PHB Synthesis Research Articles

Engineering of fast-growing Vibrio natriegens for biosynthesis of poly(3-hydroxybutyrate-co-lactate)

Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] is a highly promising valuable biodegradable material with good biocompatibility and degradability. Vibrio natriegens, owing to its fast-growth, wide substrate spectrum characteristics, was selected to produce P(3HB-co-LA). Herein, the crucial role of acetyltransferase PN96-18060 for PHB synthesis in V. natriegens was identified. Heterologous pathway of P(3HB-co-LA) was introduced into V. natriegens successfully, in addition, overexpression of the dldh gene led to 1.84 fold enhancement of the lactate content in P(3HB-co-LA). Finally, the production of P(3HB-co-LA) was characterized under different carbon sources. The lactate fraction in P(3HB-co-LA) was increased to 28.3 mol% by the modification, about 1.84 times of that of the control. This is the first successful case of producing the P(3HB-co-LA) in V. natriegens. Collectively, this study showed that V. natriegens is an attractive host organism for producing P(3HB-co-LA) and has great potential to produce other co-polymers.

Methylosinus trichosporium OB3b drives composition-independent application of biogas in poly(3-hydroxybutyrate) synthesis

Biological conversion of biogas into poly(3-hydroxybutyrate) (PHB) via Type II methanotrophs holds great promise. However, a prerequisite for widespread biogas utilization is a comprehensive understanding of the effects of biogas composition on methanotrophic PHB synthesis. Herein, we explore the effects of biogas composition on the microbial growth and PHB accumulation of Methylosinus trichosporium OB3b under five different artificial biogas conditions with varying the ratio of methane (CH4) to carbon dioxide (CO2), the two main components of raw biogas. Stoichiometric and kinetic analyses revealed that increased CO2 concentrations correlated with higher maximum specific growth rates (0.021–0.029 h−1) and maximum CH4 utilization rates (0.029–0.042 mg CH4/mg TSS-h). Besides, no significant correlation was found between the CH4-to-CO2 ratio and other growth parameters. Consistent PHB contents (20.2–25.3 wt%) were achieved, irrespective of the CH4-to-CO2 ratio. Furthermore, the ability of M. trichosporium OB3b to grow and synthesize PHB (13.2 wt%) was confirmed using pilot-scale biogas. Our findings highlight the potential of biogas as a feedstock for producing valuable biodegradable polymers, thus contributing to waste-derived fuel (WDF) innovation and environmental sustainability.

A Homolog of the Histidine Kinase RetS Controls the Synthesis of Alginates, PHB, Alkylresorcinols, and Motility in Azotobacter vinelandii

The two-component system GacS/A and the posttranscriptional control system Rsm constitute a genetic regulation pathway in Gammaproteobacteria; in some species of Pseudomonas, this pathway is part of a multikinase network (MKN) that regulates the activity of the Rsm system. In this network, the activity of GacS is controlled by other kinases. One of the most studied MKNs is the MKN-GacS of Pseudomonas aeruginosa, where GacS is controlled by the kinases RetS and LadS; RetS decreases the kinase activity of GacS, whereas LadS stimulates the activity of the central kinase GacS. Outside of the Pseudomonas genus, the network has been studied only in Azotobacter vinelandii. In this work, we report the study of the RetS kinase of A. vinelandii; as expected, the phenotypes affected in gacS mutants, such as production of alginates, polyhydroxybutyrate, and alkylresorcinols and swimming motility, were also affected in retS mutants. Interestingly, our data indicated that RetS in A. vinelandii acts as a positive regulator of GacA activity. Consistent with this finding, mutation in retS also negatively affected the expression of small regulatory RNAs belonging to the Rsm family. We also confirmed the interaction of RetS with GacS, as well as with the phosphotransfer protein HptB.

