Generic placeholder image

Current Pharmaceutical Biotechnology


ISSN (Print): 1389-2010
ISSN (Online): 1873-4316

Research Article

The Production of Biodegradable Polymers-medium-chain-length Polyhydroxyalkanoates (mcl-PHA) in Pseudomonas putida for Biomedical Engineering Applications

Author(s): Nicoleta Ene, Mariana-Gratiela Soare Vladu, Irina Lupescu, Ana-Despina Ionescu and Emanuel Vamanu*

Volume 23, Issue 8, 2022

Published on: 10 January, 2022

Page: [1109 - 1117] Pages: 9

DOI: 10.2174/1389201022666210810114117

Price: $65


Background: Polyhydroxyalkanoates (PHAs) are bacteria-synthesized biopolymers under imbalanced growth conditions. These biopolymers are acknowledged as potential biomaterials for future applications because of their characteristics of biocompatibility and biodegradability, and ability to be produced rapidly, and strong functionality of mechanical resistance. This article aims to perform microbial fermentation using the Pseudomonas putida strain to identify the quantity of biopolymers, particularly of the medium-chain-length (mcl-PHA) polyhydroxyalkanoates, based on the type and quantity of the added precursors (glucose and fatty acids).

Methods: To understand the microbial interaction and the mechanism involved in PHA biosynthesis, several methods were employed and microbial biomass was obtained using the Pseudomonas putida strain capable of producing PHA. The polymer production by acetone extraction was analyzed using the Soxhlet method, while the biopolymer purification was done via the methanol-ethanol treatment, after which the biomass estimation was done through spectrophotometric analysis. This was followed by measuring the dry weight of the cells and quantification of the biopolymer produced using the gas chromatography method (GC).

Results: The highest PHA yield was obtained using the octanoic (17 mL in 2000 mL medium) and hexanoic acids (14 mL in 2000 mL medium) as the precursors. As a result, the octanoic acid - octanoic acid, heptanoic acid – nonanoic acid, and octanoic acid - hexanoic acid were identified as the different precursors that supported the quantity of PHA obtained.

Conclusion: Among the 4 types of structurally related substrates, the Pseudomonas putida ICCF 319 strain showed a preference for the C8 sublayer for the biosynthesis of the elastomeric PHAs composed predominantly of more C8 monomers than the C6 and C10.

Keywords: Polyhydroxyalkanoates, biopolymer, pseudomonas putida, biocompatibility, fatty acids, tissue regeneration.

