Effect of Oxygen on Formation and Structure of Azotobacter vinelandii Alginate and Its Role in Protecting Nitrogenase

Abstract

The activity of nitrogenase in the nitrogen-fixing bacterium Azotobacter vinelandii grown diazotrophically under aerobic conditions is generally considered to be protected against O 2 by a high respiration rate. In this work, we have shown that a high rate of respiration is not the prevailing mechanism for nitrogenase protection in A. vinelandii grown in phosphate-limited nitrogen-free chemostat culture. Instead, the formation of alginate appeared to play a decisive role in protecting the nitrogenase that is required for cell growth in this culture. Depending on the O 2 tension and cell growth rate, the formation rate and composition of alginate released into the culture broth varied significantly. Furthermore, transmission electron microscopic analysis of cell morphology and the cell surface revealed the existence of an alginate capsule on the surface of A. vinelandii. The composition, thickness, and compactness of this alginate capsule also varied significantly. In general, increasing O 2 tension led to the formation of alginate with a higher molecular weight and a greater L-guluronic acid content. The alginate capsule was accordingly thicker and more compact. In addition, the formation of the alginate capsule was found to be strongly affected by the shear rate in a bioreactor. Based on these experimental results, it is suggested that the production of alginate, especially the formation of an alginate capsule on the cell surface, forms an effective barrier for O 2 transfer into the cell. It is obviously the quality, not the quantity, of alginate that is decisive for the protection of nitrogenase.

NifH and NifD phylogenies: an evolutionary basis for understanding nitrogen fixation capabilities of methanotrophic bacteria

“…In contrast to some other well-studied free-living diazotrophic bacteria, such as Azotobacter, the mechanisms of dinitrogenase protection from oxygen in Beijerinckia have never been investigated. Protection of dinitrogenase from oxygen in Azotobacter occurs mainly through two mechanisms: a high respiratory activity that removes oxygen at the cell surface, and formation of alginate, which increases the viscosity of the culture broth and reduces the oxygen transfer rate from the gas phase to the aqueous phase (Sabra et al, 2000). Like Azotobacter, strains of Beijerinckia also produce large amounts of polysaccharide material when grown in nitrogen-free media, so that the cultures become highly viscous.…”

Expression of the quorum-sensing regulatory protein LasR is strongly affected by iron and oxygen concentrations in cultures of Pseudomonas aeruginosa irrespective of cell density

“…Fluorescence was determined by exciting the supernatant of the culture at 400 nm for pyoverdine and 355 nm for pyochelin; the emission was measured at 460 nm for pyoverdine and 440 nm for pyochelin (McMorran et al, 2001;Ankenbauer et al, 1985). Biomass dry weight was determined gravimetrically as described previously (Sabra et al, 2000). The concentration of iron ions in the culture supernatant was separately determined as Fe 2+ and Fe 3+ by spectrophotometric assay using iron test kits (Merck) as described previously (Kim et al, 2003).…”

Alginate biopolymer production by Azotobacter chroococcum from whey degradation

“…This exopolysaccharide is essential for the encystment process, since nonmucoid strains fail to encyst (Campos, et al, 1996). In Azotobacter vinelandii, alginate protects nitrogenase from oxygen and increases nitrogen fixation (Sabra, et al, 2000). In the case of the dairy industry, one such 'waste' which is produced in enormous quantities is the whey resulting from cheese and casein manufacture.…”