A newly isolated strain for poly(3-hydroxybutyrate) production under anaerobic conditions and the key enzyme analysis

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters with great possibility to replace petroleum-based plastics, but the high costs of production associated with energy inputs have been an obstacle to their commercial application. The production of PHAs under anaerobic environments without aeration appears to be more attractive but has not been adequately investigated. A strain capable of accumulating poly(3-hydroxybutyrate) (PHB) under anaerobic conditions was isolated. The strain anaerobically accumulated PHB reached 38.7 % of cell dry weight (CDW), which was 1.9 times higher than under aerobic conditions. It was also found that the concentrations of key enzymes for PHB synthesis were higher under anaerobic conditions than under aerobic conditions. The highest PHB accumulation was 77.9 % obtained at oxygen-limited conditions and the highest PHB yield was 0.13 g PHB/g carbon obtained at anaerobic conditions. It suggests that the conditions should be well controlled according to the purpose of PHB production. This study offers a new alternative of PHAs formation.

Disrupted H2 synthesis combined with methyl viologen treatment inhibits photosynthetic electron flow to synergistically enhance glycogen accumulation in the cyanobacterium Synechocystis sp. PCC 6803.

Under nitrogen deprivation (-N), cyanobacterium Synechocystis sp. PCC 6803 exhibits growth arrest, reduced protein content, and remarkably increased glycogen accumulation. However, producing glycogen under this condition requires a two-step process with cell transfer from normal to -N medium. Metabolic engineering and chemical treatment for rapid glycogen accumulation can bypass the need for two-step cultivation. For example, recent studies indicate that individually disrupting hydrogen (H2) or poly(3-hydroxybutyrate) (PHB) synthesis, or treatment with methyl viologen (MV), effectively increases glycogen accumulation in Synechocystis. Here we explore the effects of disrupted H2 or poly(3-hydroxybutyrate) synthesis, together with MV treatment to on enhanced glycogen accumulation in Synechocystis grown in normal medium. Wild-type cells without MV treatment exhibited low glycogen content of less than 6% w/w dry weight (DW). Compared with wild type, disrupting PHB synthesis combined with MV treatment did not increase glycogen content. Disrupted H₂ production without MV treatment yielded up to 11% w/w DW glycogen content. Interestingly, when combined, disrupted H2 production with MV treatment synergistically enhanced glycogen accumulation to 51% and 59% w/w DW within 3 and 7days, respectively. Metabolomic analysis suggests that MV treatment mediated the conversion of proteins into glycogen. Metabolomic and transcriptional-expression analysis suggests that disrupted H2 synthesis under MV treatment positively influenced glycogen synthesis. Disrupted H₂ synthesis under MV treatment significantly increased NADPH levels. This increased NADPH content potentially contributed to the observed enhancements in antioxidant activity against MV-induced oxidants, O2 evolution, and metabolite substrates levels for glycogen synthesis in normal medium, ultimately leading to enhanced glycogen accumulation in Synechocystis. KEY MESSAGE: Combining disrupted hydrogen-gas synthesis and the treatment by photosynthesis electron-transport inhibitor significantly enhance glycogen production in cyanobacteria.

Boosting hydrogen production in Rhodospirillum rubrum by syngas-driven photoheterotrophic adaptive evolution

Rhodospirillum rubrum is a photosynthetic purple non-sulphur bacterium with great potential to be used for complex waste valorisation in biotechnological applications due to its metabolic versatility. This study investigates the production of hydrogen (H2) and polyhydroxyalkanoates (PHA) by R. rubrum from syngas under photoheterotrophic conditions. An adaptive laboratory evolution strategy (ALE) has been carried out to improve the yield of the process. After 200 generations, two evolved strains were selected that showed reduced lag phase and enhanced poly-3-hydroxybutyrate (PHB) and H2 synthesis compared to the parental strain. Genomic analysis of the photo-adapted (PA) variants showed four genes with single point mutations, including the photosynthesis gene expression regulator PpsR. The proteome of the variants suggested that the adapted variants overproduced H2 due to a more efficient CO oxidation through the CO-dehydrogenase enzyme complex and confirmed that energy acquisition was enhanced through overexpression of the photosynthetic system and metal cofactors essential for pigment biosynthesis.