Graphical Abstract
Dietrich, K.; Dumont, M.J.; Del Rio, L.F.; Orsat, V. Producing PHAs in the bioeconomy-towards a sustainable bioplastic. Sustain. Prod. Consum, 2017, 9, 58-70.
Takabatake, H.; Satoh, H.; Mino, T.; Matsuo, T. PHA (polyhydroxyalkanoate) production potential of activated sludge treating wastewater. Water Sci. Technol., 2002, 45(12), 119-126.
[] [PMID: 12201092]
Bugnicourt, E.; Cinelli, P.; Lazzeri, A.; Alvarez, V.A. Polyhydroxyalkanoate (PHA): Review of synthesis, characteristics, processing and potential applications in packaging. Express Polym. Lett., 2014, 11(8), 791-808.
Weng, Y.X.; Wang, X.L.; Wang, Y.Z. Biodegradation behavior of PHAs with different chemical structures under controlled composting conditions. Polym. Test., 2011, 30(4), 372-380.
Amelia, T.S.M.; Govindasamy, S.; Tamothran, A.M.; Vigneswari, S.; Bhubalan, K. Applications of PHA in agriculture; Springer, 2019, pp. 347-361.
Ong, S.Y.; Chee, J.Y.; Sudesh, K. Degradation of Polyhydroxyalkanoate (PHA): A review. J. Siber. Fed. Univ., 2017, 10(2), 211-225.
Tokiwa, Y.; Calabia, B.P. Degradation of microbial polyesters. Biotechnol. Lett., 2004, 26(15), 1181-1189.
[] [PMID: 15289671]
Santhanam, A.; Sasidharan, S. Microbial production of Polyhydroxy Alkanotes (PHA) from Alcaligens spp. and Pseudomonas oleovorans using different carbon sources. Afr. J. Biotechnol., 2010, 9(21), 3144-3150.
Singh, A.K.; Mallick, N. Enhanced production of SCL-LCL-PHA co-polymer by sludge-isolated Pseudomonas aeruginosa MTCC 7925. Lett. Appl. Microbiol., 2008, 46(3), 350-357.
[] [PMID: 18221276]
Zinn, M.; Durner, R.; Zinn, H.; Ren, Q.; Egli, T.; Witholt, B. Growth and accumulation dynamics of Poly(3-hydroxyalkanoate) (PHA) in Pseudomonas putida GPo1 cultivated in continuous culture under transient feed conditions. Biotechnol. J., 2011, 6(10), 1240-1252.
[] [PMID: 21751398]
Chen, G.Q.; Wu, Q. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials, 2005, 26(33), 6565-6578.
[] [PMID: 15946738]
Thorat Gadgil, B.S.; Killi, N.; Rathna, G.V.N. Polyhydroxyalkanoates as biomaterials. MedChemComm, 2017, 8(9), 1774-1787.
[] [PMID: 30108887]
Philip, S.; Keshavarz, T.; Roy, I. Polyhydroxyalkanoates: Biodegradable polymers with a range of applications. J. Chem. Technol. Biotechnol., 2007, 82(3), 233-247.
Singh, M.; Kumar, P.; Ray, S.; Kalia, V.C. Challenges and opportunities for customizing polyhydroxyalkanoates. Int. J. Microbiol., 2015, 55(3), 235-249.
[] [PMID: 26063933]
Nigmatullin, R.; Thomas, P.; Lukasiewicz, B.; Puthussery, H.; Roy, I. Polyhydroxyalkanoates, a family of natural polymers, and their applications in drug delivery. J. Chem. Technol. Biotechnol., 2015, 90(7), 1209-1221.
Jung, Y.C.; Bhushan, B. Contact angle, adhesion and friction properties of micro-and nanopatterned polymers for superhydrophobicity. Nanotechnology, 2006, 17(19), 4970.
Mathew, A.P.; Oksman, K.; Sain, M. Mechanical properties of biodegradable composites from Poly Lactic Acid (PLA) and Microcrystalline Cellulose (MCC). J. Appl. Polym. Sci., 2005, 97(5), 2014-2025.
Siracusa, V.; Rocculi, P.; Romani, S.; Dalla Rosa, M. Biodegradable polymers for food packaging: A review. Trends Food Sci. Technol., 2008, 19(12), 634-643.
Poltronieri, P.; Kumar, P. Polyhydroxyalkanoates (PHAs) in industrial applications. Handbook of ecomaterials; Springer International Publishing: Cham, 2017, pp. 1-30.
Gao, X.; Chen, J.C.; Wu, Q.; Chen, G.Q. Polyhydroxyalkanoates as a source of chemicals, polymers, and biofuels. Curr. Opin. Biotechnol., 2011, 22(6), 768-774.
[] [PMID: 21705209]
Masood, F. Polyhydroxyalkanoates in the food packaging industry; Academic Press, 2017, pp. 153-177.
Kourmentza, C.; Costa, J.; Azevedo, Z.; Servin, C.; Grandfils, C.; De Freitas, V.; Reis, M.A.M. Burkholderia thailandensis as a microbial cell factory for the bioconversion of used cooking oil to polyhydroxyalkanoates and rhamnolipids. Bioresour. Technol., 2018, 247, 829-837.
