Polyhydroxybutyrate Production Research Articles

Model-Based Nutrient Feeding Strategies for the Increased Production of Polyhydroxybutyrate (PHB) by Alcaligenes latus

Polyhydroxyalkanoates (PHAs) are biodegradable polymers which are considered as an effective alternative for conventional plastics due to their mechanical properties similar to the latter. However, the widespread use of these polymers is still hampered due to their higher cost of production as compared to plastics. The production cost could be overcome by obtaining high yields and productivity. The goal of the present research was to enhance the yield of polyhydroxybutyrate (PHB) with the help of two simple fed-batch cultivation strategies. In the present study, average batch kinetic and substrate limitation/inhibition study data of Alcaligenes latus was used for the development of PHB model which was then adopted for designing various off-line nutrient feeding strategies to enhance PHB accumulation. The predictive ability of the model was validated by experimental implementation of two fed-batch strategies. One such dynamic strategy of fed-batch cultivation under pseudo-steady state with respect to nitrogen and simultaneous carbon feeding strategy resulted in significantly high biomass and PHB concentration of 39.17 g/L and 29.64g/L, respectively. This feeding strategy demonstrated a high PHB productivity and PHB content of 0.6g/Lh and 75%, respectively, which were remarkably high in comparison to batch cultivation. The mathematical model can also be employed for designing various other nutrient feeding strategies.

Acid pretreatment and enzymatic saccharification of brown seaweed for polyhydroxybutyrate (PHB) production using Cupriavidus necator

The brown seaweed Sargassum sp. was used as a feedstock to produce polyhydroxybutyarte (PHB) using Cupriavidus necator PTCC 1615. In order to release monomeric sugars, dilute acid hydrolysis of Sargassum sp. biomass was followed by enzymatic saccharification. In addition, the effect of different nitrogen sources was evaluated for PHB production. The fermentation of hydrolysate with the ammonium sulfate as selected nitrogen source resulted PHB yield of 0.54±0.01g/g reducing sugar. Then, NaCl was used as external stress factor which was added to the media. Addition of 8g/L NaCl had a positive impact on high PHB yield of 0.74±0.01g/g reducing sugar. Increasing trend of NaCl concentration to 16g/L was found to inhibit the production of PHB. Based on obtained results using 20g/L of reducing sugar, at desired condition the highest cell dry weight and PHB concentrations were 5.36±0.22 and 3.93±0.24g/L, respectively. The findings of this study reveal that Sargassum sp. is a promising feedstock for biopolymer production. The characteristics of produced PHB were analyzed by FTIR, differential scanning calorimetry and 1H NMR.

Microbial production, ultrasound-assisted extraction and characterization of biopolymer polyhydroxybutyrate (PHB) from terrestrial (P. hysterophorus) and aquatic (E. crassipes) invasive weeds

This study reports synthesis of biodegradable poly(3-hydroxybutyrate) (PHB) polymer from two invasive weeds, viz. P. hysterophorus and E. crassipes. The pentose and hexose-rich hydrolyzates obtained from acid pretreatment and enzymatic hydrolysis of two biomasses were separately fermented using Ralstonia eutropha MTCC 8320 sp. PHB was extracted using sonication and was characterized using FTIR, 1H and 13C NMR and XRD. PHB content of dry cell mass was 8.1–21.6% w/w, and the PHB yield was 6.85×10−3–36.41×10−3% w/w raw biomass. Thermal properties of PHB were determined by TGA, DTG and DSC analysis. PHB obtained from pentose-hydrolyzate had glass transition temperatures of 6°–9°C, while PHB from hexose-rich hydrolyzate had maximum thermal degradation temperatures of 370°–389°C. These thermal properties were comparable to the properties of commercial PHB. Probable causes leading to differences in thermal properties of pentose and hexose-derived PHB are: extent of crystallinity and presence of impurity in the polymer matrix.

Development of polyhydroxybutyrate biosynthesis in Bacillus subtilis with combination of PHB-associated genes derived from Ralstonia eutropha and Bacillus megaterium

Abstract Polyhydroxybutyrate (PHB) is a biodegradable polymer synthesized by bacteria for carbon storage in response to environmental stress. B. subtilis is generally recognized as a safe Gram-positive bacterium and is widely used for industrial-scale production of proteins and chemicals. Owing to lack of toxic lipopolysaccharides, B. subtilis is a superior alternative to the Gram-negative bacteria presently used for PHB production. In this study, we introduced PHB synthesis genes from R. eutropha and B. megaterium into B. subtilis for PHB production. The highest yield of PHB was achieved by coexpression of phaA from R. eutropha and phaR, phaB , and phaC from B. megaterium (up to 13.02% ± 1.67% of total cell dry weight in LB medium containing 1% glucose). The results indicate that B. subtilis is potentially used as a safe vehicle for producing PHB.

