Polyhydroxybutyrate Production Research Articles

Effects of methane to oxygen ratio on cell growth and polyhydroxybutyrate synthesis in high cell density cultivation of Methylocystis sp. MJC1.

Polyhydroxybutyrate (PHB) production through CH4 conversion by methanotrophs offers a solution for greenhouse gas emissions and plastic waste concerns. In this study, we aimed to achieve high cell density cultivation of Methylocystis sp. MJC1 for efficient PHB production. Cultivating MJC1 using CH4 and air (3:7, v/v) yielded a final cell density of 52.9 g/L with a 53.7% (28.4 g/L) PHB content after 210 h, showcasing PHB mass production potential. However, long-term cultivation led to a low volumetric productivity of 0.200g/L/h. To address this, we conducted cultivation at various O2/CH4 ratios using O2 instead of air, which significantly improved the PHB productivity. Under high O2 conditions (O2/CH4 ratio of 1.5), biomass productivity increased 1.51-fold compared to that under low O2 conditions in the same time frame; however, PHB accumulation was delayed. Using an equal ratio of CH4 and O2 induced active cell growth and selective PHB production, achieving the highest PHB productivity (0.365 g/L/h) with a final cell density of 55.9 g/L and PHB content of 61.7% (34.5 g/L) in 162 h. This study highlighted the significance of the O2/CH4 ratio in CH4 conversion and PHB production by M. sp. MJC1.

Production of polyhydroxybutyrate from industrial flue gas by microbial electrosynthesis

The aim of this work was to produce the biopolymer polyhydroxybutyrate (PHB) from industrial flue gas as CO2 source and electrolysis originating hydrogen via microbial electrosynthesis. Besides the laboratory experiments, the experiments were carried out under industrial conditions directly on-site in a cogeneration plant. We were able to demonstrate that the use of flue gas as a CO2 source has no detectable negative effect on bacterial growth and PHB production in comparison to a pure gas mixture. In an electrochemical H-cell 333 ± 44 mg L−1 PHB were obtained, which corresponds to a PHB content of 43 ± 3 % of the cell dry weight. By using flue gas for the production of the biopolymer PHB, not only CO2 emissions are reduced, but also possible environmental pollution from non-biodegradable plastics. To the best of our knowledge, this is the first time that the production of PHB from flue gas has been demonstrated using Cupriavidus necator.

Biosynthesis of bioplastic polyhydroxybutyrate (PHB) from microbial isolated from paddy/sugarcane fields and fabrication of biodegradable thin film

Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA) family microbial polyester. Three Gram-negative bacteria that produce PHB, Acinetobacter baumannii, Enterobacter hormaechei, and Pseudomonas xanthomarina were isolated and cultured in nitrogen-limited minimal salt media. The pH and culture media were further optimized with various glucose and peptone as carbon and nitrogen sources to improve PHB production. FTIR, UV-vis, NMR, TGA/DSC, and XRD were used to characterize the PHB produced. The optimal pH for PHB production by Acinetobacter baumannii, Enterobacter hormaechei, and Pseudomonas xanthomarina was discovered to be 6.5, 7.5, and 7.5, respectively. P. xanthomarina produced the highest PHB (7.5gm/L) in the optimized culture media. Furthermore, PHB-starch thin films were prepared and characterized by XRD before their biodegradability was investigated. The degradation study reveals that 25% of PHB-starch blended thin film has been degraded within 30 days.

Metabolic modeling of Halomonas campaniensis improves polyhydroxybutyrate production under nitrogen limitation

