Poly (3-hydroxybutyrate) from a moderate halophile: production and modification for new applications

Abstract

Polyhydroxybutyrate (PHB) is a homopolymer belonging to the family of biopolyesters, polyhydroxyalkanoates (PHA) produced by different bacteria and archaea as intracellular granules often under conditions of metabolic stress due to a limited supply of essential nutrients. The properties of the PHAs are similar to those of polyethylene and polypropylene and can hence replace these fossil based polymers in a large variety of applications. However, because of high production cost, PHAs are still expensive for use in commodity products but due to their biodegradability and biocompatibility they can be used for high value applications. This thesis concerns investigations on the production and recovery of PHB from a moderately halophilic bacterium, Halomonas boliviensis, and modification of the biopolymer for some novel applications.

Halomonas boliviensis accumulates PHB from different carbon sources. The effect of ammonium, phosphate and yeast extract on production of cell mass and PHB content was studied using a factorial design. Batch cultivation of H. boliviensis under controlled conditions in a fermentor was studied using 2.5% (w/v) sucrose, 1.5% (w/v) yeast extract, and induced oxygen limitation which resulted in the cell dry weight of 14 g l-1 and PHB content of 54 wt% after 19 h of cultivation. Addition of casaminoacids to the medium led to a slight increase in the cell dry weight to 14.4 g l-1 in 17 h but reduced the PHB content to 52 wt%, indicating that reduced nitrogen levels promote the polymer accumulation in the bacteria.

Extraction of PHB from H. boliviensis cells was investigated using various chemicals to rapidly lyses the cells under mild conditions. Treatment with alkali, acids and detergents, respectively, at 30 degree for 1 hour resulted in quantitative recovery of the polymer. The sugar based detergent, octyl maltoside gave a higher polymer recovery than the ionic detergent, SDS.

Native PHB is a hydrophobic, stiff and brittle polymer, and needs to be modified for several applications, especially those relating to biomolecules and cells. To enable covalent modification, PHB scaffolds, prepared by thermally induced phase separation, were subjected to limited hydrolysis by the enzyme lipase and the generated carbonyl groups were coupled to ligands bearing amino groups using carbodiimide and N-hydroxysulfosuccinide. Coupling of the polycation, polyethyleneimine (PEI) and lysozyme, respectively were investigated. The coupling was confirmed by fourier transform infrared analysis, which showed amide bands absorption peak around 1651 cm-1 and 1540 cm-1 attributed to Amide I and amide II bands, respectively. The modified scaffolds exhibited significantly increased water absorption capacity. PHB-PEI scaffolds adsorbed DNA by ionic interactions and the bound DNA could be eluted at high ionic strength, demonstrating that the biodegradable conjugate could be used as an alternative matrix to purify DNA from complex mixtures. PHB-lysozyme scaffolds exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. PHB-lysozyme complexes were also prepared to evaluate the controlled release of the protein. Hydrophobic ion pairing of lysozyme with sodium oleate prior to complexing with PHB resulted in high recovery of the enzyme activity released over a one week period. These modifications show the potential applications of PHB based conjugates in molecular biology and as antimicrobial materials.

Abstract (Swedish)

Popular Abstract in English

Polyhydroxybutyrate (PHB) is a homopolymer belonging to the family of biopolyesters, polyhydroxyalkanoates (PHA) produced by different bacteria and archaea as intracellular granules often under conditions of metabolic stress due to a limited supply of essential nutrients. The properties of the PHAs are similar to those of polyethylene and polypropylene and can hence replace these fossil based polymers in a large variety of applications. However, because of high production cost, PHAs are still expensive for use in commodity products but due to their biodegradability and biocompatibility they can be used for high value applications. This thesis concerns investigations on the production and recovery of PHB from a moderately halophilic bacterium, Halomonas boliviensis, and modification of the biopolymer for some novel applications.

Halomonas boliviensis accumulates PHB from different carbon sources. The effect of ammonium, phosphate and yeast extract on production of cell mass and PHB content was studied using a factorial design. Batch cultivation of H. boliviensis under controlled conditions in a fermentor was studied using 2.5% (w/v) sucrose, 1.5% (w/v) yeast extract, and induced oxygen limitation which resulted in the cell dry weight of 14 g l-1 and PHB content of 54 wt% after 19 h of cultivation. Addition of casaminoacids to the medium led to a slight increase in the cell dry weight to 14.4 g l-1 in 17 h but reduced the PHB content to 52 wt%, indicating that reduced nitrogen levels promote the polymer accumulation in the bacteria.

Extraction of PHB from H. boliviensis cells was investigated using various chemicals to rapidly lyses the cells under mild conditions. Treatment with alkali, acids and detergents, respectively, at 30 degree for 1 hour resulted in quantitative recovery of the polymer. The sugar based detergent, octyl maltoside gave a higher polymer recovery than the ionic detergent, SDS.

Native PHB is a hydrophobic, stiff and brittle polymer, and needs to be modified for several applications, especially those relating to biomolecules and cells. To enable covalent modification, PHB scaffolds, prepared by thermally induced phase separation, were subjected to limited hydrolysis by the enzyme lipase and the generated carbonyl groups were coupled to ligands bearing amino groups using carbodiimide and N-hydroxysulfosuccinide. Coupling of the polycation, polyethyleneimine (PEI) and lysozyme, respectively were investigated. The coupling was confirmed by fourier transform infrared analysis, which showed amide bands absorption peak around 1651 cm-1 and 1540 cm-1 attributed to Amide I and amide II bands, respectively. The modified scaffolds exhibited significantly increased water absorption capacity. PHB-PEI scaffolds adsorbed DNA by ionic interactions and the bound DNA could be eluted at high ionic strength, demonstrating that the biodegradable conjugate could be used as an alternative matrix to purify DNA from complex mixtures. PHB-lysozyme scaffolds exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. PHB-lysozyme complexes were also prepared to evaluate the controlled release of the protein. Hydrophobic ion pairing of lysozyme with sodium oleate prior to complexing with PHB resulted in high recovery of the enzyme activity released over a one week period. These modifications show the potential applications of PHB based conjugates in molecular biology and as antimicrobial materials.

• author: Munoz, Marlene
• supervisor: Rajni Hatti-Kaul
• opponent: Professor K. Sudesh, Kumar, School of Biological Sciences, University Sains Malaysia, Penang, Malaysia
• organization: Biotechnology
• publishing date: 2011
• type: Thesis
• publication status: published
• subject: Industrial Biotechnology
• keywords: Biopolymers, polyhydroxyalkanoates, polyhydroxybutyrate, moderate halophile, Halomonas boliviensis, fermentation, recovery, thermally induced phase separation, scaffolds, covalent modification, hydrophobic ion pairing, polyethylenemine, lysozyme.
• pages: 124 pages
• defense location: Lecture hall B, Center for Chemistry and Chemical Engineering, Getingevägen 60, Lund University Faculty of Engineering
• defense date: 2011-10-18 10:30:00
• ISBN: 978-91-89627-77-2
• language: English
• LU publication?: yes
• id: c738b9e9-7ba6-4df2-bdaf-54c65b28e006 (old id 2199128)
• date added to LUP: 2016-04-04 13:29:16
• date last changed: 2018-11-21 21:14:18