Disrupted biosynthesis of glycogen and PHB combined with physicochemical factors increased bioproduction of γ-aminobutyric acid and δ-aminolevulinic acid in cyanobacteria

In Synechocystis sp. PCC 6803, redirecting the metabolic flow from upstream substrate for carbon metabolism i.e. glyceraldehyde-3-phosphate (the glycogen synthesis precursor) and poly(3-hydroxybutyrate) (PHB), towards ALA or GABA has not been studied. The PHB synthesis, one of the pathways consuming carbon via glycolysis pathway, was disrupted to generate the strain, ΔP, and another adjacent carbon utilizing pathway that is responsible for glycogen synthesis was also disrupted in the earlier mutant strain to generate ΔGP with the aim to increase carbon flux towards precursor metabolites for GABA and ALA biosynthesis. The ΔP strain showed an increase in intracellular glutamate and ALA levels (∼19.5 and 2.5 ng g−1 DCW, respectively), whereas intracellular GABA levels were found increased (up to 7 ng g−1 DCW) in ΔGP strain as compared to the wild type strain (WT). Modified nutrient conditions including glucose, glutamate and LA supplementation as well as N-deprivation led to several fold increase in GABA and ALA levels in ΔP and ΔGP strains in comparison to those observed in WT, with the maximum GABA and ALA accumulation (∼262 and 48 ng g−1 DCW, respectively) noticed in ΔP. Abiotic stresses including cold and UV irradiation also significantly improved the intracellular GABA and ALA accumulation in mutant strains. Increased biosynthesis of GABA and ALA in mutant strains was found correlated with the increased mRNA levels of genes involved in GABA, ALA and TCA cycle metabolism. Further organic acids profiling revealed that the disruption of two pathways redirects carbon routes to TCA cycle without affecting the viability of cells. This strategy might be applicable in strain development for enhancing other high value bioproducts synthesized by connected metabolic pathways that utilize carbon sources diverted from non-essential metabolism.

Computational Analysis of the Tripartite Interaction of Phasins (PhaP4 and 5)-Sigma Factor (σ24)-DNA of Azospirillum brasilense Sp7.

Azospirillum brasilense Sp7 produces PHB, which is covered by granule-associated proteins (GAPs). Phasins are the main GAPs. Previous studies have shown phasins can regulate PHB synthesis. When A. brasilense grows under stress conditions, it uses sigma factors to transcribe genes for survival. One of these factors is the σ24 factor. This study determined the possible interaction between phasins and the σ24 factor or phasin-σ24 factor complex and DNA. Three-dimensional structures of phasins and σ24 factor structures were predicted using the I-TASSER and SWISS-Model servers, respectively. Subsequently, a molecular docking between phasins and the σ24 factor was performed using the ClusPro 2.0 server, followed by molecular docking between protein complexes and DNA using the HDOCK server. Evaluation of the types of ligand-receptor interactions was performed using the BIOVIA Discovery Visualizer for three-dimensional diagrams, as well as the LigPlot server to obtain bi-dimensional diagrams. The results showed the phasins (Pha4Abs7 or Pha5Abs7)-σ24 factor complex was bound near the -35 box of the promoter region of the phaC gene. However, in the individual interaction of PhaP5Abs7 and the σ24 factor, with DNA, both proteins were bound to the -35 box. This did not occur with PhaP4Abs7, which was bound to the -10 box. This change could affect the transcription level of the phaC gene and possibly affect PHB synthesis.

Analysis of heterologous expression of phaCBA promotes the acetoin stress response mechanism in Bacillus subtilis using transcriptomics and metabolomics approaches.