Analysis of the fermentation strategy and its impact on the economics of the production process of PHB (polyhydroxybutyrate)

PHB (polyhydroxybutyrate) is a biodegradable polymer that can replace petrochemical polymers due to its properties and applications. However, its commercial use remains limited because of its high production cost. In this work, factors affecting the PHB fermentation reactor and its impact on the production cost are studied. The production process considers the sucrose fermentation with Azohydromonas australica, followed by the PHB purification and drying. The proposed methodology is divided into two stages: firstly, the analysis of the reactor where it is necessary to define the operation mode and the operating conditions to achieve high product yield and productivity; and secondly, the analysis of production process using technical, economic and environmental criteria to evaluate the process conceptual design. The proposed methodology can reduce complex efforts to achieve an efficient fermentation and validate the design of PHB production process, thereby enabling a biopolymer production with a competitive cost and low environmental impact. The best operation mode for the fermentation reactor was fed-batch, whose feeding strategy allowed to achieve satisfactory technical-economic-environmental results for the PHB production plant: product yield of 0.36g PHB/g sucrose, productivity of 1.6g/L/h, production cost of 2.6USD/kg PHB, return on investment of 34.2%, payback period of 2.9yr, emissions of 1.7kg CO2/kg PHB, energy consumption of 10.1MJ/kg PHB and process requirement of 16.9kg water/kg PHB.

Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai.

This study was focused on the polyhydroxybutyrate (PHB) accumulation property of Bacillus aryabhattai isolated from environment. Twenty-four polyhydroxyalkanoate (PHA) producers were screened out from sixty-two environmental bacterial isolates based on Sudan Black B colony staining. Based on their PHA accumulation property, six promising isolates were further screened out. The most productive isolate PHB10 was identified as B. aryabhattai PHB10. The polymer production maxima were 3.264 g/L, 2.181 g/L, 1.47 g/L, 1.742 g/L and 1.786 g/L in glucose, fructose, maltose, starch and glycerol respectively. The bacterial culture reached its stationary and declining phases at 18 h and 21 h respectively and indicated growth-associated PHB production. Nuclear Magnetic Resonance (NMR) spectra confirmed the material as PHB. The material has thermal stability between 30 and 140 °C, melting point at 170 °C and maximum thermal degradation at 287 °C. The molecular weight and poly dispersion index of the polymer were found as 199.7 kDa and 2.67 respectively. The bacterium B. aryabhattai accumulating PHB up to 75% of cell dry mass utilizing various carbon sources is a potential candidate for large scale production of bacterial polyhydroxybutyrate.

Poly-hydroxybutyrate (PHB) production by Halomonas elongata BK AG 18 indigenous from salty mud crater at central Java Indonesia

Poly-hydroxybutyrate (PHB) production by Halomonas elongata BK AG 18 indigenous from salty mud crater at central Java Indonesia

Bioconversion of fish solid waste into PHB using Bacillus subtilis based submerged fermentation process

ABSTRACTCurrently, one of the major problem affecting the world is solid waste management, predominantly petroleum-based plastic and fish solid waste (FSW). However, it is very difficult to reduce the consumption of plastic as well as fish products, but it is promising to convert FSW to biopolymer to reduce eco-pollution. On account of that, the bioconversion of FSW extract to polyhydroxybutyrate (PHB) was undertaken by using Bacillus subtilis (KP172548). Under optimized conditions, 1.62 g/L of PHB has been produced by the bacterium. The purified compound was further characterized by advanced analytical technologies to elucidate its chemical structure. Results indicated that the biopolymer was found to be PHB, the most common homopolymer of polyhydroxyalkanoates (PHAs). This is the first report demonstrating the efficacy of B. subtilis to utilize FSW extract to produce biopolymer. The biocompatibility of the PHB against murine macrophage cell line RAW264.7 demonstrated that, it was comparatively less toxic, favourable for surface attachment and proliferation in comparison with poly-lactic acid (PLA) and commercially available PHB. Thus, further exploration is highly indispensable to use FSW extract as a substrate for production of PHB at pilot scale.

