Poly-β-hydroxybutyrate Content Research Articles

Poly-β-hydroxybutyrate production by Synechocystis MT_a24 in a raceway pond using urban wastewater.

Poly-β-hydroxybutyrate (PHB) is a potential source of biodegradable plastics that are environmentally friendly due to their complete degradation to water and carbon dioxide. This study aimed to investigate PHB production in the cyanobacterium Synechocystis sp. PCC6714 MT_a24 in an outdoor bioreactor using urban wastewater as a sole nutrient source. The culture was grown in a thin-layer raceway pond with a working volume of 100 L, reaching a biomass density of up to 3.5g L-1 of cell dry weight (CDW). The maximum PHB content was found under nutrient-limiting conditions in the late stationary phase, reaching 23.7 ± 2.2% PHB per CDW. These data are one of the highest reported for photosynthetic production of PHB by cyanobacteria, moreover using urban wastewater in pilot-scale cultivation which multiplies the potential of sustainable cultivation approaches. Contamination by grazers (Poterioochromonas malhamensis) was managed by culturing Synechocystis in a highly alkaline environment (pH about 10.5) which did not significantly affect the culture growth. Furthermore, the strain MT_a24 showed significant wastewater nutrient remediation removing about 72% of nitrogen and 67% of phosphorus. These trials demonstrate that the photosynthetic production of PHB by Synechocystis sp. PCC6714 MT_a24 in the outdoor thin-layer bioreactor using urban wastewater and ambient carbon dioxide. It shows a promising approach for the cost-effective and sustainable production of biodegradable carbon-negative plastics. KEY POINTS: • High PHB production by cyanobacteria in outdoor raceway pond • Urban wastewater used as a sole source of nutrients for phototrophic growth • Potential for cost-effective and sustainable production of biodegradable plastics.

Co-utilization of glucose and xylose for the production of poly-β-hydroxybutyrate (PHB) by Sphingomonas sanxanigenens NX02

BackgroundPoly-β-hydroxybutyrate (PHB), produced by a variety of microbial organisms, is a good substitute for petrochemically derived plastics due to its excellent properties such as biocompatibility and biodegradability. The high cost of PHB production is a huge barrier for application and popularization of such bioplastics. Thus, the reduction of the cost is of great interest. Using low-cost substrates for PHB production is an efficient and feasible means to reduce manufacturing costs, and the construction of microbial cell factories is also a potential way to reduce the cost.ResultsIn this study, an engineered Sphingomonas sanxanigenens strain to produce PHB by blocking the biosynthetic pathway of exopolysaccharide was constructed, and the resulting strain was named NXdE. NXdE could produce 9.24 ± 0.11 g/L PHB with a content of 84.0% cell dry weight (CDW) using glucose as a sole carbon source, which was significantly increased by 76.3% compared with the original strain NX02. Subsequently, the PHB yield of NXdE under the co-substrate with different proportions of glucose and xylose was also investigated, and results showed that the addition of xylose would reduce the PHB production. Hence, the Dahms pathway, which directly converted D-xylose into pyruvate in four sequential enzymatic steps, was enhanced by overexpressing the genes xylB, xylC, and kdpgA encoding xylose dehydrogenase, gluconolactonase, and aldolase in different combinations. The final strain NX02 (ΔssB, pBTxylBxylCkdpgA) (named NXdE II) could successfully co-utilize glucose and xylose from corn straw total hydrolysate (CSTH) to produce 21.49 ± 0.67 g/L PHB with a content of 91.2% CDW, representing a 4.10-fold increase compared to the original strain NX02.ConclusionThe engineered strain NXdE II could co-utilize glucose and xylose from corn straw hydrolysate, and had a significant increase not only in cell growth but also in PHB yield and content. This work provided a new host strain and strategy for utilization of lignocellulosic biomass such as corn straw to produce intracellular products like PHB.

Modeling of poly-β-hydroxybutyrate production by Bacillus subtilis and its use for feed-forward bioreactor studies.