[] [PMID: 30060419]
Pardo‐Ibáñez, P.; Lopez‐Rubio, A.; Martínez‐Sanz, M.; Cabedo, L.; Lagaron, J.M. Keratin-polyhydroxyalkanoate melt‐compounded composites with improved barrier properties of interest in food packaging applications. J. Appl. Polym. Sci., 2014, 131(4), 39947.
Cavaliere, C.; Montone, C.M.; Capriotti, A.L.; La Barbera, G.; Piovesana, S.; Rotatori, M.; Valentino, F.; Laganà, A. Extraction of polycyclic aromatic hydrocarbons from polyhydroxyalkanoates before gas chromatography/mass spectrometry analysis. Talanta, 2018, 188, 671-675.
[] [PMID: 30029430]
Hiraishi, A.; Khan, S.T. Application of polyhydroxyalkanoates for denitrification in water and wastewater treatment. Appl. Microbiol. Biotechnol., 2003, 61(2), 103-109.
[] [PMID: 12655451]
Jamil, N.; Ahmed, N. Production of biopolymers by Pseudomonas aeruginosa isolated from marine source. Braz. Arch. Biol. Technol., 2008, 51(3), 457-464.
Zhang, F.; Keasling, J. Biosensors and their applications in microbial metabolic engineering. Trends Microbiol., 2011, 19(7), 323-329.
[] [PMID: 21664818]
Mohiuddin, M.; Kumar, B.; Haque, S. Biopolymer composites in photovoltaics and photodetectors; Elsevier, 2017, pp. 459-486.
Dwivedi, R.; Pandey, R.; Kumar, S.; Mehrotra, D. Poly Hydroxyalkanoates (PHA): Role in bone scaffolds. J. Oral Biol. Craniofac. Res., 2020, 10(1), 389-392.
[] [PMID: 31754599]
Zadorojnâi, L.; Zadorojnîi, A. Hyaluronic acid: obtaining, properties and application. Chem. J. Moldova, 2012, 7(2), 57-66.
Struszczyk, M.H. Global requirements for medical applications of chitin and its derivatives; Polish Chitin Society: Lodz, 2006. Monograph XI
Lu, X.; Wang, L.; Yang, Z.; Lu, H. Strategies of polyhydroxyalkanoates modification for the medical application in neural regeneration/nerve tissue engineering. Adv. Biosci. Biotechnol., 2013, 4(06), 731-740.
Anjum, A.; Zuber, M.; Zia, K.M.; Noreen, A.; Anjum, M.N.; Tabasum, S. Microbial production of Polyhydroxyalkanoates (PHAs) and its copolymers: A review of recent advancements. Int. J. Biol. Macromol., 2016, 89, 161-174.
[] [PMID: 27126172]
Chardron, S.; Bruzaud, S.; Lignot, B.; Elain, A.; Sire, O. Characterization of bionanocomposites based on medium chain length polyhydroxyalkanoates synthesized by Pseudomonas oleovorans. Polym. Test., 2010, 29(8), 966-971.
Huijberts, G.N.; Eggink, G.; de Waard, P.; Huisman, G.W.; Witholt, B. Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers. Appl. Environ. Microbiol., 1992, 58(2), 536-544.
[] [PMID: 1610179]
Cruz, M.V.; Freitas, F.; Paiva, A.; Mano, F.; Dionísio, M.; Ramos, A.M.; Reis, M.A. Valorization of fatty acids-containing wastes and byproducts into short- and medium-chain length polyhydroxyalkanoates. N. Biotechnol., 2016, 33(1), 206-215.
[] [PMID: 26047553]
Freitas, F.; Alves, V.D.; Pais, J.; Costa, N.; Oliveira, C.; Mafra, L.; Hilliou, L.; Oliveira, R.; Reis, M.A. Characterization of an extracellular polysaccharide produced by a Pseudomonas strain grown on glycerol. Bioresour. Technol., 2009, 100(2), 859-865.
[] [PMID: 18713662]
Mojaveryazdia, F.S.; Zainb, N.A.B.M.; Rezaniac, S. Production of biodegradable polymers (PHA) through low cost carbon sources. Int. J. Chem. Environ., 2013, 4(3), 181-188.
Solaiman, D.K.; Ashby, R.D.; Foglia, T.A. Rapid and specific identification of medium-chain-length polyhydroxyalkanoate synthase gene by polymerase chain reaction. Appl. Microbiol. Biotechnol., 2000, 53(6), 690-694.
[] [PMID: 10919328]
Shang, L.; Jiang, M.; Chang, H.N. Poly(3-hydroxybutyrate) synthesis in fed-batch culture of Ralstonia eutropha with phosphate limitation under different glucose concentrations. Biotechnol. Lett., 2003, 25(17), 1415-1419.
[] [PMID: 14514042]
Verlinden, R.A.; Hill, D.J.; Kenward, M.A.; Williams, C.D.; Radecka, I. Bacterial synthesis of biodegradable polyhydroxyalkanoates. J. Appl. Microbiol., 2007, 102(6), 1437-1449.