Acetoin, a versatile platform chemical and popular food additive, poses a challenge to the biosafety strain Bacillus subtilis when produced in high concentrations due to its intrinsic toxicity. Incorporating the PHB synthesis pathway into Bacillus subtilis 168 has been shown to significantly enhance the strain's acetoin tolerance. This study aims to elucidate the molecular mechanisms underlying the response of B. subtilis 168-phaCBA to acetoin stress, employing transcriptomic and metabolomic analyses. Acetoin stress induces fatty acid degradation and disrupts amino acid synthesis. In response, B. subtilis 168-phaCBA down-regulates genes associated with flagellum assembly and bacterial chemotaxis, while up-regulating genes related to the ABC transport system encoding amino acid transport proteins. Notably, genes coding for cysteine and D-methionine transport proteins (tcyB, tcyC and metQ) and the biotin transporter protein bioY, are up-regulated, enhancing cellular tolerance. Our findings highlight that the expression of phaCBA significantly increases the ratio of long-chain unsaturated fatty acids and modulates intracellular concentrations of amino acids, including L-tryptophan, L-tyrosine, L-leucine, L-threonine, L-methionine, L-glutamic acid, L-proline, D-phenylalanine, L-arginine, and membrane fatty acids, thereby imparting acetoin tolerance. Furthermore, the supplementation with specific exogenous amino acids (L-alanine, L-proline, L-cysteine, L-arginine, L-glutamic acid, and L-isoleucine) alleviates acetoin's detrimental effects on the bacterium. Simultaneously, the introduction of phaCBA into the acetoin-producing strain BS03 addressed the issue of insufficient intracellular cofactors in the fermentation strain, resulting in the successful production of 70.14g/L of acetoin through fed-batch fermentation. This study enhances our understanding of Bacillus's cellular response to acetoin-induced stress and provides valuable insights for the development of acetoin-resistant Bacillus strains.

Poly(3-hydroxybutyrate) Production from Lignocellulosic Wastes Using Bacillus megaterium ATCC 14581.

This study aimed to analyze the production of poly(3-hydroxybutyrate) (PHB) from lignocellulosic biomass through a series of steps, including microwave irradiation, ammonia delignification, enzymatic hydrolysis, and fermentation, using the Bacillus megaterium ATCC 14581 strain. The lignocellulosic biomass was first pretreated using microwave irradiation at different temperatures (180, 200, and 220 °C) for 10, 20, and 30 min. The optimal pretreatment conditions were determined using the central composite design (CCD) and the response surface methodology (RSM). In the second step, the pretreated biomass was subjected to ammonia delignification, followed by enzymatic hydrolysis. The yield obtained for the pretreated and enzymatically hydrolyzed biomass was lower (70.2%) compared to the pretreated, delignified, and enzymatically hydrolyzed biomass (91.4%). These hydrolysates were used as carbon substrates for the synthesis of PHB using Bacillus megaterium ATCC 14581 in batch cultures. Various analytical methods were employed, namely nuclear magnetic resonance (1H-NMR and13C-NMR), electrospray ionization mass spectrometry (EI-MS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and thermogravimetric analysis (TGA), to identify and characterize the extracted PHB. The XRD analysis confirmed the partially crystalline nature of PHB.

Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli

Utilizing anaerobic metabolisms for the production of biotechnologically relevant products presents potential advantages, such as increased yields and reduced energy dissipation. However, lower energy dissipation may indicate that certain reactions are operating closer to their thermodynamic equilibrium. While stoichiometric analyses and genetic modifications are frequently employed in metabolic engineering, the use of thermodynamic tools to evaluate the feasibility of planned interventions is less documented. In this study, we propose a novel metabolic engineering strategy to achieve an efficient anaerobic production of poly-(R)-3-hydroxybutyrate (PHB) in the model organism Escherichia coli. Our approach involves re-routing of two-thirds of the glycolytic flux through non-oxidative glycolysis and coupling PHB synthesis with NADH re-oxidation. We complemented our stoichiometric analysis with various thermodynamic approaches to assess the feasibility and the bottlenecks in the proposed engineered pathway. According to our calculations, the main thermodynamic bottleneck are the reactions catalyzed by the acetoacetyl-CoA β-ketothiolase (EC 2.3.1.9) and the acetoacetyl-CoA reductase (EC 1.1.1.36). Furthermore, we calculated thermodynamically consistent sets of kinetic parameters to determine the enzyme amounts required for sustaining the conversion fluxes. In the case of the engineered conversion route, the protein pool necessary to sustain the desired fluxes could account for 20% of the whole cell dry weight.