Successful scale-up of Bacillus subtilis culture for poly-β-hydroxybutyrate (PHB) production was performed in 5-l stirred-tank reactor using batch, fed-batch, and two-stage culture strategies. The kinetics of biomass production, substrate consumption, and PHB production were established in the stirred tank bioreactor in all the studies. A mathematical model was developed to investigate the role of limiting substrate on overall culture metabolism. A fed-batch strategy was predicted on the basis of computer simulations, for maximum PHB production. This was performed by extrapolation of batch model for predicting the feeding rate and suitable time of feeding. Substrate inhibition was studied and the substrate inhibition terms were incorporated in the model. The maximum cell biomass concentration in batch culture (24h) and fed-batch culture (30h) was 1.79 ± 0.03g/l on dry cell weight (DCW) basis and 1.66 ± 0.050g/l on DCW basis and the corresponding PHB content was 68.71% and 85.54% of DCW, respectively. Glucose was found to be the major limiting nutrient during the bioreactor culture. A two-stage culture, where cells were first grown in stage I in LBG media containing excess carbon and thereafter in stage II in OM media, showed biomass production of 1.95 ± 0.045g/l at 4h and PHB production of 93.33% of DCW at 16h. A 9% increase in growth and 25% increase in PHB yield were obtained using two-stage culture with computer-simulated feeding strategy in the 5l reactor. Oxygen limitation was overcome in modified two-stage culture to obtain a PHB production of 98% at 30h. KEY POINTS: • Polyhydroxybutyrate production was studied in a 5-l stirred-tank bioreactor using HPLC • Mathematical model-assisted fed-batch strategy was implemented in bioreactor • Two-stage fed-batch cultivation was implemented and PHB production was 93% of dry weight in Gram-positive bacteria.

Inactivation of phosphate regulator (SphU) in cyanobacterium Synechocystis sp. 6803 directly induced acetyl phosphate pathway leading to enhanced PHB level under nitrogen-sufficient condition

Poly-β-hydroxybutyrate (PHB) is a biodegradable and biocompatible polymer that has potential in the fields of environmental, agricultural, and biomedical sciences. Cyanobacteria are considered an excellent source of PHB by bioconversion of CO2. This study aimed to prolong PHB production under nitrogen-sufficient condition in the model cyanobacterium Synechocystis sp. PCC 6803. Interestingly, the lack of phosphate regulator (SphU) enabled the mutant strain (ΔSphU) to have the ability to accumulate phosphate with higher expression of Pho regulon. When strain ΔSphU was cultured in nitrogen complete medium for 14 days, the PHB granules were more extensively accumulated in the ΔSphU strain than in the wild type. Photosynthesis activity slightly increased in ΔSphU strain, with no significant difference in chlorophyll a content between wild-type and ΔSphU strain in nitrogen-containing medium, indicating that the higher PHB content (14.57% (w/w) cell dry weight) was not influent of chlorosis. The RT-qPCR analysis revealed that genes involved in PHB biosynthesis and acetyl phosphate pathway were more upregulated in ΔSphU strain. Moreover, the level of acetate production in ΔSphU cells was higher than that in the wild type, suggesting that the deletion of the phosphate regulator could directly induce PHB metabolism by activation of the acetyl phosphate pathway. This research provides better understanding of PHB production regulation in cyanobacteria which are a promising hosts for industrial production of biodegradable plastics.

Statistical Optimization of Poly-β-Hydroxybutyrate Biosynthesis Using the Spent Mushroom Substrate by Bacillus tequilensis PSR-2

Poly-β-hydroxybutyrate (PHB) belonging to the polyhydroxyalkanoates family is a natural polyester used as a biodegradable plastic for various commercial applications. In this study, soil samples from the vegetable oil processing industry were used to screen for PHB-producing bacteria using Sudan black B staining. Among the isolated bacteria, PHB positive PSR-2 isolate was chosen as a potent PHB producer. The phylogenetic tree revealed that the PSR-2 isolate has a high 16S rRNA gene sequence similarity of 99.9% with Bacillus tequilensis. The PHB content of 2.8 ± 0.09 g/L was produced by PSR-2 isolate in 48 h in a nutrient broth medium containing 1% glucose compared to the PHB production of 1.6 ± 0.08% by the reference strain, Bacillus circulans. Taguchi method was used to optimize PHB production using the alkali-pretreated spent mushroom substrate of sugarcane bagasse (SMS-SB) as an additional carbon substrate along with other energy sources. The optimized factors in the contribution of PHB production from the highest- to the lowest-ranking are as follows: alkali-pretreated SMS-SB, glucose, glycerol, peptone, ammonium chloride, and potassium dihydrogen phosphate at 30 oC, pH 7.0, which resulted in the production of 12.4 ± 0.95 g/L PHB was higher than the predicted value of 11.59 g/L. The synthesized PHB was characterized using Ultraviolet–visible spectrophotometry, Fourier transform infrared spectrometry, differential scanning calorimetry, thermogravimetric analysis, nuclear magnetic resonance spectroscopy, and gas chromatography-mass spectrometry. The results revealed the presence of hydroxyl (–OH), methyl (–CH3), methine (=CH–), methylene (–CH2–) and ester carbonyl (>C=O) groups, which confirmed the PHB structure. Thus, alkali-pretreated SMS-SB plays a significant role as an energy substrate for the production of PHB. This gives the knowledge to utilize cost-effective lignocellulosic agro-waste materials as a feedstock for the sustainable production of biodegradable PHB for many biomedical applications.

