Polyhydroxyalkanoates (PHAs) as Biomaterials in Tissue Engineering: Production, Isolation, Characterization
Abstract
:1. Introduction
2. Structure and Properties of Polyhydroxyalkanoates
- (a)
- The structure of the radicals attached to the carbon atoms with the R configuration in the skeleton of the polymer chain; these radicals represent the side chain of monomeric hydroxy acids;
- (b)
- The number and structure of the monomers in the polymer chain.
- PHA is made up of monomers with 3–5 carbon atoms and called PHA with short side chains, scl-PHA (short-chain-length—PHA);
- PHA is composed of monomers with 6–14 C atoms and called PHA with medium side chains, mcl-PHA (medium-chain-length—PHA);
- PHA is composed of mixed monomers, both with a short side chain (3–5 C atoms) and a long one (6–14 C atoms), called scl-mcl-PHA and later discovered in the first two categories.
3. Production PHA
3.1. Biological Synthesis
3.1.1. Metabolic Pathways for PHA Biosynthesis
3.1.2. Microorganisms Producing PHA
3.1.3. Cultivation and Product Biosynthesis Media
- Substrates that support both cell growth and Poly (3HA) production;
- Substrates that support cell growth but not the production of Poly (3HA);
- Substrates that do not support cell growth but support the production of Poly (3HA).
3.1.4. Fermentation Bioprocess
4. Isolation and Purification
4.1. Solvent Extraction
4.2. Digestion of Non-PHA Cell Mass (NPCM)
Method | Chemical | Conditions | Purity and Recovery | Reference |
---|---|---|---|---|
Solvent extraction | Chloroform | Mixing continuously at 25 °C for 12 h | Purity: 94.0–96.0% Recovery: 65–70% | [130] |
Methylene chloride | Mixing continuously t 25 °C for 12 h | Purity: 95–98% Recovery: 24–25% | [130] | |
1,2-Dichloroethane | Mixing continuously at 25 °C for 12 h | Purity: 93–98% Recovery: 66–70% | [130] | |
Acetone | Continuous mixing at 120 °C, 7 bar for 20 min under anaerobic conditions, followed by filtering hot solution and cooling it to 4 °C to precipitate polymer | Purity: 98.4% Recovery: 96.8% | [129] | |
Medium-chain-length alcohols | In continuous stirred tank reactors, a multi-stage extraction technique is used. Cool the extract to recover the polymer after removing the cell debris | Purity: >98.0% Recovery: 95.0% | [131] | |
Hypochlorite digestion | Sodium hypochlorite | Biomass concentration: 10–40 g/L; pH: 8–13.6; Temperature: 0–25 °C; Digestion time: 10 min–6 h; Hypochlorite concentration: 1–10.5% weight/volume (w/v) | Purity: 90–98.0% Recovery: 90–95% | [132] |
Sodium hypochlorite and chloroform | Biomass concentration: 1% (w/v); Temperature: 30 °C; Digestion time: 1 h; Hypochlorite concentration: 3–20% (v/v) | Purity: 86.0% Recovery: NG Purity: 93.0% Recovery: NG | [133] | |
Enzyme digestion | Trypsin, bromelain, pancreatin | Digestion with 2% trypsin (50 °C, pH 9.0, 1 h) or 2% bromelain (50 °C, pH 4.75, 10 h) or 2% pancreatin (50 °C, pH 8.0, 8 h), followed by centrifugation then washing with 0.85% saline solution | Purity: 87.7–90.3% Recovery: NG | [134] |
4.3. Purification of PHA
5. Characterization, Methods and Results
5.1. Monomeric Composition and Molecular Distribution
5.2. Thermal Properties
5.3. Crystallinity
5.4. Mechanical Properties
5.5. Biocompatibility and Biodegradability
6. Brief Review of PHA Biomedical Applications
7. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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X | R (Radical) | Monomer Name | Monomer Add. | Polymer Name | Polymer Add. |
---|---|---|---|---|---|
1 | H | 3-hydroxypropionate | 3HP | Poly-(3-hydroxypropionate) | 3PHP |
CH3– | 3-hydroxybutyrate | 3HB | Poly-(3-hydroxybutyrate) | 3PHB | |
CH3–CH2– | 3-hydroxyvalerate | 3HV | Poly-(3-hydroxyvalerate) | 3PHV | |
CH3–CH2–CH2– | 3-hydroxycaproate | 3HC | Poly-(3-hydroxyhexanoate) | 3PHC | |
CH3–(CH2)2–CH2– | 3-hydroxyheptanoate | 3HH | Poly-(3-hydroxyheptanoate) | 3PHH | |
CH3–(CH2)3–CH2– | 3-hydroxyoctanoate | 3HO | Poly-(3-hydroxyoctanoate) | 3PHO | |
