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Production scale and PHA Productivity

Keywords: Archaea; Bacteria; Biopolymers; Extremophiles; Halophiles; Polyhydroxyalkanoate; Psychrophiles; Thermophiles

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Polyhydroxyalkanoates - AOCS Lipid Library

AB - Polyhydroxyalkanoate (PHA) granules with core-shell layered microstructure were synthesized in Ralstonia eutropha using periodic feeding of valeric acid into a growth medium containing excess fructose. The O2 consumption and CO2 evolution rates, determined by off-gas mass spectrometry, have been used as sensitive measures to indicate the type of nutrients utilized by R. eutropha during PHA synthesis. Domains of poly-3-hydroxybutyrate (PHB) were formed during polymer storage conditions when only fructose was present. Feeding of valeric acid (pentanoic acid) resulted in the synthesis of hydroxyvalerate (HV) monomers, forming a poly-3-hydroxybutyrate-co-valerate (PHBV) co-polymer. The synthesis of desired polymer microstructures was monitored and controlled using online mass spectrometry (MS). The respiratory quotient (RQ) was unique to the type of polymer being synthesized due to increased O2 consumption during PHBV synthesis. MS data was used as the control signal for nutrient feeding strategies in the bioreactor. The core-shell structures synthesized were verified in cells using transmission electron microscopy after thin sectioning and staining with RuO4. It was demonstrated that the synthesis of core-shell microstructures can be precisely controlled utilizing a MS feedback control system.

Chemical and pharmacokinetic properties and preliminary pharmacotoxicological data.

Figure 2: Metalophil production strain Cupriavidus necator DSM 545 with PHA granules (bright intracellular inclusions) imaged by STEM, magnification x 30,000. By curtesy by E. Ingolić, FELMI-ZFE Graz.

Polyhydroxyalkanoate (pha): review of synthesis ..

Table 1: Overview on most promising PHA production processes by extremophiles.

Polyhydroxyalkanoate-degrading PHA microorganisms secrete PHA depolymerases, which hydrolyze the polymer extracellularly to water-soluble products and utilize the hydrolysis products as carbon and energy sources for growth [, ].

18 Rev1)[1] : Aliphatic, alicyclic and aromatic saturated and unsaturated tertiary alcohols, aromatic tertiary alcohols and their esters from chemical groups 6 and 8
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Biosynthesis of polyhydroxyalkanoate (PHA) copolymer …

Static cultivation on poly[styrene] biofilms biofilm vs. shaking flask cultures PHA productivity not reported

N2 - The role of glycogen in the uptake of acetate in anaerobic-aerobic activated sludge without enhanced biological phosphorus removal were investigated. Although the polyphosphate content of the sludge was minimized by lowering the phosphorus feeding concentration, significant acetate uptake and accumulation of polyhydroxyalkanoates (PHAs) were observed in proportion to glycogen consumption under anaerobic conditions. The results of anaerobic inhibition studies, which showed suppressive effects on acetate uptake by a glycolysis inhibitor (iodoacetate) but not by a membrane ATPase inhibitor (N,N′-dicyclohexyl carbodiimide), supported an assumption that glycogen degradation through glycolysis supplies the required ATP and reducing power for PHA synthesis from acetate and consumed glycogen. Under subsequent aerobic conditions, the accumulated PHAs were depleted and the consumed glycogen recovered to the same level as that at the start of the anaerobic phase. Iodoacetate also inhibited the recovery of glycogen under aerobic conditions, suggesting that nearly 50% of the PHAs depleted was used for glycogen synthesis through reversed glycolysis.

µmax: Maximum Specific Growth Rate; 16S rRNA: 16 Svedberg rRibosomal RNA Sequencing; 3HB: 3-HydroxyButyrate; 3HD: 3-HydroxyDecanoate; 3HDD: 3HydroxyDocecanoate; 3HHx: 3-HydroxyHexanoate; 3HN: 3-HydroxyNonanoate; 3HO: 3-HydroxyOctanoate; 3HV: 3HydroxyValerate; 3UD: 3-HydroxyUnDecanoate; 4HB: 4-HydroxyButyrate; AC: Acetyl-CoA synthetase; BOD: Biochemical Oxygen Demand; b-PHA: Blocky Structured PolyhydroxyAlkanoate; CBF: Cyclic Batch Fermentation; CDM: Cell Dry Mass; CFBF: Cyclic Fed-Batch Fermentation; COD: Chemical Oxygen Demand; CT: CoA Transferase; DSC: Differential Scanning Calorimetry; ECS: Extruded Corn Starch; EPS: Extracellular Polysaccharides; ERB: Extruded Rice Bran; FT-IR: Fourier Transform Infrared Spectroscopy; GBL: γ-ButyroLactone; GC-MS: Gas Chromatography coupled to Mass Spectroscopy; Hm: Melting Enthalpyk; Da: 103 Dalton; LPS: Lipopolysaccharides; MCL: Medium Chain Length; MW: Weight Average Molecular Mass; NADH: Nicotinamide Adenine Dinucleotide; NADPH: Nicotinamide Adenine Dinucleotide Phosphate; NMR: Nuclear Magnetic Resonance; PCR: Polymerase Chain Reaction; PHA: PolyHydroxyAlkanoate; PHB: Poly (3-HydroxyButyrate); PHBHV: Poly (3-HydroxyButyrate-co-3-HydroxyValerate); PHV: Poly (3-HydroxyValerate); Pi: Polydispersity; qp: Specific Production rate; scl: Short Chain Length; SDS: Sodium Dodecyl Sulfate; SEM: Scanning Electron Microscopy; SSCAs: Star-Shaped Cell Aggregates; STEM: Scanning Transmission Electron Microscopy; TCA: Citric Acid Cycle; TDS: Total Dissolved Solids; TEM: Transmission Electron Microscopy; Td: Onset of polymer Decomposition Temperature; Tg: Glass Transition Temperature; Tm: Melting Temperature; Xc: Degree of Crystallinity

