Poly-β-hydroxybutyrate Production 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.

Artificial Soil-Like Material Enhances CO2 Bio-Valorization into Chemicals in Gas Fermentation.

Gas fermentation offers a carbon-neutral route for producing industrial feedstocks using autotrophic microbes to convert carbon dioxide (CO2) in waste gases, such as industrial emissions and biogas, into valuable chemicals or biofuels. However, slow microbial metabolism owing to low gaseous solubility causes significant challenges in gas fermentation. Although chemical or genetic manipulations have been explored to improve gas fermentation, they are either nonsustainable or complex. Herein, an artificial soil-like material (SLM) inspired by natural soil was fabricated to improve the growth and metabolism ofCupriavidus necatorfor enhanced poly-β-hydroxybutyrate (PHB) biosynthesis from CO2 and hydrogen (H2). Porous SLM comprises low-cost nanoclay, boehmite, and starch and serves as a biocarrier to facilitate the colonization of bacteria and delivery of CO2 to bacteria. With 3.0 g/L SLM addition, the solubility of CO2 in water increased by ∼4 times and biomass and PHB production boosted by 29 and 102%, respectively, in the 24 h culture. In addition, a positive modulation was observed in the metabolism of PHB biosynthesis. PHB biosynthesis-associated gene expression was found to be enhanced in response to the SLM addition. The concentrations of intermediates in the metabolic pathway of PHB biosynthesis, such as pyruvate and acetyl-CoA, as well as reducing energy (ATP and NADPH) significantly increased with SLM addition. SLM also demonstrated the merits of easy fabrication, high stability, recyclability, and plasticity, thereby indicating its considerable potential for large-scale application in gas fermentation.

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.

Light and oxygen facilitating the directly treatment food wastewater and poly-β-hydroxybutyrate, 5-aminolevulinic acid, pigment productions by Rubrivivax gelatinosus.

Rubrivivax gelatinosus has the advantage of using wastewater to realize biomass recovery. However, they still cannot be applied large scale because they cannot directly treat the wastewater containing macromolecular organics. Thus, this article investigated the effects of light-oxygen conditions on R. gelatinosus by directly recycling wastewater containing macromolecular organics to produce biomass, poly-β-hydroxybutyrate (PHB), 5-aminolevulinic acid (5-ALA), and pigment. Results showed that R. gelatinosus directly treated the macromolecule organic (soybean protein and starch) wastewaters and achieved biomass recovery under light-anaerobic and light-micro-oxygen in six conditions. Chemical oxygen demand, protein, and starch removals for two wastewaters all reached above 70%. Renewable bio-resources such as biomass, PHB, 5-ALA, and pigment production were 10 times the initial content. Theoretical analysis indicated that light activated the synthesis of protease and amylase. However, oxygen concentration decided the number of enzymes. When oxygen was at micro-oxygen or anaerobic, the aforementioned expression and synthesis were conducted. In summary, this study expanded the viewpoint ignored by traditional theory. It was realized that R. gelatinosus directly treated wastewater and accumulated nutrients (biomass, PHB, pigment, and 5-ALA) for recycling, which reduced the secondary pollution of excess sludge into the environment.

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.

Neural networks and genetic algorithm as robust optimization tools for modeling the microbial production of poly-β-hydroxybutyrate (PHB) from Brewers' spent grain.

This research work has been carried out to establish the combinatorial impact of various fermentation medium constituents, used for poly-β hydroxybutyrate (PHB) biosynthesis. Model development was performed with an optimized medium composition that enhanced the biosynthesis of PHB from the biowaste material Brewers' spent grain (BSG). The latter was used as a carbon substrate in submerged fermentation with Bacillus sphaericus NCIM 2478. Three independent variables: BSG, yeast extract (YE), and salt solution concentration (SS) and one dependent variable (amount of PHB produced) were assigned. A total of 35 microbial fermentation trials were conducted by which a nonlinear mathematical relationship was established in terms of neural network model between independent and dependent variables. The resulting artificial neural networks (ANNs) model for this process was further optimized using a global genetic algorithm optimization technique, which predicted the maximum production of PHB (916.31mg/L) at a concentration of BSG (50.12g/L), concentration of YE (0.22g/L), and concentration of SS (24.06%, v/v). The experimental value of the quantity of PHB (concentration ∼916mg/L) was found to be very close to the value predicted by the ANN-GA model approach.