CH3–(CH2)4–CH2– | 3-hydroxynonanoate | 3HN | Poly-(3-hydroxynonanoate) | 3PHN | |
CH3–(CH2)5–CH2– | 3-hydroxydecanoate | 3HD | Poly-(3-hydroxydecanoate) | 3PHD | |
CH3–(CH2)6–CH2– | 3-hydroxyundecanoate | 3HUD | Poly-(3-hydroxyundecanoate) | 3PHUD | |
CH3–(CH2)7–CH2– | 3-hydroxydodecanoate | 3HDD | Poly-(3-hydroxydodecanoate) | 3PHDD | |
2 | H | 4-hydroxybutyrate | 4HB | Poly-(4-hydroxybutyrate) | 4PHB |
3 | H | 5-hydroxyvalerate | 5HV | Poly-(5-hydroxyvalerate) | 5PHB |
Microorganism | Carbon Source | PHA Content (% Cell Dry Mass) | PHA Monomer or Polymer | References |
---|---|---|---|---|
Alcaligenes latus | Sucrose, mart, soy waste, milk waste sesame oil | 31.0 | P3HB | [55,56] |
mAzotobacter chroococcum | wastewater from olive oil mills | 80 | P3HB P[HB-co-HV] | [39,57] |
Azotobacter beijerinckii | Glucose | 24.8 | P3HB | [58] |
Bacillus megaterium | Citric acid, glucose, glycerol, succinic acid, octanoic acid | 3.0–48.0 | P3HB, scl-mcl-PHA, mcl-PHA | [59] |
various Bacillus spp. type strains | Acetate, valerate 3-hydroxybutyrate, propionate, sucrose, | 2.2–47.6 | 3HB, 3HV, 3HHx | [39,60] |
Corynebacterium glutamicum | Acetic acid, citric acid, glucose, glycerol, succinic acid | 4.0–32.0 | P3HB, mcl-PHA | [59] |
Corynebacterium hydrocarboxydans | Acetate, glucose | 8.0–21.0 | 3HB, 3HV | [18] |
Cupriavidus necator (formerly Hydrogenomonas eutropha, Alcaligenes eutrophus, Ralstonia eutropha and Wautersia eutropha) | Glucose | 76.0 | P3HB | [61] |
Potato starch, saccharified Waste | 46.0 | P3HB | [62] | |
Escherichia coli mutants | Glucose, glycerol, palm oil, sucrose, molasses | (UHMV)P3HB | [39,40] | |
Halomonas boliviensis | Hydrolyzed starch, maltose | 56.0 | P3HB | [39,63] |
Haloferax mediterranei | Whey sugars | 72.8 | P-(3HB-co-3HV) | [64] |
Pseudomonas aeruginosa | Glucose, technical oleic acid, waste free fatty acids, waste free flying oil | 25.0 | mcl-PHAs | [39,49,65] |
Pseudomonas fluorescens | Citric acid, glucose, fatty acids | 28.17–39.01 | mcl-PHA | [65,66] |
Pseudomonas mendocina | 1,3-Butanediol, octanoate | 13.5–19.3 | scl-mcl-PHA | [67] |
Pseudomonas oleovorans | 4-Hydroxyhexanoic acid | 18.6 | scl-mcl-PHA | [39,51,56] |
Pseudomonas putida | Glucose, octanoic acid, undecenoic acid | 61.8–67.1 | mcl-PHA | [65,68] |
Pseudomonas putida KT2440 | Glucose | 32.1 | mcl-PHA | [69] |
4-Hydroxyhexanoic acid | 25.3–29.8 | mcl-PHA | [70] | |
Nonanoic acid | 26.8–75.4 | mcl-PHA | [71] | |
Pseudomonas stutzeri | Glucose, soybean oil, alcohols, alkanoates | 21–65 | mcl-PHA | [39,72] |
Thermus thermophiles | Whey | 35.6 | scl-mcl-PHA | [73] |
Various Streptomyces spp. type culture | Glucose, malt, soy waste, sesame oil | 1.2–82.0 | P3HB | [39,60] |
No | Strains | C8 (g/L) | Fermentation Final | ||
---|---|---|---|---|---|
pH | OD 1 | DC 2 (g/L) | |||
1 | P.putida | 8.51 | 7.74 | 0.559 | 1.86 |
2 | P.putida:B.subtilis BSP (3:1) | 8.51 | 7.55 | 0.599 | 3.96 |
3 | P.putida:B.subtilis BSV (3:1) | 8.51 | 7.60 | 0.562 | 3.93 |
Characteristics | Method | Typical Conditions | Reference |
---|---|---|---|
PHA monomeric composition | Gas chromatography (GC) | In GC-FID analysis, a BP-20 polar capillary column was used. This column or an HP-5MS capillary column could be used in GS-MS chromatography. | [141,142] |
Liquid chromatography (LC) | A UV detector at 210 nm and an ion-exclusion organic acid analysis column are used in high-performance liquid chromatography. For HPLC-MS analysis, a C18 column is used for separation. The source parameters are optimized to obtain the dominant ions for all compounds and keep them constant throughout the analysis. | [143,144] | |