Isolation of new Paraburkholderia strains for polyhydroxybutyrate production.

Polyhydroxyalkanoates (PHAs) are bioplastics that can serve as substitutes for petroleum-based plastics with the advantages of being biodegradable, biocompatible, and biobased. The microbial production of polyhydroxyalkanoates is generally conducted in the presence of sugar mixes rich in monosaccharides. In this study, molecular and cultural approaches based on forest soils enriched with hydrocarbon complexes led to the identification and isolation of microbial strains affiliated with Paraburkholderia sp. that dominated the microbial communities that are recognized among the top polyhydroxyalkanoates producers. The genome sequencing of those isolated affiliated strains showed that compared to the reference type strain of their species, they harbored more gene copies of the enzymes involved in PHB synthesis. The microbial conversion of sugar mixes for the newly isolated strains showed a higher PHB production (g/L) and content (%) than was exhibited by the reference strain type of that genus Paraburkholderia for PHB production (P. sacchari LMG 19450T).

Polyhydroxybutyrate production by recombinant Escherichia coli based on genes related to synthesis pathway of PHB from Massilia sp. UMI-21

BackgroundPolyhydroxybutyrate (PHB) is currently the most common polymer produced by natural bacteria and alternative to conventional petrochemical-based plastics due to its similar material properties and biodegradability. Massilia sp. UMI-21, a newly found bacterium, could produce PHB from starch, maltotriose, or maltose, etc. and could serve as a candidate for seaweed-degrading bioplastic producers. However, the genes involved in PHB metabolism in Massilia sp. UMI-21 are still unclear.ResultsIn the present study, we assembled and annotated the genome of Massilia sp. UMI-21, identified genes related to the metabolism of PHB, and successfully constructed recombinant Escherichia coli harboring PHB-related genes (phaA2, phaB1 and phaC1) of Massilia sp. UMI-21, which showed up to 139.41% more product. Also, the vgb gene (encoding Vitreoscilla hemoglobin) was introduced into the genetically engineered E. coli and gained up to 117.42% more cell dry weight, 213.30% more PHB-like production and 44.09% more product content. Fermentation products extracted from recombinant E. coli harboring pETDuet1-phaA2phaB1-phaC1 and pETDuet1-phaA2phaB1-phaC1-vgb were identified as PHB by Fourier Transform Infrared and Proton nuclear magnetic resonance spectroscopy analysis. Furthermore, the decomposition temperature at 10% weight loss of PHB extracted from Massilia sp. UMI-21, recombinant E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb was 276.5, 278.7 and 286.3 °C, respectively, showing good thermal stability.ConclusionsHerein, we presented the whole genome information of PHB-producing Massilia sp. UMI-21 and constructed novel recombinant strains using key genes in PHB synthesis of strain UMI-21 and the vgb gene. This genetically engineered E. coli strain can serve as an effective novel candidate in E. coli cell factory for PHB production by the rapid cell growth and high PHB production.

Identification of novel coenzyme Q10 biosynthetic proteins Coq11 and Coq12 in Schizosaccharomyces pombe

Coenzyme Q (CoQ) is an essential component of the electron transport system in aerobic organisms. CoQ10 has ten isoprene units in its quinone structure and is especially valuable as a food supplement. However, the CoQ biosynthetic pathway has not been fully elucidated, including synthesis of the p-hydroxybenzoic acid (PHB) precursor to form a quinone backbone. To identify the novel components of CoQ10 synthesis, we investigated CoQ10 production in 400 Schizosaccharomyces pombe gene-deleted strains in which individual mitochondrial proteins were lost. We found that deletion of coq11 (an S.cerevisiae COQ11 homolog) and a novel gene designated coq12 lowered CoQ levels to ∼4% of that of the WT strain. Addition of PHB or p-hydroxybenzaldehyde restored the CoQ content and growth and lowered hydrogen sulfide production of the Δcoq12 strain, but these compounds did not affect the Δcoq11 strain. The primary structure of Coq12 has a flavin reductase motif coupled with an NAD+ reductase domain. We determined that purified Coq12 protein from S.pombe displayed NAD+ reductase activity when incubated with ethanol-extracted substrate of S.pombe. Because purified Coq12 from Escherichia coli did not exhibit reductase activity under the same conditions, an extra protein is thought to be necessary for its activity. Analysis of Coq12-interacting proteins by LC-MS/MS revealed interactions with other Coq proteins, suggesting formation of a complex. Thus, our analysis indicates that Coq12 is required for PHB synthesis, and it has diverged among species.