Poly-hydroxybutyrate (PHB) is an environmentally friendly alternative for conventional fossil fuel-based plastics that is produced by various microorganisms. Large-scale PHB production is challenging due to the comparatively higher biomanufacturing costs. A PHB overproducer is the haloalkaliphilic bacterium Halomonas campaniensis, which has low nutritional requirements and can grow in cultures with high salt concentrations, rendering it resistant to contamination. Despite its virtues, the metabolic capabilities of H. campaniensis as well as the limitations hindering higher PHB production remain poorly studied. To address this limitation, we present HaloGEM, the first high-quality genome-scale metabolic network reconstruction, which encompasses 888 genes, 1528 reactions (1257 gene-associated), and 1274 metabolites. HaloGEM not only displays excellent agreement with previous growth data and experiments from this study, but it also revealed nitrogen as a limiting nutrient when growing aerobically under high salt concentrations using glucose as carbon source. Among different nitrogen source mixtures for optimal growth, HaloGEM predicted glutamate and arginine as a promising mixture producing increases of 54.2% and 153.4% in the biomass yield and PHB titer, respectively. Furthermore, the model was used to predict genetic interventions for increasing PHB yield, which were consistent with the rationale of previously reported strategies. Overall, the presented reconstruction advances our understanding of the metabolic capabilities of H. campaniensis for rationally engineering this next-generation industrial biotechnology platform.Key pointsA comprehensive genome-scale metabolic reconstruction of H. campaniensis was developed.Experiments and simulations predict N limitation in minimal media under aerobiosis.In silico media design increased experimental biomass yield and PHB titer.

ATP-free in vitro biotransformation of starch-derived maltodextrin into poly-3-hydroxybutyrate via acetyl-CoA

In vitro biotransformation (ivBT) facilitated by in vitro synthetic enzymatic biosystems (ivSEBs) has emerged as a highly promising biosynthetic platform. Several ivSEBs have been constructed to produce poly-3-hydroxybutyrate (PHB) via acetyl-coenzyme A (acetyl-CoA). However, some systems are hindered by their reliance on costly ATP, limiting their practicality. This study presents the design of an ATP-free ivSEB for one-pot PHB biosynthesis via acetyl-CoA utilizing starch-derived maltodextrin as the sole substrate. Stoichiometric analysis indicates this ivSEB can self-maintain NADP+/NADPH balance and achieve a theoretical molar yield of 133.3%. Leveraging simple one-pot reactions, our ivSEBs achieved a near-theoretical molar yield of 125.5%, the highest PHB titer (208.3 mM, approximately 17.9 g/L) and the fastest PHB production rate (9.4 mM/h, approximately 0.8 g/L/h) among all the reported ivSEBs to date, and demonstrated easy scalability. This study unveils the promising potential of ivBT for the industrial-scale production of PHB and other acetyl-CoA-derived chemicals from starch.

PHB Production by Bacillus megaterium LSRB 0103 Using Cornstarch and Urea.

Polyhydroxybutyrates (PHBs) are biopolymers that are good green alternative for synthetic carbon-based polymers, and are also one of the most researched members of the Polyhydroxyalkanoates (PHA) family. In this study, a gram-positive bacterial strain Bacillus megaterium LSRB 0103 was isolated from Pallikaranai Marshland, Chennai, India. Primary screening using Sudan Black dye revealed the presence of intracellular PHB granules. Minimal Davis Media (MDM) which was used or PHB production gave a yield of 0.7107g/L. Subsequently, using response surface methodology (RSM), a central composite design (CCD) model was designed for media optimization having cornstarch, urea, and pH as independent variables. The findings of the CCD model were fitted into a second-order polynomial equation. The RSM model predicted the maximum PHB yield of 0.93g/L, at these independent variable levels, cornstarch, 5g/L; urea, 2.1g/L; and pH 7.0; while the experimental PHB yield was 0.94g/L, with a percentage error of 1.1%. This study is the first-time report of production of PHB by Bacillus megaterium using cornstarch and urea as substrate.

Metabolic flux analysis and metabolic engineering for polyhydroxybutyrate (PHB) production

Metabolic flux analysis and metabolic engineering for polyhydroxybutyrate (PHB) production

Poly(3-hydroxybutyrate) production from cassava processing wastes by Paracoccus sp. through high cell density cultivation: Effects of substrates limitation and kinetic analysis