PHA polymeric composition | Nuclear magnetic resonance (NMR) | Chemical changes were expressed in ppm relative to the remaining chloroform signals as an internal reference (1H NMR: 7.26 ppm; 13C NMR: 77.0 ppm). At 499.883 MHz, a 1H NMR spectrum was acquired using the following parameters: 6.7 s 90° pulse duration, 4112 Hz spectral width, 64k data points, 24 scans, and a relaxation delay of 20 s. 13C NMR spectrum was recorded at 125.709 MHz with 1H WALTZ decoupling. Other parameters were chosen as follows: 6.45 s 45° pulse length, 25,510 Hz spectral width, 64 k datapoints, 24,000 scans, relaxation delay 10 s, and decoupling field 2.5 kHz | [145] |
Matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) | The MALDI-TOF mass spectra is using a delay extraction procedure with ion detection in linear mode: 25 kV applied after 2600 ns with a potential gradient of 454 V/mm and a wire voltage of 25 V. The laser irradiation was slightly above the threshold to prevent polymer fragmentation: 106 W/cm2, and each spectrum can have an average of 32 laser pulses. | [146] | |
Molecular distribution | Gel permeation chromatography (GPC) | Samples were diluted to a concentration of 0.5 mg/mL in chloroform and placed in an orbital shaker for 16 h. To facilitate dissolution, samples were heated to 60 °C for 5 min, when necessary. HPLC-grade chloroform was used as the mobile phase, and samples were processed at a flow rate of 1 mL/min. The detector temperature was set to 45 °C. | [147] |
Thermal properties | Differential scanning calorimetry (DSC) | The samples were evaluated under dry nitrogen. 6–8 mg samples were enclosed in hermetic aluminum pans, equilibrated at 70 °C, and held isothermally for 5 min. They were then heated to 100 °C at a rate of 5 °C/min, kept isothermally for 3 min, and then cooled to 70 °C at a rate of 5 °C/min. Finally, the samples were reheated to 100 °C at a rate of 5 °C/min. While calculating the percentage crystallinity, the fusion heat on cold crystallization was calculated using the heat flux of melting from the second heat cycle. | [148] |
Thermogravimetric analysis (TGA) | A sample was placed on platinum pan for each analysis. A nitrogen atmosphere was used at, 50 mL/min, for analysis. Furnace temperature was set from 0 °C to 800 °C at a heating ramp of 10 °C/min. Temperature accuracy can be maintained ±0.25 °C. | [149] | |
Crystallinity | Fourier transform infrared spectroscopy (FTIR) | The samples are a mixt of 5 mg PHA with 100 mg of KBr and pelletized. The infrared spectra were obtained in the 4000 to 400 cm−1 wavenumber range. Sample was melt at 100 °C for 2 min in FTIR hot stage under the protection of dry nitrogen gas. The amorphous sample was then quenched to selected temperature by a flow of liquid nitrogen for isothermal melt-crystallization. Afterward, the isothermally crystallized samples were heated again at 1 °C/min. | [150,151] |
X-ray diffraction | The samples were size of 10 mm × 10 mm for testing. The diffractometer with Cu-Kα radiation, wavelength = 1.542 Å, scanning from 10° to 50° in 2θ at a scanning speed of 10°/min. | [152] | |
Mechanical properties | Mechanical testing machine | Film strips: 135 mm × 22 mm, were tested with static load cell; maximum load of 5KN (Rating = ± 50 N; Max Torque = ± 1.5 N m) for a temperature range of −29 to 82 °C was used. A 125 mm initial gap separation and a separation rate of 12.5 mm min−1 were used for tensile testing at room temperature. | [153] |
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Miu, D.-M.; Eremia, M.C.; Moscovici, M. Polyhydroxyalkanoates (PHAs) as Biomaterials in Tissue Engineering: Production, Isolation, Characterization. Materials 2022, 15, 1410. https://doi.org/10.3390/ma15041410
Miu D-M, Eremia MC, Moscovici M. Polyhydroxyalkanoates (PHAs) as Biomaterials in Tissue Engineering: Production, Isolation, Characterization. Materials. 2022; 15(4):1410. https://doi.org/10.3390/ma15041410
Chicago/Turabian StyleMiu, Dana-Maria, Mihaela Carmen Eremia, and Misu Moscovici. 2022. "Polyhydroxyalkanoates (PHAs) as Biomaterials in Tissue Engineering: Production, Isolation, Characterization" Materials 15, no. 4: 1410. https://doi.org/10.3390/ma15041410