinity were observed after blending in comparison to the standard PHB. The melting temperature (Tm), melting enthalpy (∆Hf), and crystallinity (Xc) of the blended PHB sample were found to be 109.4 °C, 64.58 J/g, and 44.23%, respectively.

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.

Biosynthesis of poly-β-hydroxybutyrate (PHB) with a high molecular mass by a mutant strain of Azotobacter vinelandii (OPN)

The aim of this study was to characterize the influence of the aeration conditions on the production of PHB and its molecular mass in a mutant strain of Azotobacter vinelandii (OPN), which carries a mutation on ptsN, the gene encoding enzyme IIANtr, previously shown to increase the accumulation of PHB. Cultures of A. vinelandii wild-type strain OP and its mutant derivative strain OPN were grown in 500-mL flasks, containing 100 and 200 mL of PY sucrose medium. PHB production and its molecular mass were analyzed at the end of the culture. The molecular mass (MM) was significantly influenced by the aeration conditions and strain used. A polymer with a higher molecular weight was produced under low aeration conditions for both strains. A maximal molecular mass of 2,026 kDa (equivalent to 3,670 kDa measured by GPC) was obtained with strain OPN cultured under low-aeration conditions, reaching a value two-fold higher than that obtained from the parental strain OP (MM = 1,013 kDa) grown under the same conditions. Aeration conditions and the ptsN mutation influence the molecular mass of the PHB produced by A. vinelandii affecting in turn its physico-chemical properties.