Screening of Poly-β-hydroxybutyrate (PHB) Producing Bacteria and yeasts from locally vendored ‘Whey’ biowaste samples

Whey- a byproduct of the manufacture of cheese has several commercial uses. The present study was undertaken to explore the microbial load of the whey and the possible existence of biopolymer (PHB) producing microorganisms in this byproduct. Whey samples collected from different vendors in the locality of Mirpur, Dhaka were brought into laboratory and the microbial load was determined by serial dilution technique using nutrient agar and YEPD medium. From 30 colonies, 32 strains were preliminarily isolated. Out of 32 isolates, fifteen exhibited pink fluorescence on mineral medium, indicating the presence of PHB production inside cells. Among these, seven strains were identified as members of the genera Candida tropicalis; based on molecular characterization. The remaining 8 isolates were bacteria. It is interesting to note that, yeast isolates were obtained from locally vendored samples whereas bacterial strains were predominant in commercial samples. This work can be considered as the first step in the development of an ecofriendly bio-refinery pathway for cheese industry.

An Eco-Friendly Conversion of Aquaculture Suspended Solid Wastes Into High-Quality Fish Food by Improving Poly-β-Hydroxybutyrate Production

The aquaculture industry is vital in providing a valuable protein food source for humans, but generates a huge amount of solid and dissolved wastes that pose great risks to the environment and aquaculture sustainability. Suspended solids (in short SS), one of the aquaculture wastes, are very difficult to be treated due to their high organic contents. The bioconversion from wastewater, food effluents, and activated sludge into poly-β-hydroxybutyrate (PHB) is a sustainable alternative to generate an additional income and could be highly attractive to the agricultural and environmental management firms. However, little is known about its potential application in aquaculture wastes. In the present study, we first determined that 7.2% of SS was PHB. Then, the production of PHB was increased two-fold by the optimal fermentation conditions of wheat bran and microbial cocktails at a C/N ratio of 12. Also, the PHB-enriched SS showed a higher total ammonia nitrogen removal rate. Importantly, we further demonstrated that the PHB-enriched SS as a feed could promote fish growth and up-regulate the expression of the immune-related genes. Our study developed an eco-friendly and simple approach to transforming problematic SS wastes into PHB-enriched high-quality food for omnivorous fish, which will increase the usage efficiency of SS and provide a cheaper diet for aquatic animals.

Two-Stage Polyhydroxyalkanoates (PHA) Production from Cheese Whey Using Acetobacter pasteurianus C1 and Bacillus sp. CYR1.

Cheese whey (CW) can be an excellent carbon source for polyhydroxyalkanoates (PHA)-producing bacteria. Most studies have used CW, which contains high amounts of lactose, however, there are no reports using raw CW, which has a relatively low amount of lactose. Therefore, in the present study, PHA production was evaluated in a two-stage process using the CW that contains low amounts of lactose. In first stage, the carbon source existing in CW was converted into acetic acid using the bacteria, Acetobacter pasteurianus C1, which was isolated from food waste. In the second stage, acetic acid produced in the first stage was converted into PHA using the bacteria, Bacillus sp. CYR-1. Under the condition of without the pretreatment of CW, acetic acid produced from CW was diluted at different folds and used for the production of PHA. Strain CYR-1 incubated with 10-fold diluted CW containing 5.7 g/L of acetic acid showed the higher PHA production (240.6 mg/L), whereas strain CYR-1 incubated with four-fold diluted CW containing 12.3 g/L of acetic acid showed 126 mg/L of PHA. After removing the excess protein present in CW, PHA production was further enhanced by 3.26 times (411 mg/L) at a four-fold dilution containing 11.3 g/L of acetic acid. Based on Fourier transform infrared spectroscopy (FT-IR), and 1H and 13C nuclear magnetic resonance (NMR) analyses, it was confirmed that the PHA produced from the two-stage process is poly-β-hydroxybutyrate (PHB). All bands appearing in the FT-IR spectrum and the chemical shifts of NMR nearly matched with those of standard PHB. Based on these studies, we concluded that a two-stage process using Acetobacter pasteurianus C1 and Bacillus sp. CYR-1 would be applicable for the production of PHB using CW containing a low amount of lactose.