Polyhydroxybutyrate (PHB) is bioplastic that has been recognized as a viable alternative to petroleum-based ones, owing to its biodegradability and biocompatibility. However, its use has long been hampered by its high selling price, caused mainly by using costly fermentation feedstock. In the present study, cassava pulp (CP) was investigated as an alternative low-cost carbon source for a high cell density cultivation of Paracoccus sp. KKU01 for PHB production. CP was hydrolyzed enzymatically, and the resulting hydrolysate was used as the base medium to optimize the bacterial growth conditions, i.e., medium compositions and dissolved oxygen setpoints, attaining as high as 40.9 ± 0.0 g/L of biomass. The optimum growth conditions were subsequently used in 4 fed-batch fermentation regimes, and Regime 2, where complete medium (CP hydrolysate supplemented with other nutrients) was fed twice followed by twice feedings of concentrated CP hydrolysate (CPH), was found to give highest biomass and PHB production of 114.5 ± 1.3 and 47.8 ± 3.9 g/L, respectively, with 41.8 ± 2.9% PHB content and yield of 0.12 kg/kg-CP. PHB productivity was 0.80 ± 0.06 g/(L·h). Logistic and Luedeking-Piret models were used successfully to describe the profiles of bacterial growth and PHB production, respectively. Curve fitting using the Luedeking-Piret model revealed that PHB synthesis and PHB turnover occurred simultaneously during the process. Overall, the results demonstrated that cassava processing wastes are feasible feedstock for PHB production by Paracoccus sp. KKU01.

Influence of Aeration Rate on Uncoupled Fed Mixed Microbial Cultures for Polyhydroxybutyrate Production

The use of residual streams as feedstock for the production of polyhydroxyalkanoates (PHAs) is growing steadily, as it allows the valorization of waste and nutrients otherwise disposed of and the potential production of a biodegradable bioplastic. To date, the environmental and economic costs associated with this process limit its scale-up, which is why it is important to identify possible solutions and optimize the costliest steps. With this in mind, a laboratory-scale sequenced batch reactor (SBR, 5 L) was constructed to allow the selection of a mixed microbial culture able to convert volatile fatty acids (VFAs) into PHA. The reactor is fed with synthetic water containing VFAs, ammonium, phosphate, and micronutrients, typical compounds of fermented streams of certain wastes, such as cheese whey, food waste, or wastewater sludge. The biomass selected and produced by this first reactor is sent to an accumulation reactor, which is fed with a solution rich in VFAs, allowing the accumulation of PHAs. The role of aeration and its impacts on the main process parameters were analyzed. Three scenarios corresponding to different aeration rates were analyzed: 0.08, 0.16, and 0.32 vvm. The SBR was operated at an organic load rate of 600 mgCOD L−1d−1, under a dynamic feeding regime (feast–famine) and a short hydraulic retention time (HRT; 1 day). The results obtained showed that a value of 0.32 enabled better selection and better settling of the sludge. Furthermore, a potential correlation between aeration rate and VFA and NH4+ consumption rates was identified. The resulting biomass was able to accumulate up to 0.15 ± 0.02 g PHAgVSS−1.

Kinetic model for polyhydroxybutyrate production by Bacillus aryabhattai MH997667.1 in a batch culture using different agitation speeds

Background Slow degradation of synthetic plastics represents a high environmental hazard; therefore, there is an essential requirement to substitute them with eco-friendly products. Polyhydroxybutyrate (PHB) is a biodegradable biopolymer and also has several industrial, agricultural, and medical applications. Scaling up the production of PHB is still a problem due to the numerous parameters tangled in the fermentation processes. Objective The present work seeks to scale up polyhydroxybutyrate production by Bacillus aryabhattai MH997667.1 from shaken flasks to a 5L-bench top bioreactor with previously optimized media by applying a batch fermentation strategy. Materials and methods Different agitation speeds (200, 250 and 300 rpm) were tested in a 5L-bench-top bioreactor with a working volume of 3L. A kinetic model (logistic and Luedeking–Piret) that describes the microbial biomass and polyhydroxybutyrate (PHB) production is used to expect the performance of batch fermentation of Bacillus aryabhattai MH997667.1. Results and conclusion The optimum PHB yield (2.32 g/l) was expressed at 250 rpm agitation after 28 hours of fermentation. The experimental data were also fitted with the logistic and Luedeking–Piret equations for growth and PHB formation, respectively. The mathematical model proposed for batch fermentation revealed that the simulated data showed a good fit with the experimental results obtained during the first 24 h of PHB production at 250 rpm, where the productivity was 0.095 g L−1h−1. Our data suggest that agitation speed had a significant effect on PHB production and the 250 rpm agitation speed is the optimum speed for PHB production using Bacillus aryabhattai MH997667.1 in batch fermentation.

