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

Abstract

This study investigated the effect of various cultivation conditions (sucrose/phosphate concentrations, aeration level) on alginate biosynthesis using the bacterial producing strain Azotobacter vinelandii 12 by the full factorial design (FFD) method and physicochemical properties (e.g., rheological properties) of the produced bacterial alginate. We demonstrated experimentally the applicability of bacterial alginate for tissue engineering (the cytotoxicity testing using mesenchymal stem cells (MSCs)). The isolated synthesis of high molecular weight (Mw) capsular alginate with a high level of acetylation (25%) was achieved by FFD method under a low sucrose concentration, an increased phosphate concentration, and a high aeration level. Testing the viscoelastic properties and cytotoxicity showed that bacterial alginate with a maximal Mw (574 kDa) formed the densest hydrogels (which demonstrated relatively low cytotoxicity for MSCs in contrast to bacterial alginate with low Mw). The obtained data have shown promising prospects in controlled biosynthesis of bacterial alginate with different physicochemical characteristics for various biomedical applications including tissue engineering.

Promising substitute of inconsistent algal alginates: exploring the biocompatible properties of di-O-acetylated, poly-L-guluronate-deficient alginate from soil bacterium Pseudomonas aeruginosa CMG1418

“…Similarly, the MTT assay revealed that none of the test concentrations of CMG1418 alginate and its partial acid hydrolysate inhibited the proliferation of the MCF-7 cell line, and the proliferating cells were 100% viable with active metabolism similar to untreated cells ( P > 0.05) ( Table 2 ). Since CMG1418 alginate did not show any cytotoxic or antimetabolic activities, these results provide experimental evidence of its biocompatible, nonimmunogenic, and nontoxic nature for safe use (Sachan et al, 2009 ; Dudun et al, 2022 ).…”

Bacterial Alginate Biosynthesis and Metabolism

“…Disaccharide repeating unit Producing organism The production of tailor-made alginate in order to obtain polymers with different molecular weights, M/G ratio, and O-acetyl content to suit specific applications has been intensively investigated [41][42][43] as it poses as a potential commercial interest for the use-driven design of these polysaccharides in different industrial and medical applications [44][45][46]. Altering fermentation parameters and growth conditions in order to induce alginate production and or structural modifications during microbial cultivation has been extensively evaluated, especially for Azotobacter vinelandii [47][48][49][50][51][52] due to its non-pathogenic characteristics, which makes this bacterium suitable for biotechnological processes.…”

“…However, there is still much to be unveiled on the genetics and enzymatic regulatory complex for these bacteria, especially for A. vinelandii, which is considered a generally recognized as safe (GRAS) microorganism, with much potential for the biotechnological industry. Therefore, a broad understanding of all the biochemical and regulatory aspects of alginate biosynthesis, combined with the testing of culture conditions and molecular biology tools is paramount for the development of optimal growth methods and strains able to produce this exopolysaccharide in vivo or with post-production in vitro modifications to achieve defined G/M composition, molecular weight and ideal tailor-made physicochemical properties [46,[105][106][107][108].…”

Changes in the Gut Microbiota Composition during Implantation of Composite Scaffolds Based on Poly(3-hydroxybutyrate) and Alginate on the Large-Intestine Wall

“…Based on our earlier published work [19], the cultivation of A. vinelandii 12 for optimal synthesis of PHB with molecular mass in the range from 250 to 600 kDa was performed on liquid Burke's medium in the presence of low concentrations of sucrose and phosphate, as well as a high level of aeration (210 rpm). Optimal synthesis of ALG was also performed when the bacteria were grown on liquid Burke's medium but in the presence of high concentrations of phosphate [19]. Cultivation was carried out in 750 mL shake flasks (volume of nutrient medium 200 mL) at constant pH = 7.2 in the medium and at a temperature of 28 • C.…”

“…The supernatant was then obtained by centrifugation at 11,000× g for 30 min. The supernatant was precipitated with 3 volumes of chilled 80% ethanol at 4400 g for 15 min; at the end, the obtained precipitate was lyophilized for 24 h. The final step for purification of alginate was to dissolve the precipitate in 1 M NaCl solution and perform a dialysis on the resulting solution against 1 L of 0.1 M NaCl for 30 h. To obtain purified alginate, the supernatants after dialysis were again precipitated with 3 volumes of ethanol and the precipitates were lyophilized [19].…”