Phenotypic dynamics in polyphosphate and glycogen accumulating organisms in response to varying influent C/P ratios in EBPR systems

This study employed molecular tools and single cell Raman micro-spectroscopy techniques to reveal the single cell- and population-level phenotypic dynamics and changes in functionally relevant organisms, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), in response to influent loading readily biodegradable carbon to phosphorus ratio (C/P) changes in enhanced biological phosphorus removal (EBPR) systems. The results, for the first time, provided direct and cellular evidence confirming the adaptive anaerobic metabolic pathway shifts in PAOs in response to influent loading variations. Increase in influent readily biodegradable carbon to phosphorus (C/P) ratio from 20 to 50 led to nearly 50% decline in polyphosphate content and drastic rise of intracellular polyβhydroxybutyrate (PHB) to polyphosphate (polyP) ratio by nearly 6 times in PAOs, indicating corresponding diminishing reliance on polyP hydrolysis for energy as P becomes limiting. Influent carbon availability surge also impacted the intracellular carbon polymers in GAOs, with significant increase in the mean PHB content level but no observed changes in the intracellular glycogen level. Furthermore, the Raman-based quantification of differentiated intracellular polymer content associated with PAOs and GAOs, revealed new insights into the quantitative shift in intracellular carbon storage distribution between the two populations and their variations between the two carbon polymers (PHB, Glycogen). In summary, this investigation revealed high-resolution cellular level information regarding the metabolic flexibility in PAOs, phenotypic stoichiometry changes and carbon flux and distribution among PAOs and GAOs, in response to influent loading conditions. The new information will contribute to improvement in mechanistic EBPR modeling and design.

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 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.

Pilot-scale production of poly-β-hydroxybutyrate with the cyanobacterium Synechocytis sp. CCALA192 in a non-sterile tubular photobioreactor

The biopolymer poly-β-hydroxybutyrate (PHB) can be used as a promising bioplastic. It has a broad range of applications and is degraded relatively rapidly by soil organisms. Like many prokaryotes, the cyanobacterium Synechocystis sp. CCALA192 produces this biopolymer as a storage compound, especially under nutrient limitation.In a 200-L tubular photobioreactor, we cultivated Synechocystis sp. CCALA192 semi-continuously over a period of 75 days with CO2 as sole carbon source. A two-stage cultivation strategy was performed, where after 5–7 days nitrogen was depleted and the culture started to produce PHB and gradually turned from blue-green to yellow. After 16–20 days, 90% of the culture were harvested and the residual 10% were used as inoculum for the following cycle. The harvested culture had an average biomass concentration of 1.0 g/L with an average PHB content of 12.5% of cell dry weight. After restarting with fresh nutrients, the yellow culture turned blue-green again and degraded the PHB within 24–48 h. When nitrogen of the medium was consumed, PHB was produced again and the cycle continued. In the late stage of each production cycle, a ripening process was observed, where no CO2 was consumed but the PHB concentration was still rising at the expense of the existing glycogen rich biomass.Establishing a stable Synechocystis sp. CCALA192 culture under non-sterile conditions turned out to be difficult, as this small unicellular organism is very sensitive and easily grazed by protozoa. Therefore, a special cultivation strategy with partially anoxic conditions was necessary.

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.

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.