Production of eco-friendly PHB-based bioplastics by Pseudomonas aeruginosa CWS2020 isolate using poultry (chicken feather) waste.

Nowadays, the accumulation of non-degradable plastics and other disposed wastes leads to environmental pollution across the world. The production of eco-friendly and cost-effective poly-β-hydroxybutyrate (PHB) could be a better alternative to conventional petroleum-based plastics and prevent environmental pollution. Besides, the area in and around Namakkal, Tamil Nadu, India is well known for poultries, currently facing the number of environmental issues due to the accumulation of chicken feather waste. This study focused on the production of eco-friendly PHB by recycling poultry (chicken feather) waste as the substrate. The native PHB producers were screened from the chicken waste disposal site in Namakkal by Sudan black B staining method. Further, the potent bacterial isolate was identified as Pseudomonas aeruginosa (NCBI accession MF18889) by phenotypic and genotypic characteristics. The PHB production media with chicken feather waste was statistically optimized by response surface methodology. The dry weight of PHB produced under optimized condition (15.96g/L chicken feather waste, 37°C temperature, 19.8g/L glucose and 6.85 pH) was found to be 4.8g/L. Besides, PHB was characterized and confirmed by thin-layer chromatography, Fourier-transform infrared spectroscopy and Gas chromatography-mass spectrometry analysis. Thus, this study concludes that poultry waste could be a complex nitrogen source for improving the growth of PHB producers and substantially increasing the yield of PHB, and it will be an eco-friendly and low-cost production in bioprocess technology.

Biodegradable polyhydroxyalkanoates production from wheat straw by recombinant Halomonas elongata A1

Waste straw bio-transformation of high value-added macromolecule polyhydroxyalkanoates (PHAs) was significance to environmental sustainable development. As a member of the PHA family, poly-β-hydroxybutyrate (PHB) could be synthesized by Halomonas elongata A1 with maximal yields of 22.8% and 11.8% of bacterial weights using glucose and carboxymethyl cellulose as carbon sources, respectively. To improve PHB production, we generated three recombinant strains, the H. elongata P2 with highest PHB biosynthesis ability. When wheat straw, mixed substrate and oleic acid were individually used as single carbon source, the maximal PHA polymer accumulation in the H. elongata P2 reached 5.2%, 16.5% and 27.5%, respectively, after 84 h of cultivation. This hardness, toughness and crystallization properties of the PHA macromolecule altered dependent on starting substrates, when analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). In terms of the hardness and roughness, the PHA produced from mixed substrates was much softer than that from wheat straw but harder than that from oleic acid. The long-chain carbon improved the softness and strength of the produced PHA. Our data indicate that economical substrates, such as straw and waste oil, can be used in the synthesis of multi-functional plastic products with biodegradable properties.

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.

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.

Production of Biodegradable Polymer from Agro-Wastes in Alcaligenes sp. and Pseudomonas sp.