Erythromycin treatment under a specific nitrogen supply affects carbon metabolism and increases poly(3-hydroxybutyrate) and glycogen accumulation in cyanobacterium Synechocystis sp. PCC 6803

Under an adequate nitrate supply (BG11), cyanobacterium Synechocystis sp. PCC 6803 has high protein content but a low storage of glycogen and poly(3-hydroxybutyrate) (PHB). In contrast, when deprived of nitrogen (N), protein synthesis was repressed and the accumulation of glycogen and PHB was remarkably increased. The repressed protein synthesis caused by sulfur deprivation or treatment with chloramphenicol was also found to slightly enhance PHB accumulation in Synechocystis. Thus, the repressed protein synthesis is associated with the increased PHB and glycogen storage. Here, we examined two other antibiotics that inhibit protein synthesis, erythromycin (EM) and streptomycin (SM) for their effect on the accumulation of PHB and glycogen in Synechocystis. Under N cultivation, 10 μM EM treatment significantly increased the PHB level to 18 % (w/w cell dry weight [DW]) compared to 3 % (w/w DW) in the untreated cells; however, SM treatment did not increase the PHB level. Under BG11 cultivation, exposure to 0.7 μM SM and 5 μM EM enhanced glycogen accumulation up to 15 % and 41 % (w/w DW) respectively, compared to only 7 % (w/w DW) in the untreated cells. The EM treatment also remarkably reduced the total protein and phycobiliprotein contents. Results from metabolomic analysis suggested that EM treatment caused protein degradation into amino acids, some of which were metabolized into substrates that can be used for PHB and glycogen synthesis. This was substantiated by the result showing that EM treated cells had significantly increased levels of both the PHB-synthesizing substrates (acetyl-CoA, acetoacetyl-CoA, and 3-hydroxybutyryl-CoA) when cultured under N, and the glycogen-synthesizing substrates (fructose-1,6-BP, fructose-6-P, glucose-6-P, glucose-1-P, and ADP-glucose) when cultured in BG11. Therefore, EM treatment might be applicable for enhancing the accumulation of these two biopolymers in other cyanobacteria.

Engineering Escherichia coli for Poly-β-hydroxybutyrate Production from Methanol.

The naturally occurring one-carbon assimilation pathways for the production of acetyl-CoA and its derivatives often have low product yields because of carbon loss as CO2. We constructed a methanol assimilation pathway to produce poly-3-hydroxybutyrate (P3HB) using the MCC pathway, which included the ribulose monophosphate (RuMP) pathway for methanol assimilation and non-oxidative glycolysis (NOG) for acetyl-CoA (precursor for PHB synthesis) production. The theoretical product carbon yield of the new pathway is 100%, hence no carbon loss. We constructed this pathway in E. coli JM109 by introducing methanol dehydrogenase (Mdh), a fused Hps-phi (hexulose-6-phosphate synthase and 3-phospho-6-hexuloisomerase), phosphoketolase, and the genes for PHB synthesis. We also knocked out the frmA gene (encoding formaldehyde dehydrogenase) to prevent the dehydrogenation of formaldehyde to formate. Mdh is the primary rate-limiting enzyme in methanol uptake; thus, we compared the activities of three Mdhs in vitro and in vivo and then selected the one from Bacillus methanolicus MGA3 for further study. Experimental results indicate that, in agreement with the computational analysis results, the introduction of the NOG pathway is essential for improving PHB production (65% increase in PHB concentration, up to 6.19% of dry cell weight). We demonstrated that PHB can be produced from methanol via metabolic engineering, which provides the foundation for the future large-scale use of one-carbon compounds for biopolymer production.