In this work, we studied the effect of the capability to accumulate PHAs on biosurfactant production and microbial attachment to hydrocarbons (MATH).
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    Additionally, polyhydroxyalkanoate (PHA) accumulation was proven to improve tolerance to stressful conditions.

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Production of Polyhydroxyalkanoate (PHA) …

Polyhydroxyalkanoic acids (PHAs) are a significant type of biodegradable plastics, since they possess properties similar to conventional plastics. They are completely biodegradable but may be melted and modelled, making them ideal for use in consumer products. , displays the typical PHA structure, as well as the structures of most important PHAs: poly (3-hydroxybutyrate) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) [].

Metabolic modelling of polyhydroxyalkanoate …

In the context of alkaliphily, as described for H. campestris, also cyanobacterial strains were found which preferably accumulate PHA under elevate pH-values. This is especially the case for Spirulina platensis; for this organism, the optimum pH-value for both PHA production and degradation is reported in the rather strong alkaline range of 9 to 11 [73].

in the biosynthesis of polyhydroxyalkanoate ..

Han et al. [76] were the first who experimentally investigated the genes responsible for archaeal PHA synthesis. The authors revealed that the haloarchaeon Haloarcula marismortui, isolated from the Dead Sea, was able to accumulate up to 21 wt.-% PHB in CDM in minimal medium containing excessive glucose, and identified the adjacent genes phaEHm and phaCHm which encode two subunits of a Class-III PHA synthase and are directed by a single promoter upstream of the transcriptional start site; the genes were constitutively expressed under both nutritionally balanced and nutrient-limited conditions. Remarkably, PhaCHm turned out to be, in contrast to PhaEHm, strongly connected to the PHA granules. The introduction of phaEHm or phaCHm into Haloarcula hispanica, a strain harboring highly homologous phaECHh genes and widely used in haloarchaeal studies, particularly for isolating haloviruses, boosted PHB synthesis in this recombinant organism; coexpression of both genes resulted in highest PHB production. It is worth to mnetion that the knockout of phaECHh genes in H. hispanica resulted in a total termination of PHA synthesis. PHA accumulation capability and PHA synthase activity was successfully restored by complementation with phaECHm genes. These outcomes demonstrated the importance of phaEC genes for PHA accumulation in Archaea. Later, Ding et al. [77] carried out the sequencing of the genome of a H. hispanica ssp., and observed significant differences from the published gene sequence of the model strain H. hispanica ATCC 33960.

Polyhydroxyalkanoate containing amide group, sulfonic ..

In addition, Halomonascampaniensis LS21, a halophile and in parallel alkalophile eubacterium, was isolated and cultivated in an open, non-sterile, continuously operated PHA production process. This process was based on alkaline seawater and artificial carbonaceous kitchen waste, predominately consisting of polysaccharides, lipids, and proteins. PHB was selected as model product of the strain during long-term cultivation to assess the general viability of open and long-term cultivation of industrially relevant bacteria, and to investigate genetic engineering of this organism to further enhance the process. Both the wild type strain and a recombinant H. campaniensis LS21, additionally equipped with the PHB synthesis genes phbCAB, were farmed in a continuous process for 65 days in artificial seawater-based medium. Under extremely saline (27 g/L NaCl) and highly alkaline (pH-value 10) conditions and a moderate temperature of 37 °C according to the strain´s optimum values, the genetically engineered strain achieved about 70 wt.-% PHB in CDM, in contrast to 26 wt.-% achieved by the wild type organism. Despite the open process regime, both cultures remained monoseptic. Extracellular hydrolytic enzymes were excreted during the entire cultivation, which enabled them to convert the mixed substrates. Until the end of the cultivation, the plasmid carrying phbCAB genes maintained stable in the recombinant cells. Combined with its expedient susceptibility for genetic improvement, H. campaniensis LS21 definitely might constitute a powerful cellular factory for cost- and energy-efficient production of PHA or extremozymes from inexpensive feedstocks [72].

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