Effect of light-dark cycles on hydrogen and poly-β-hydroxybutyrate production by a photoheterotrophic culture and Rhodobacter capsulatus using a dark fermentation effluent as substrate

A Rhodobacter capsulatus strain and a photoheterotrophic culture (IZT) were cultivated to produce hydrogen under different light-dark cycles. A dark fermentation effluent (DFE) was used as substrate. It was found that IZT culture had an average cumulative hydrogen production (Paccum H2) of 1300±43mLH2L−1 under continuous illumination and light-dark cycles of 30 or 60min. In contrast, R. capsulatus reduced its Paccum H2 by 20% under 30:30min light-dark cycles, but tripled its poly-β-hydroxybutyrate (PHB) content (308±2mgPHB gdw−1) compared to continuous illumination. The highest PHB content by IZT culture was 178±10mgPHB gdw−1 under 15:15min light-dark cycles. PCR-DGGE analysis revealed that the IZT culture was mainly composed of Rhodopseudomonas palustris identified with high nucleotide similarity (99%). The evaluated cultures might be used for hydrogen and PHB production. They might provide energy savings by using light-dark cycles and DFE valorization.

Agroindustrial residues and energy crops for the production of hydrogen and poly-β-hydroxybutyrate via photofermentation

The present study was aimed at assessing the biotransformation of dark fermented agroindustrial residues and energy crops for the production of hydrogen and poly-β-hydroxybutyrate (PHB), in lab-scale photofermentation. The investigation on novel substrates for photofermentation is needed in order to enlarge the range of sustainable feedstocks. Dark fermentation effluents of ensiled maize, ensiled giant reed, ensiled olive pomace, and wheat bran were inoculated with Rhodopseudomonas palustris CGA676, a mutant strain suitable for hydrogen production in ammonium-rich media. The highest hydrogen producing performances were observed in wheat bran and maize effluents (648.6 and 320.3mLL−1, respectively), both characterized by high initial volatile fatty acids (VFAs) concentrations. Giant reed and olive pomace effluents led to poor hydrogen production due to low initial VFAs concentrations, as the original substrates are rich in fiber. The highest PHB content was accumulated in olive pomace effluent (11.53%TS), ascribable to magnesium deficiency.

Coupled effects of methane monooxygenase and nitrogen source on growth and poly-β-hydroxybutyrate (PHB) production of Methylosinus trichosporium OB3b

The coupled effects of nitrogen source and methane monooxygenase (MMO) on the growth and poly-β-hydroxybutyrate (PHB) accumulation capacity of methanotrophs were explored. The ammonia-supplied methanotrophs expressing soluble MMO (sMMO) grew at the highest rate, while N2-fixing bacteria expressing particulate MMO (pMMO) grew at the lowest rate. Further study showed that more hydroxylamine and nitrite was formed by ammonia-supplied bacteria containing pMMO, which might cause their slightly lower growth rate. The highest PHB content (51.0%) was obtained under nitrogen-limiting conditions with the inoculation of nitrate-supplied bacteria containing pMMO. Ammonia-supplied bacteria also accumulated a higher content of PHB (45.2%) with the expression of pMMO, while N2-fixing bacteria containing pMMO only showed low PHB production capacity (32.1%). The maximal PHB contents of bacteria expressing sMMO were low, with no significant change under different nitrogen source conditions. The low MMO activity, low cell growth rate and low PHB production capacity of methanotrophs continuously cultivated with N2 with the expression of pMMO were greatly improved in the cyclic NO3−N2 cultivation regime, indicating that long-term deficiency of nitrogen sources was detrimental to the activity of methanotrophs expressing pMMO.

Enhancing photo-fermentative hydrogen production performance of Rhodobacter capsulatus by disrupting methylmalonate-semialdehyde dehydrogenase gene

Abstract The effects of mmsA disruption in Rhodobacter capsulatus SB1003 on the pigment content, poly-β-hydroxybutyrate (PHB) content and photo-fermentative hydrogen production were studied. A transposon mutant assigned ZY29 with mutation on mmsA and an mmsA partially deleted mutant assigned ZYDM9 ( mmsA − ) were obtained. The mutants showed reduced pigment and PHB content, and improved photo-fermentative hydrogen production performance. The hydrogen yield and maximum hydrogen production rate of ZYDM9 were 4675 ± 76 mL/L and 92.4 ± 2.6 mL/(Lh), which increased by 22.8% and 20% compared with that of the WT, respectively. In the meantime, its pigment and PHB content were 20.7 ± 0.1 nmol/mg and 54.5 ± 2.6 mg/g-dcw, which reduced by 34.2% and 43.4% compared with that of WT, respectively. The optimum parameters for hydrogen production were initial pH 7.0, carbon nitrogen ratio (g/g) 3.0:0.5, and light intensity 800 ± 35 W/m 2 .

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.