Developing a green and sustainable process for enhanced PHB production by Azohydromonas australica

Abstract Reports suggest that Azohydromonas australica DSM 1124 is an eminent Poly 3-Hydroxy Butyrate (PHB) producer capable of utilizing different carbon sources with insufficient lactose assimilation. The main idea behind the present study was the adaptation of A. australica to a defined optimal lactose concentration through lactose enrichment technique and thereby enhancement in PHB production. Herein; statistical medium optimization was done using synthetic lactose as carbon substrate and microorganism was adapted on lactose containing medium (up to 2% w/v). Results clearly showed that a maximum PHB of 2.07±0.2 g L −1 was obtained from 4.71±0.3 g L −1 of dried cell biomass with 8.5 fold increase in PHB production by an adapted strain as against wild type strain after optimization in 72 h. The process could be scaled up in 7 L bioreactor with nearly same amount of PHB produced. The utilization of lactose by A. australica would pave the way to use cheap carbon substrate such as cheese whey in order to reduce the overall cost and to make the process cost-effective.

Comparison of metabolite profiling of Ralstonia eutropha H16 phaBCA mutants grown on different carbon sources

Metabolite profiling was conducted to monitor the metabolic differences of the Ralstonia eutropha H16, polyhydroxybutyrate (PHB) producing strain, when grown on different sole carbon sources and to identify the effect of the deletion of the PHB production pathway. The combination of rapid filtration and boiling ethanol extraction successfully extracted intracellular metabolites. Accumulation of acetyl-CoA was detected when the strain was grown on fructose. Whereas, lower adenylate energy charge (AEC) ratio and cell mass was identified on the cells grown on acetate. No significant difference was detected between the wild type and mutant strain except metabolites in the PHB synthetic pathway. Increased PHB production is expected on fructose due to the accumulation of AcCoA, while it is more difficult to improve the production on acetate because of lower metabolic precursor level and AEC ratio.

Production of polyhydroxybutyrate (PHB) of high and ultra-high molecular weight byAzotobacter vinelandiiin batch and fed-batch cultures

BACKGROUND Polyhydroxybutyrate (PHB) is a biopolymer with thermo-mechanical properties like those of plastics produced by the petrochemical industry. These properties are improved in PHBs of high or ultra-high weight average molecular weight. Azotobacter vinelandii OPNA is a potential producer of large quantities of PHB, having ultra-high molecular weight by fed-batch production strategies. RESULTS In batch and fed-batch cultures at different carbon–nitrogen molar ratios (10, 14 and 18), the OPNA strain produced PHB of high and ultra-high weight average molecular weight, with values between 2.3 and 6.6 MDa. In this strain, the weight average molecular weight was highly dependent on the initial carbon–nitrogen ratio, reaching the highest value (6.6 MDa) in batch cultures conducted with a carbon–nitrogen ratio of 18 and the lowest (2.3) with a carbon–nitrogen ratio of 10. On the other hand, using a two feeding-pulses fed-batch strategy, the highest global PHB volumetric productivity (0.56 g L−1 h−1) and polymer concentration (27.6 g L−1) were obtained. CONCLUSION The OPNA strain accumulated up to 80% of PHB of ultra-high molecular weight as a function of the initial carbon–nitrogen molar ratio. Using a simple fed-batch strategy of two feeding-pulses was possible to produce up to 27 g L−1 of PHB. © 2016 Society of Chemical Industry

Isolation, Cloning and Sequencing of Poly 3-Hydroxybutyrate Synthesis Genes from Local Strain of Bacillus cereus mm7 and Expression them in E. coli

Poly 3-hydroxybutyrate (PHB) is the most known degradable biopolymers, produced by some genera of bacteria under unfavorable growth conditions. Isolation, cloning, and expression of PHB genes from PHB producing Bacillus cereuswas achieved. A highest Poly 3-hydroxybutyrate producer bacterium was selected and identified as Bacillus cereus MM7, based on morphological and physiological characters in addition to 16S rRNA gene sequences. The four genes of Bacillus cereus MM7 that involved in Poly 3-hydroxybutyrate production were successfully amplified using specific primers which were designed depending on the sequence alignment of the known PHA biosynthetic genes from Bacillus cereus, cloned in E. coli DH5α and completely sequenced. Moreover, the expression of the four genes responsible for PHA biosynthetic in E. coli XL1-Blue and E coli MJ 109 was assayed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the current study Bacillus cereus was able to synthesize PHB. PHB genes were isolated, cloned and expressed in E. coli DH5α. The PHB encoding proteins were analyzed in SDS-PAGE and the PHB corresponding protein bands were analyzed using mass spectrometry to nominate their amino acid sequence.

Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction

BackgroundMicrobial consortia composed of autotrophic and heterotrophic species abound in nature, yet examples of synthetic communities with mixed metabolism are limited in the laboratory. We previously engineered a model cyanobacterium, Synechococcus elongatus PCC 7942, to secrete the bulk of the carbon it fixes as sucrose, a carbohydrate that can be utilized by many other microbes. Here, we tested the capability of sucrose-secreting cyanobacteria to act as a flexible platform for the construction of synthetic, light-driven consortia by pairing them with three disparate heterotrophs: Bacillus subtilis, Escherichia coli, or Saccharomyces cerevisiae. The comparison of these different co-culture dyads reveals general design principles for the construction of robust autotroph/heterotroph consortia.ResultsWe observed heterotrophic growth dependent upon cyanobacterial photosynthate in each co-culture pair. Furthermore, these synthetic consortia could be stabilized over the long-term (weeks to months) and both species could persist when challenged with specific perturbations. Stability and productivity of autotroph/heterotroph co-cultures was dependent on heterotroph sucrose utilization, as well as other species-independent interactions that we observed across all dyads. One destabilizing interaction we observed was that non-sucrose byproducts of oxygenic photosynthesis negatively impacted heterotroph growth. Conversely, inoculation of each heterotrophic species enhanced cyanobacterial growth in comparison to axenic cultures. Finally, these consortia can be flexibly programmed for photoproduction of target compounds and proteins; by changing the heterotroph in co-culture to specialized strains of B. subtilis or E. coli we demonstrate production of alpha-amylase and polyhydroxybutyrate, respectively.ConclusionsEnabled by the unprecedented flexibility of this consortia design, we uncover species-independent design principles that influence cyanobacteria/heterotroph consortia robustness. The modular nature of these communities and their unusual robustness exhibits promise as a platform for highly-versatile photoproduction strategies that capitalize on multi-species interactions and could be utilized as a tool for the study of nascent symbioses. Further consortia improvements via engineered interventions beyond those we show here (i.e., increased efficiency growing on sucrose) could improve these communities as production platforms.

Efficient polyhydroxybutyrate production from Bacillus juice substrate thuringiensis using sugarcane.

The present study focused on the screening and optimization of biopolymer polyhydroxybutyrate (PHB) production by Bacillus spp. using cost-effective substrates. Among 602 local Bacillus isolates, Bacillus thuringiensis B417-5 produced the highest amount of PHB (2.278 g/L, 60.07% of dry cell weight, DCW). 1H NMR and FTIR analyses of the extracted polymer revealed the characteristic peaks of PHB. The optimization results showed that the highest PHB accumulation (2.768 g/L, 72.08% of DCW) was achieved when culturing B. thuringiensis B417-5 in a nitrogen-deficient medium containing 1% total sugar from sugarcane juice and 0.5% yeast extract, with a pH of 7.0 and an incubation temperature of 37 °C for 48 h. B. thuringiensis B417-5 can thus be considered a good candidate for large-scale production of PHB. We are reporting for the first time that sugarcane juice is a promising carbon source for economical PHB production by B. thuringiensis.

Isolation and Identification of PolyHydroxyButyrate (PHB) producing bacteria from Sewage sample

Plastics and synthetic polymers are synthesized from nonrenewable resources like petrochemicals and persist in the environment long after intended use, resulting into problems of solid waste management and global environmental pollution. Hence, an alternative source such as Polyhydroxyalkanoates that are biodegradable, linear polyesters produced primarily by bacteria which can be used as an effective thermoplastic, and has many characteristics similar to those of standard commercial plastics like polypropylene. Aliphatic polyester ,poly3-hydroxy butyrate was discovered and identified as a granular component in bacterial cells.PHB can grow in a wide variety of natural environments and is the reserve polymer found in many species of bacteria found in nature, e.g. in soil, sea water, sewage waste or compost. In this present study high PHB producing strains were isolated from sewage sample. Five strains were showing PHB granules with Sudan Black B staining. The five strains were labeled as strain 2, 4, 5, 9 and 11. Further, they were morphologically and biochemically characterized. Growth profiles were studied for all these strains and were found that the PHB was produced maximum after 48 hrs at 37°C of incubation. Strain 2 showed high PHB production among the five strains isolated. The sugarcane molasses used in the medium for PHB production accounted for the least production cost.

