Alginate production and alg8 gene expression by Azotobacter vinelandii in continuous cultures

Abstract

Alginates are polysaccharides that are used as thickening agents, stabilizers, and emulsifiers in various industries. These biopolymers are produced by fermentation with a limited understanding of the processes occurring at the cellular level. The objective of this study was to evaluate the effects of agitation rate and inlet sucrose concentrations (ISC) on alginate production and the expression of the genes encoding for alginate-lyases (algL) and the catalytic subunit of the alginate polymerase complex (alg8) in chemostat cultures of Azotobacter vinelandii ATCC 9046. Increased alginate production (2.4 g l⁻¹) and a higher specific alginate production rate (0.1 g g⁻¹ h⁻¹) were obtained at an ISC of 15 g l⁻¹. Carbon recovery of about 100% was obtained at an ISC of 10 g l⁻¹, whereas it was close to 50% at higher ISCs, suggesting that cells growing at lower sucrose feed rates utilize the carbon source more efficiently. In each of the steady states evaluated, an increase in algL gene expression was not related to a decrease in alginate molecular weight, whereas an increase in the molecular weight of alginate was linked to higher alg8 gene expression, demonstrating a relationship between the alg8 gene and alginate polymerization in A. vinelandii for the first time. The results obtained provide a possible explanation for changes observed in the molecular weight of alginate synthesized and this knowledge can be used to build a recombinant strain able to overexpress alg8 in order to produce alginates with higher molecular weights.

Evaluation of Gene Expression and Alginate Production in Response to Oxygen Transfer in Continuous Culture of Azotobacter vinelandii

“…In disagreement with these results, Díaz-Barrera et al . [10] reported that in chemostat cultures fed sucrose (15 g l −1 ), a percentage of carbon (20 and 35%) was diverted to acetate and malate, which were released into the culture medium. This different behavior could be attributed to the non-nitrogen-fixation conditions used by Díaz-Barrera et al .…”

“…This different behavior could be attributed to the non-nitrogen-fixation conditions used by Díaz-Barrera et al . [10], compared to the nitrogen-fixing conditions used in this work. In light of this observation, further research must be carried out to evaluate how nitrogen fixation conditions affect metabolic fluxes in A .…”

“…The inoculum for the bioreactor was prepared in 500 ml Erlenmeyer flasks with 100 ml of culture medium, as described previously [10]. The initial pH was adjusted to 7.0 using 2 M NaOH.…”

“…[10]. Sucrose concentration was determined by hydrolysis with β-fructofuranosidase, followed by the determination of reducing sugars with dinitrosalicylic acid (DNS) reagent [13].…”

“…Similarly, Díaz-Barrera et al . [10] showed that increased alginate molecular weight produced by A. vinelandii continuous cultures can be linked to higher alg8 gene expression.…”