Methanotrophs mediated biogas valorization: Sustainable route to polyhydroxybutyrate production

This study explores the ability of methanotrophs to convert biogas into biopolymers, addressing H2S as a limitation in the utilization of biogas as a carbon source for bioconversion. Transcriptomic analysis was conducted to understand the growth and changes in the expression patterns of Type I and II methanotrophs under varying H2S concentrations. Results suggested that Type II methanotrophs can possess a native H2S utilization pathway. Both Type I and II methanotrophs were evaluated for their growth and polyhydroxybutyrate (PHB) production from biogas. Methylocystis sp. MJC1 and Methylocystis sp. OK1 exhibited a maximum biomass production of 4.0 and 4.5 gDCW/L, respectively, in fed-batch culture, aligning with the transcriptome data. Furthermore, Methylocystis sp. MJC1 produced 2.9 g PHB/L from biogas through gas fermentation. These findings underscore biogas-based biotechnology as an innovative solution for environmental and industrial challenges with further optimization and productivity enhancement research expected to broaden the potential in this field.

Polyhydroxybutyrate (PHB)-Based sustainable bioplastic derived from Bacillus sp. KE4 isolated from kitchen waste effluent

Petrochemical-derived plastics pose a major threat to the biosphere and the environment due to their acute or long-term toxicity. Therefore, to ensure sustainability, it is essential to find an effective replacement for synthetic plastics with natural and ecofriendly biomaterial. In this context, production of polyhydroxybutyrate (PHB) was produced by the Bacillus sp. KE4 using glucose as the carbon source, and a maximum dry cell weight (DCW) of 4.4 g/l with a PHB concentration of 3.1 g/l, PHB yield of 0.15 g/g was obtained, and the PHB content was 70.4% of the DCW. The extracted biopolymer was characterized using FTIR, GC-MS, 1H NMR, and 13C NMR which confirmed the structure of the biopolymer as PHB. The findings of TGA, DTG, DTA and DSC studies corresponded to PHB's excellent thermal stability. PHB biopolymer has a melting temperature of 168.7 °C and a maximum degradation temperature of 273 °C. The weight average molar mass (Mw), number average molar mass (Mn), and polydispersity index (PDI) of the extracted PHB was 1,55,436 g mol−1; 1,43,258 g mol−1; and 1.08, respectively. The XRD analysis validated the material's partly crystalline nature. FESEM-EDS and XPS were employed to determine the elemental composition of the purified PHB film. The Young's modulus, tensile strength and elongation at break showed rigid and brittle nature of PHB. Using open windrow composting method, the extracted PHB biopolymer film was assessed to determine its biodegradability. Within 28 days of soil burial, it was found that the biopolymer film had degraded by 61.34%. Cytotoxicity assessment in HepG2 cells indicated that PHB is non-cytotoxic in nature. The results have showed that the biomaterial is environmentally benign, biodegradable, and biocompatible; therefore, it may find its diverse application covering packaging, medicine, agriculture and allied fields.

Metabolic and physiological adaptations of microalgal growth-promoting bacterium Azospirillum brasilense growing under biogas atmosphere: a microarray-based transcriptome analysis.

Using microalgal growth-promoting bacteria (MGPB) to improve the cultured microalga metabolism during biotechnological processes is one of the most promising strategies to enhance their benefits. Nonetheless, the culture condition effect used during the biotechnological process on MGPB growth and metabolism is key to ensure the expected positive bacterium growth and metabolism of microalgae. In this sense, the present research study investigated the effect of the synthetic biogas atmosphere (75% CH4-25% CO2) on metabolic and physiological adaptations of the MGPB Azospirillum brasilense by a microarray-based transcriptome approach. A total of 394 A. brasilense differentially expressed genes (DEGs) were found: 201 DEGs (34 upregulated and 167 downregulated) at 24h and 193 DEGs (140 upregulated and 53 downregulated) under the same conditions at 72h. The results showed a series of A. brasilense genes regulating processes that could be essential for its adaptation to the early stressful condition generated by biogas. Evidence of energy production is shown by nitrate/nitrite reduction and activation of the hypothetical first steps of hydrogenotrophic methanogenesis; signal molecule modulation is observed: indole-3-acetic acid (IAA), riboflavin, and vitamin B6, activation of Type VI secretion system responding to IAA exposure, as well as polyhydroxybutyrate (PHB) biosynthesis and accumulation. Moreover, an overexpression of ipdC, ribB, and phaC genes, encoding the key enzymes for the production of the signal molecule IAA, vitamin riboflavin, and PHB production of 2, 1.5 and 11 folds, respectively, was observed at the first 24h of incubation under biogas atmosphere Overall, the ability of A. brasilense to metabolically adapt to a biogas atmosphere is demonstrated, which allows its implementation for generating biogas with high calorific values and the use of renewable energies through microalga biotechnologies.