[] [PMID: 17578408]
Steinbüchel, A.; Lütke-Eversloh, T. Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms. Biochem. Eng. J., 2003, 16(2), 81-96.
Chen, X.; Shi, J.; Chen, Y.; Xu, X.; Xu, S.; Wang, Y. Tolerance and biosorption of copper and zinc by Pseudomonas putida CZ1 isolated from metal-polluted soil. Can. J. Microbiol., 2006, 52(4), 308-316.
[] [PMID: 16699581]
Raghavan, P.U.M.; Vivekanandan, M. Bioremediation of oil-spilled sites through seeding of naturally adapted Pseudomonas putida. Int. Biodeterior. Biodegradation, 1999, 44(1), 29-32.
Vladu, M.; Lakatos, E.S.; Ene, N.; Popa, O.; Babeanu, N. The potential applications of Bacillus sp. and Pseudomonas sp. strains with antimicrobial activity against phytopathogens, in waste oils and the bioremediation of hydrocarbons. Catalysts, 2019, 9(11), 959.
Karanth, N.G.K.; Deo, P.G.; Veenanadig, N.K. Microbial production of biosurfactants and their importance. Curr. Sci., 1999, 116-126.
Panaitescu, D.M.; Lupescu, I.; Frone, A.N.; Chiulan, I.; Nicolae, C.A.; Tofan, V.; Stefaniu, A.; Somoghi, R.; Trusca, R. Medium chain-length polyhydroxyalkanoate copolymer modified by bacterial cellulose for medical devices. Biomacromolecules, 2017, 18(10), 3222-3232.
[] [PMID: 28892612]
Eremia, M.C.; Lupescu, I.; Vladu, M.; Petrescu, M.; Savoiu, G.; Stefaniu, A.; Spiridon, M. Studies on poly-3-hydroxyoctanoate biosynthesis by a consortium of microorganisms. An. Univ. Ovidius Constanta Ser. Chim., 2016, 27(1), 44-47.
Soare, M.G.; Tomulescu, C.; Petrescu, M.M.; Lupescu, I.; Moscovici, M.; Popa, O.; Băbeanu, N. Antimicrobial activity of newly isolated Bacillus sp. and Pseudomonas sp. strains and their potential use as biocontrol agents. Sci. Bull. Ser. F Biotechnol., 2017, XXI, 81-86.
Cohen, S.S. Growth requirements of bacterial viruses. Bacteriol. Rev., 1949, 13(1), 1-24.
[] [PMID: 16350126]
Locatelli, G.O.; Finkler, L.; Finkler, C.L.L. Orange and passion fruit wastes characterization, substrate hydrolysis and cell growth of Cupriavidus necator, as proposal to converting of residues in high value added product. An. Acad. Bras. Cienc., 2019, 91(1)e20180058
[] [PMID: 30994757]
Kellerhals, M.B.; Kessler, B.; Witholt, B. Closed-loop control of bacterial high-cell-density fed-batch cultures: Production of mcl-PHAs by Pseudomonas putida KT2442 under single-substrate and cofeeding conditions. Biotechnol. Bioeng., 1999, 65(3), 306-315.
[<306:AID-BIT8>3.0.CO;2-0] [PMID: 10486129]
Lupescu, I.; Eremia, M.C.; Savoiu, G.V.; Spiridon, M.; Panaitescu, D.; Nicolae, C.; Stefaniu, A. Comparative studies on isolation of medium-chain-length Polyhydroxyalkanoates produced by Pseudomonas spp. strains. Rev. Chim, 2016, 67(10), 1957-1962.
Sun, Z.; Ramsay, J.; Guay, M.; Ramsay, B. Enhanced yield of medium-chain-length polyhydroxyalkanoates from nonanoic acid by co-feeding glucose in carbon-limited, fed-batch culture. J. Biotechnol., 2009, 143(4), 262-267.
[] [PMID: 19632279]
Jiang, X.; Sun, Z.; Marchessault, R.H.; Ramsay, J.A.; Ramsay, B.A. Biosynthesis and properties of medium-chain-length polyhydroxyalkanoates with enriched content of the dominant monomer. Biomacromolecules, 2012, 13(9), 2926-2932.
[] [PMID: 22871146]
Follonier, S.; Riesen, R.; Zinn, M. Pilot-scale production of functionalized mcl-PHA from grape pomace supplemented with fatty acids. Chem. Biochem. Eng. Q., 2015, 29(2), 113-121.
Guzik, M.W.; Narancic, T.; Ilic-Tomic, T.; Vojnovic, S.; Kenny, S.T.; Casey, W.T.; Duane, G.F.; Casey, E.; Woods, T.; Babu, R.P.; Nikodinovic-Runic, J.; O’Connor, K.E. Identification and characterization of an acyl-CoA dehydrogenase from Pseudomonas putida KT2440 that shows preference towards medium to long chain length fatty acids. Microbiology, 2014, 160(Pt 8), 1760-1771.
[] [PMID: 24794972]