The present study was aimed to evaluate the suitability of agro-wastes and crude vegetable oils for the cost-effective production of poly-β-hydroxybutyrate (PHB), to evaluate growth kinetics and PHB production in Alcaligenes faecalis RZS4 and Pseudomonas sp. RZS1 with these carbon substrates and to study the biodegradation of PHB accumulated by these cultures. Alcaligenes faecalis RZS4 and Pseudomonas sp. RZS1 accumulates higher amounts of PHB corn (79.90% of dry cell mass) and rice straw (66.22% of dry cell mass) medium respectively. The kinetic model suggests that the Pseudomonas sp. RZS1 follows the Monod model more closely than A. faecalis RZS4. Both the cultures degrade their PHB extract under the influence of PHB depolymerase. Corn waste and rice straw appear as the best and cost-effective substrates for the sustainable production of PHB from Alcaligenes faecalis RZS4 and Pseudomonas sp. RZS1. The biopolymer accumulated by these organisms is biodegradable in nature. The agro-wastes and crude vegetable oils are good and low-cost sources of nutrients for the growth and production of PHB and other metabolites. Their use would lower the production cost of PHB and the low-cost production will reduce the sailing price of PHB-based products. This would promote the large-scale commercialization and popularization of PHB as an ecofriendly bioplastic/biopolymer.

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.

Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering

BackgroundRhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides.ResultsHere we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ΔphbB strains under the same conditions is only slightly affected.ConclusionsIn this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.

Polyhydroxybutyrate Production from Natural Gas in A Bubble Column Bioreactor: Simulation Using COMSOL.

In this study, the simulation of microorganism ability for the production of poly-β-hydroxybutyrate (PHB) from natural gas (as a carbon source) was carried out. Based on the Taguchi algorithm, the optimum situations for PHB production from natural gas in the columnar bubble reactor with 30 cm length and 1.5 cm diameter at a temperature of 32 °C was evaluated. So, the volume ratio of air to methane of 50:50 was calculated. The simulation was carried out by COMSOL software with two-dimensional symmetric mode. Mass transfer, momentum, density-time, and density-place were investigated. The maximum production of biomass concentration reached was 1.63 g/L, which shows a 10% difference in contrast to the number of experimental results. Furthermore, the consequence of inlet gas rate on concentration and gas hold up was investigated Andres the simulation results were confirmed to experimental results with less than 20% error.

Optimization of Poly-β-Hydroxybutyrate (PHB) Production by an Egyption strain of Rhizobium fabae F44 using Response Surface Methodology

The present work is aimed to isolate, purify, identify a newly isolate Rhizobium fabae from different regions in Egypt then, produce and optimize Poly-β-Hydroxybutyrate polymer (PHB) production by the selected isolate Rhizobium fabae F44 using a statistical approach of response surface methodology (RSM). Rhizobium fabae F44 isolate was picked up from Sharkia Governorate in Egypt and then identified by the 16SrRNA gene as Rhizobium fabae. Eleven different variables which affect the production of PHB polymer were screened by Plackett–Burman statistical design approach. Maximization of PHB production was adjusted by the terms of The Face Central Composite Design (FCCD) of RSM and assessed at three coded levels (–1, 0, +1). One way Anova was used to statistically analyze all obtained results in relation to post hoc multiple comparison analysis performed by Tukey’s HSD. Appulses of nutritional and physical factors (two carbon sources, two nitrogen sources, mineral salts, pH, temperature, incubation time, inoculum size and agitation rate) were studied using Plackett-Burman design method. 58 isolates were obtained from the different 9 governorates with 100 % infection plant technique. The 16SrRNA showed a similarity of 99.56 (%) to Rhizobium fabae F44. Out of all tested significant media components, sucrose, yeast extract, agitation rate had the highest significant effects on the response as for PHB production, with confidence level > 98% and were further optimized using FCCD. Predicted Maximum PHB production was observed as (78.51%) which in near the mid-point (0) values (concentrations) of sucrose which reached (25 g/l) and yeast extract of (0.5 g/l) for 48 hrs. at 150 rpm agitation rate. The observed experimental value reached 87.5u/ml was very close result to the predicted one validating the model. So, Response Surface Methodology is an effective statistical approach which can substitute the use of one variable at a time approach due to its adequacy and efficiency to optimize PHB production by Rhizobium fabae F44.