A dynamic model of growth phase of bio-conversion of methane to polyhydroxybutyrate using dynamic flux balance analysis.

Biological conversion of waste methane to biodegradable plastics is a way of reducing their production cost. This study addresses the computational modeling of the growth phase reactor of the process of polyhydroxybutyrate production. The model was used for investigating the effect of gas recycling and inlet gas retention time on the reactor performance. The model was run by the use of a genome-scale metabolic network of Methylocystis hirsuta in a dynamic flux balance analysis framework. The reactor has been modeled for two separate feeding scenarios: a pure methane feed and a biogas feed. The mass transfer coefficient parameter was predicted as a function of superficial gas velocities by the regression of data from published experiments. The results show an increase of removal efficiency by 38% and biomass concentration by 2.8g/L with the increase of gas recycle ratio from 0 to 30 at the empty bed residence time of 60 .

Polyhydroxybutyrate (PHB) bioplastic characterization from the isolate Pseudomonas stutzeri PSB1 synthesized using potato peel feedstock to combat solid waste management

The global fossil-based plastic industry is at a crucial point, requiring bio-based polymers. Polyhydroxybutyrate (PHB) is formed under nutritional stress conditions; however, efficient bacteria and cheap substrates are needed. This study found that potato peel, a cheap and abundant carbon source, can improve PHB production. PHB crotonic assay screened 11 Sudan Black B and Nile red positive isolates for PHB production. PSB1, a Pseudomonas stutzeri PSB1 (MN539619) yields 2.39 g/L maximal PHB. PHB generation from high-carbon agro-wastes using Pseudomonas stutzeri PSB1was 3.09 g/L for potato peel. Plackett-Burman and CCD, a statistical optimization method, increased PHB production. After optimizing all CCD variables, potato peel, Urea, and RPM were found to increase PHB production (6.1 g/L). The optimized parameters extracted 3.98 g/L of PHB from 6.36 g/L of dry biomass of Pseudomonas stutzeri PSB1. FT-IR, NMR, TGA, and DSC were used to characterize and compare the extracted PHB biopolymer to Sigma Aldrich PHB powder. Extracted PHB-bioplastic was tested for tensile strength and Elongation at break. Based on the above criteria, the recovered PHB biopolymer from Pseudomonas stutzeri PSB1 is a good alternative to agricultural mulch films and non-biodegradable plastics.

Photosynthetic species composition determines bioplastics production in microbiomes: Strategy to enrich cyanobacteria PHB-producers

The aim of this study was to set the operating mode in regards to nutrients, temperature and light to use as a strategy to enrich a microbiome rich in cyanobacteria in polyhydroxybutyrate (PHB)-producers in order to enhance the biopolymer production. Alternate growth and accumulation phases were conducted for 197 days in a 3 L photobioreactor. Although, presence of green algae potentially reduced PHB production, the microbiome produced up to 22 % dry cell weight (dcw) PHB. Results highlighted the important role of culture composition and suggested that this methodology could be applied to a robust microbiome rich in cyanobacteria to boost PHB production.

Techno-Economic Feasibility of Polyhydroxybutyrate (PHB) Production from Corn Stover

Techno-Economic Feasibility of Polyhydroxybutyrate (PHB) Production from Corn Stover

Bioprospecting of Polyhydroxy butyrate (PHB) from Bacillus thuringiensis KUMBNGBT-41 by using Agro-industrial wastes

The diverse group of chemicals used in making of plastic is known to be highly toxic and poses a serious threat to the biosphere. These substances besides hitting hard to ecosystem cause an array of problems like birth defects, cancer, damage of nervous and immune systems. The present research work was aimed to isolate and characterize PHB producing bacteria from landfill soil sample collected from Sanehally, Chitradurga district and qualitatively screened by using viable staining techniques. Quantitative estimation was examined by using solvent extraction method. The PHB producing bacteria were characterized based on morphological, biochemical and molecular identification and confirmed as Bacillus thuringiensis strain KUMBNGBT-41 (Accession No. MW040075). Growth parameters were optimized for biomass production using various conditions such as media-nutrient broth, incubation time-72hrs, temperature-37˚C, pH-7.0, carbon source-glucose, nitrogen source-ammonium chloride and carbon to nitrogen ratio as 4:1. The low-cost production media was used for PHB production by cheaper substrates in submerged fermentation. PHB was analysed and quantified by using UV-Visible spectrophotometer at 200-600nm.