Le Meur, S.; Zinn, M.; Egli, T.; Thöny-Meyer, L.; Ren, Q. Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440. BMC Biotechnol., 2012, 12(1), 53.
[] [PMID: 22913372]
Sun, Z.; Ramsay, J.A.; Guay, M.; Ramsay, B.A. Carbon-limited fed-batch production of medium-chain-length polyhydroxyalkanoates from nonanoic acid by Pseudomonas putida KT2440. Appl. Microbiol. Biotechnol., 2007, 74(1), 69-77.
[] [PMID: 17063330]
Jiang, X.J.; Sun, Z.; Ramsay, J.A.; Ramsay, B.A. Fed-batch production of MCL-PHA with elevated 3-hydroxynonanoate content. AMB Express, 2013, 3(1), 50.
[] [PMID: 23987136]
Ward, P.G.; O’Connor, K.E. Bacterial synthesis of polyhydroxyalkanoates containing aromatic and aliphatic monomers by Pseudomonas putida CA-3. Int. J. Biol. Macromol., 2005, 35(3-4), 127-133.
[] [PMID: 15811466]
Lee, S.H.; Kim, J.H.; Mishra, D.; Ni, Y.Y.; Rhee, Y.H. Production of medium-chain-length polyhydroxyalkanoates by activated sludge enriched under periodic feeding with nonanoic acid. Bioresour. Technol., 2011, 102(10), 6159-6166.
[] [PMID: 21463934]
Miura, T.; Ishii, D.; Nakaoki, T. Production of Poly (3-hydroxyalkanoate) s by Pseudomonas putida cultivated in a glycerol/nonanoic acid-containing medium. J. Polym. Environ., 2013, 21(3), 760-765.
Eggink, G.; De Waard, P.; Huijberts, G.N.M. The role of fatty acid biosynthesis and degradation in the supply of substrates for poly (3-hydroxyalkanoate) formation in Pseudomonas putida. FEMS Microbiol. Rev., 1992, 9(2-4), 159-163.
Magdouli, S.; Brar, S.K.; Blais, J.F.; Tyagi, R.D. How to direct the fatty acid biosynthesis towards polyhydroxyalkanoates production? Biomass Bioenergy, 2015, 74, 268-279.
Tsuge, T.; Taguchi, K.; Seiichi, T.; Doi, Y. Molecular characterization and properties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa: Metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid beta-oxidation. Int. J. Biol. Macromol., 2003, 31(4-5), 195-205.
[] [PMID: 12568928]
Tainio, M.; Jovanovic Andersen, Z.; Nieuwenhuijsen, M.J.; Hu, L.; de Nazelle, A.; An, R.; Garcia, L.M.T.; Goenka, S.; Zapata-Diomedi, B.; Bull, F.; Sá, T.H. Air pollution, physical activity and health: A mapping review of the evidence. Environ. Int., 2021, 147105954
[] [PMID: 33352412]
Priatna, D.; Monk, K.A. The results of applied research for solutions to environmental problems, expected! Indonesian J. Appl. Environ. Studies, 2021, 2(1), 5-11.
Laherrère, J. Future of oil supplies. Energ. Explor. Exploitation, 2003, 21(3), 227-267.
Meereboer, K.W.; Misra, M.; Mohanty, A.K. Review of recent advances in the biodegradability of Polyhydroxyalkanoate (PHA) bioplastics and their composites. Green Chem., 2020, 22(17), 5519-5558.
Koller, M. Biodegradable and biocompatible polyhydroxy-alkanoates (PHA): Auspicious microbial macromolecules for pharmaceutical and therapeutic applications. Molecules, 2018, 23(2), 362.
[] [PMID: 29419813]
Vu, D.H.; Åkesson, D.; Taherzadeh, M.J.; Ferreira, J.A. Recycling strategies for polyhydroxyalkanoate-based waste materials: An overview. Bioresour. Technol., 2020, 298122393
[] [PMID: 31757612]
Brigham, C.J.; Riedel, S.L. The potential of polyhydroxyalkanoate production from food wastes. Appl. Food Biotechnol., 2018, 6(1), 7-18.
Elmowafy, E.; Abdal-Hay, A.; Skouras, A.; Tiboni, M.; Casettari, L.; Guarino, V. Polyhydroxyalkanoate (PHA): applications in drug delivery and tissue engineering. Expert Rev. Med. Devices, 2019, 16(6), 467-482.
[] [PMID: 31058550]
Mukheem, A.; Hossain, M.; Shahabuddin, S.; Muthoosamy, K.; Manickam, S.; Sudesh, K.; Saidur, R.; Sridewi, N.; Campus, N.M. Bioplastic polyhydroxyalkanoate (pha): Recent advances in modification and medical applications; Prepr. Org, 2018.
Ali, I.; Jamil, N. Polyhydroxyalkanoates: Current applications in the medical field. Front. Biol., 2016, 11(1), 19-27.

Rights & Permissions Print Export Cite as
© 2023 Bentham Science Publishers | Privacy Policy