Poly-β-hydroxybutyrate production and management of cardboard industry effluent by new Bacillus sp. NA10

Abstract Background In the present study, we aim to utilize the ecological diversity of soil for the isolation and screening for poly β-hydroxybutyrate (PHB)-accumulating bacteria and production of cost-effective bioplastic using cardboard industry effluent. Results A total of 120 isolates were isolated from different soil samples and a total of 62 isolates showed positive results with Nile blue A staining, a specific dye for PHB granules and 27 isolates produced PHB using cardboard industry effluent. The selected isolate NA10 was identified as Bacillus sp. NA10 by studying its morphological, biochemical, and molecular characteristics. The growth pattern for the microorganism was studied by logistic model and exactly fitted in the model. A maximum cell dry weight (CDW) of 7.8 g l−1 with a PHB concentration of 5.202 g l−1 was obtained when batch cultivation was conducted at 37°C for 72 h, and the PHB content was up to 66.6% and productivity was 0.072 g l−1 h−1 in 2.0 L fermentor. Chemical characterization of the extracted PHB was done by H1NMR, Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), Gas chromatography–mass spectrometry (GC-MS) analysis to determine the structure, melting point, and molecular mass of the purified PHB. The polymer sheet of extracted polymer was prepared by blending the polymer with starch for packaging applications. Conclusions The isolate NA10 can be a good candidate for industrial production of PHB from cardboard industry waste water cost-effectively and ecofriendly.

Relationship between cell growth, hydrogen production and poly-β-hydroxybutyrate (PHB) accumulation by Rhodopseudomonas palustris WP3-5

Rhodopseudomonas palustris WP3-5 accumulated PHB in growth phase, and PHB could be utilized as another carbon and energy source when its content reached maximum value. Microorganism ceased growing when nitrogen source was exhausted, and used excessive energy to produce more hydrogen in the stationary phase. Rps. palustris WP3-5 could not synthesize PHB under low concentration of carbon source because substrate was degraded rapidly, and there were no difference of hydrogen production between wild-type strain and its PHB synthase-deficient mutant Rps. palustris M23. When concentration of carbon source was three times higher, cumulated hydrogen volume and substrate conversion efficiency of Rps. palustris WP3-5 were better than Rps. palustris M23. This result coincided with other experiments in different culture conditions. But when using malate as carbon source, Rps. palustris WP3-5 had lower substrate degrading rate and cumulated hydrogen volume, and PHB was not accumulated. Rps. palustris WP3-5 used most energy to produce hydrogen in the stationary phase and PHB content was below 10% cell dry weight, accounting for less than 5% of the substrate electrons utilized. This portion of energy might partially be redistributed to synthesize soluble microbial product (SMP). Therefore, the competition relationship between hydrogen production and PHB accumulation was insignificant.

Production and characterization of poly-β-hydroxybutyrate (PHB) polymer from Aulosira fertilissima

Biopolymers such as polyhydroxyalkanoates (PHAs) are a class of secondary metabolites with promising importance in the field of environmental, agricultural, and biomedical sciences. To date, high-cost commercial production of PHAs is being carried out with heterotrophic bacterial species. In this study, a photoautotrophic N2-fixing cyanobacterium, Aulosira fertilissima, has been identified as a potential source for the production of poly-β-hydroxybutyrate (PHB). An accumulation up to 66% dry cell weight (dcw) was recorded when the cyanobacterium was cultured in acetate (0.3%) + citrate (0.3%)-supplemented medium against 6% control. Aulosira culture supplemented with 0.5% citrate under P deficiency followed by 5 days of dark incubation also depicted a PHB accumulation of 51% (dcw). PHB content of A. fertilissima reached up to 77% (dcw) under P deficiency with 0.5% acetate supplementation. Optimization of process parameters by response surface methodology resulted into polymer accumulation up to 85% (dcw) at 0.26% citrate, 0.28% acetate, and 5.58 mg L−1 K2HPO4 for an incubation period of 5 days. In the A. fertilissima cultures pre-grown in fructose (1.0%)-supplemented BG 11 medium, when subjected to the optimized condition, the PHB pool boosted up to 1.59 g L−1, a value ∼50-fold higher than the control. A. fertilissima is the first cyanobacterium where PHB accumulation reached up to 85% (dcw) by manipulating the nutrient status of the culture medium. The polymer extracted from A. fertilissima exhibited comparable material properties with the commercial polymer. As compared with heterotrophic bacteria, carbon requirement in A. fertilissima for PHB production is lower by one order magnitude; thus, low-cost PHB production can be envisaged.