Biosynthesis of poly(3-hydroxybutyrateco-3-hydroxy-4-methylvalerate) by Strain Azotobacter chroococcum 7B

Abstract

Production of novel polyhydroxyalkanoates (PHAs), biodegradable polymers for biomedical applications, and biomaterials based on them is a promising trend in modern bioengineering. We studied the ability of an effective strain-producer Azotobacter chroococcum 7B to synthesize not only poly(3-hydroxybutyrate) homopolymer (PHB) and its main copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), but also a novel copolymer, poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB4MV). For the biosynthesis of PHB copolymers, we used carboxylic acids as additional carbon sources and monomer precursors in the chain of synthesized copolymers. The main parameters of these polymers’ biosynthesis were determined: strain-producer biomass yield, polymer yield, molecular weight and monomer composition of the synthesized polymers, as well as the morphology of A. chroococcum 7B bacterial cells. The physico-chemical properties of the polymers were studied using nuclear magnetic resonance spectroscopy (NMR), differential scanning calorimetry (DSC), contact angle test, and other methods. In vitro biocompatibility of the obtained polymers was investigated using stromal cells isolated from the bone marrow of rats with the XTT cell viability test. The synthesis of the novel copolymer PHB4MV and its chemical composition were demonstrated by NMR spectroscopy: the addition of 4-methylvaleric acid to the culture medium resulted in incorporation of 3-hydroxy-4-methylvalerate (3H4MV) monomers into the PHB polymer chain (0.6 mol%). Despite the low molar content of 3H4MV in the obtained copolymer, its physico-chemical properties were significantly different from those of the PHB homopolymer: it has lower crystallinity and a higher contact angle, i.e. the physico-chemical properties of the PHB4MV copolymer containing only 0.6 mol% of 3H4MV corresponded to a PHBV copolymer with a molar content ranging from 2.5% to 7.8%. In vitro biocompatibility of the obtained PHB4MV copolymer, measured in the XTT test, was not statistically different from the cell growth of PHB and PHBV polymers, which make its use possible in biomedical research and development.

Biosynthesis and properties of P(3HB‐co‐3HV‐co‐3H4MV) produced by using the wild‐type strain Cupriavidus eutrophus B‐10646

“…Supplementation of the fructose‐containing culture medium for growing the recombinant strain R. eutropha PHB −4 with leucine at a concentration of 10 g L −1 caused cell concentration to increase to 7.1–7.3 g L −1 but copolymer content to drop to 29–36% of CDM; the 3H4MV molar fraction was no higher than 3.0 mol% . The wild‐type strain Azotobacter chroococcum 7B cultivated on sucrose supplemented with 4MV synthesized rather high contents of the copolymer (71–79% of CDM), but cell concentration was no more than 3.7 g L −1 , and the molar fraction of 3H4MV was extremely low (0.14–0.60 mol%) . Thus, synthesis of P(3HB‐ co ‐3 HV‐ co ‐3H4MV) containing a large molar fraction of 3H4MV in the culture producing high cell concentrations and copolymer content seems problematic.…”

“…C x of fractionated P(3HB‐ co ‐3H4MV) films decreased from 60 to 13% as the 3H4MV fraction increased from 0 to 39 mol%. The results reported by Bonartsev et al . suggesting a decrease in C x of P(3HB‐ co ‐3 HV‐ co ‐3H4MV) with a very low content of 3H4MV (0.14–0.6 mol%) to 49% seem dubious.…”

“…13 The wild-type strain Azotobacter chroococcum 7B cultivated on sucrose supplemented with 4MV synthesized rather high contents of the copolymer (71-79% of CDM), but cell concentration was no more than 3.7 g L −1 , and the molar fraction of 3H4MV was extremely low (0.14-0.60 mol%). 26 Thus, synthesis of P(3HB-co-3 HV-co-3H4MV) containing a large molar fraction of 3H4MV in the culture producing high cell concentrations and copolymer content seems problematic.…”

Competitive Biosynthesis of Bacterial Alginate Using Azotobacter vinelandii 12 for Tissue Engineering Applications

“…To date, the use of bacterial alginates and polyhydroxyalkanoates (PHAs) has attracted researchers’ attention whose main research focuses are tissue engineering and biopharmacology [ 1 , 2 ]. PHAs are semi-crystalline hydrophobic polyesters, where the crystalline phase dominates over the amorphous [ 3 ]. Alginates exist as hydrophilic unbranched exopolysaccharides with two uronic acid monomers: (1-4) -β- d -Mannuronic acid (M) and its C5 epimer α- l -Guluronic acid (G) [ 4 ].…”

“…These polymers are biocompatible and biodegradable and can be used to successfully develop 3D constructs for tissue engineering and regenerative surgeries [ 5 ]. Since biosynthesis and bacterial PHA physicochemical properties studies were discussed in detail, this study will be mainly focused on bacterial alginates [ 3 , 6 ]. The distinctive ability of this polymer is the ionotropic interaction of guluronic residues with bivalent calcium cations leading to hydrogel formation [ 7 ].…”