Identification, Biodegradation and bio-evaluation of biopolymer produced from Bacillus thuringenesis

Production and characterization of polyhydroxybutyrate (PHB) produced by Bacillus sp. isolated from EgyptMohamed A. Hassan, Elsayed K. Bakhiet, Salah G. Ali, Hussien R. Hussien

Production and characterization of polyhydroxybutyrate fromVibrio harveyiMCCB 284 utilizing glycerol as carbon source

Production and characterization of polyhydroxybutyrate (PHB) from moderately halophilic bacterium Vibrio harveyi MCCB 284 isolated from tunicate Phallusia nigra. Twenty-five bacterial isolates were obtained from tunicate samples and three among them exhibited an orange fluorescence in Nile red staining indicating the presence of PHB. One of the isolates, MCCB 284, which showed rapid growth and good polymer yield, was identified as V. harveyi. The optimum conditions of the isolate for the PHB production were pH 8·0, sodium chloride concentration 20gl-1 , inoculum size 0·5% (v/v), glycerol 20gl-1 and 72h of incubation at 30°C. Cell dry weight (CDW) of 3·2gl-1 , PHB content of 2·3gl-1 and final PHB yield of 1·2gl-1 were achieved. The extracted PHB was characterized by FTIR, NMR and DSC-TGA techniques. An isolate of V. harveyi that could effectively utilize glycerol for growth and PHB accumulation was obtained from tunicate P. nigra. PHB produced was up to 72% based on CDW. This is the first report of an isolate of V. harveyi which utilizes glycerol as the sole carbon source for PHB production with high biomass yield. This isolate could be of use as candidate species for commercial PHB production using glycerol as the feed stock or as source of genes for recombinant PHB production or for synthetic biology.

Autoinduced AND Gate Controls Metabolic Pathway Dynamically in Response to Microbial Communities and Cell Physiological State.

Quorum sensing (QS) systems have been widely applied in biotechnology and synthetic biology that require coordinated, community-level behaviors. Meanwhile, the cell physiological state is another key parameter that affects metabolic pathway regulation. Here, we designed an autoinduced AND gate that responds to both microbial communities and the cell physiological state. A series of tunable QS systems in response to different cell densities were obtained through random mutagenesis of LuxR and optimization of the luxRI promoter; the corresponding suitable stationary phase sensing system was selected after monitoring the fluorescence process during cell growth. The application of the final synthetic device was demonstrated using the polyhydroxybutyrate (PHB) production system. The AND gate system increased PHB production by 1-2-fold in Escherichia coli. This synthetic logic gate is a tool for developing a general dynamic regulation system in metabolic engineering in response to complex signals, without using a specific sensor.

Biodiversity of Bacteria Isolated from Different Soils

The aim of this study was to determine the biodiversity of PHB producing bacteria isolated from soils where fruit and vegetable are cultivated (onion, grape, olive, mulberry and plum) in Aydın providence. Morphological, cultural, biochemical, and molecular methods were used for bacteria identification. These isolated bacteria were identified by 16S rRNA sequencing and using BLAST. The following bacteria Bacillus thuringiensis (6), B acillus cereus (8), Bacillus anthrachis (1), Bacillus circulans (1), Bacillus weihenstephanensis (1), Pseudomonas putida (1), Azotobacter chroococcum (1), Brevibacterium frigoritolerans (1), Burkholderia sp. (1), Staphylococcus epidermidis (1), Streptomyces exfoliatus (1), Variovorax paradoxus (1) were found. The Maximum Likelihood method was used to produce a molecular phylogenetic analysis and a phylogenetic tree was constructed. These bacteria can produce polyhydroxybutyrate (PHB) which is an organic polymer with commercial potential as a biodegradable thermoplastic. PHB can be used instead of petrol derivated non-degradable plastics. For this reason, PHB producing microorganisms are substantial in industry.

GacA regulates the PTSNtr-dependent control of cyst formation in Azotobacter vinelandii.

Azotobacter vinelandii forms cysts resistant to desiccation and produces polyhydroxybutyrate (PHB), alginate and alkylresorcinols (ARs) that are components of mature cysts. The expression of genes involved in the synthesis of these compounds is under the control of the GacA-RsmA global regulatory system where the RsmA protein represses the translation of mRNAs involved in the synthesis of these polymers. The synthesis of PHB and ARs is also controlled by the Nitrogen-regulated phosphotransferase system (PTSNtr) global regulatory system. When unphosphorylated, the Enzyme IIANtr (EIIANtr) protein impairs the synthesis of PHB and ARs. Here we show that cells of gacA mutants, as well as mutants that carry the EIIANtr protein in its unphosphorylated state, have similar encysting negative phenotypes. Interestingly, we found that in the gacA mutant strain, the EIIANtr protein was present in its unphosphorylated state. These data indicated that in addition to the GacA-RsmA system, GacA controls polymer synthesis and encystment by controlling the phosphorylation of the EIIANtr, revealing a previously unrecognized link between GacA and PTSNtr.