Sustainable Synthesis of Biopolymer Polyhydroxybutyrate (PHB) from Agro-residue by Brevibacterium casei with Emphasis on Degradation Analysis

The Polyhydroxybutyrate (PHB) polymer is a biodegradable microbial polyester that is intracellularly accruing due to the depletion of nitrogen and phosphorous resources and an increase in carbon supply. As part of this research investigation, Sudan Black B staining, fermentation, chloroform-sodium hypochlorite solvent-based extraction, and characterization of extracted PHB were used to isolate and identify organisms capable of producing PHB. Brevibacterium casei (OQ519751) was used to synthesize PHB biopolymer from agro-residues (orange peel, mangosteen peel, sugarcane bagasse, water hyacinth, and jackfruit peel). Using the Response Surface Methodology (RSM) and the Central Composite Design (CCD) has proven to be highly effective for optimizing PHB synthesis. The optimal conditions determined through RSM allowed Brevibacterium casei to produce significant amounts of PHB when compared to an unoptimized medium. The model demonstrated statistical significance, as indicated by the F-value of 19.96 with an associated p-value of <0.0001. Furthermore, with an optimized pH level of 7, temperature of 37°C, and yeast extract as the nitrogen source, the carbon source water hyacinth was found to synthesize an enhanced quantity of a PHB yield of 1.29 g/L from 2.2 g/L of dry biomass (58.63%). PHB characterization was done with the aid of FTIR (Fourier-transform infrared spectroscopy), XRD (X-ray diffraction), and TGA (Thermogravimetric analysis) analysis. The degradation study of PHB films was performed by soil burial method and morphological changes were scrutinized by SEM analysis. The results reveal that utilizing water hyacinth as a feedstock employs an enhanced production of PHB. This is the first report to synthesize maximum yield of PHB from Brevibacterium casei using water hyacinth as a substrate for production.

Exogenous Trehalose Improves Growth, Glycogen and Poly-3-Hydroxybutyrate (PHB) Contents in Photoautotrophically Grown Arthrospira platensis under Nitrogen Deprivation.

Glycogen and poly-3-hydroxybutyrate (PHB) are excellent biopolymer products from cyanobacteria. In this study, we demonstrate that nitrogen metabolism is positively influenced by the exogenous application of trehalose (Tre) in Arthrospira platensis under nitrogen-deprived (-N) conditions. Cells were cultivated photoautotrophically for 5 days under -N conditions, with or without the addition of exogenous Tre. The results revealed that biomass and chlorophyll-a content of A. platensis experienced enhancement with the addition of 0.003 M and 0.03 M Tre in the -N medium after one day, indicating relief from growth inhibition caused by nitrogen deprivation. The highest glycogen content (54.09 ± 1.6% (w/w) DW) was observed in cells grown for 2 days under the -N + 0.003 M Tre condition (p < 0.05), while the highest PHB content (15.2 ± 0.2% (w/w) DW) was observed in cells grown for 3 days under the -N + 0.03 M Tre condition (p < 0.05). The RT-PCR analysis showed a significant increase in glgA and phaC transcript levels, representing approximately 1.2- and 1.3-fold increases, respectively, in A. platensis grown under -N + 0.003 M Tre and -N + 0.03 M Tre conditions. This was accompanied by the induction of enzyme activities, including glycogen synthase and PHA synthase with maximal values of 89.15 and 0.68 µmol min-1 mg-1 protein, respectively. The chemical structure identification of glycogen and PHB from A. platensis was confirmed by FTIR and NMR analysis. This research represents the first study examining the performance of trehalose in promoting glycogen and PHB production in cyanobacteria under nitrogen-deprived conditions.