Optimizing heterotrophic nitrification process: The significance of demand-driven aeration and organic matter concentration

Heterotrophic nitrification and aerobic denitrification (HNAD) sludge were successfully acclimated. The effects of organics and dissolved oxygen (DO) on nitrogen and phosphorus removal by the HNAD sludge were investigated. The nitrogen can be heterotrophically nitrified and denitrified in the sludge at a DO of 6 mg/L. The TOC/N (total organic carbon to nitrogen) ratio of 3 was found to result in removal efficiencies of over 88% for nitrogen and 99% for phosphorus. The use of demand-driven aeration with a TOC/N ratio of 1.7 improved nitrogen and phosphorus removal from 35.68% and 48.17% to 68% and 93%, respectively. The kinetics analysis generated an empirical formula, Ammonia oxidation rate = 0.08917·(TOC·Ammonia)0.329·Biomass0.342. The nitrogen, carbon, glycogen, and poly-β-hydroxybutyric acid (PHB) metabolism pathways of HNAD sludge were constructed using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The findings suggest that heterotrophic nitrification precedes aerobic denitrification, glycogen synthesis, and PHB synthesis.

Hybrid synthesis of polyhydroxybutyrate bioplastics from carbon dioxide

We report a hybrid chem-biological system, coupling photovoltaic hydrogen production, chemical CO2hydrogenation, an artificial carbon conversion pathway and the natural PHB synthesis pathway, which drove a completely closed-loop economy for PHB production.

Effect of acute vs chronic stress on Polyhydroxybutyrate production by indigenous cyanobacterium

Fossil-based plastic has become a global-threat due to its high stability and transformation into more lethal forms such as micro plastics with time. An alternative should be found to combat this global enemy. Polyhydroxybutyrate or PHB can be such an alternative to plastic. Present study explores the synthesis of PHB in Neowollea manoromense, using two different cultivation-approaches: acute and chronic stress. This study has used 6 carbon sources and 3 different level of phosphate to study the accumulation of PHB along with lipid, carbohydrate, and proteins. Highest PHB in chronic-stress was achieved under glucose supplementation without phosphate at 21st day (156.5 ± 22.5 μg/mg), whereas in acute-stress, it was achieved under acetate without phosphate (91.0 ± 2.7 μg/mg). Despite higher accumulation in chronic-stress, high PHB productivity was achieved in acute-stress. Principal Component Analysis suggests that all the variables were positively correlated with each other. Here we first report PHB accumulation in Neowollea manoromense. This study highlights that acute-stress can be a powerful tool in establishment of a sustainable cyanobacteria based bio refinery for PHB production.

Coupling continuous poly(3-hydroxybutyrate) synthesis with piperazine-contained wastewater treatment: Fermentation performance and microbial contamination deciphering

Open poly(3-hydroxybutyrate) (PHB) fermentation is of great potential, and batch PHB synthesis with piperazine as the nitrogen switch has been realized. However, it is vital to explore the feasibility of continuous PHB fermentation with piperazine-contained wastewater remediation collaboratively. Here, an aerobic membrane bioreactor was constructed for consecutive PHB synthesis. The removal efficiency of piperazine decreased from 100 % to 82.6 % after three cycles, meanwhile, the PHB concentration was 0.39 g·L−1, 0.18 g·L−1, and undetected for each cycle. Microbial community analysis showed that Proteobacteria, Actinobacteriota, and Bacteroidota were the main contaminating microbes. Furthermore, three metagenome-assembled genomes related to Flavobacterium collumnare, Herbaspirillum aquaticum, and Microbacterium enclense were identified as the dominant contaminating strains. These microbes obtained nitrogenous substrates transformed by Paracoccus sp. TOH, such as amino acids and dissolved organic matter, as nutrient for accumulation. This study verified the practicability of coupling continuous PHB synthesis with industrial wastewater treatment and revealed the derivation mechanism of contaminating species, which could provide a reference for the targeted nitrogen release gene knockout of functional PHB fermentation chassis.