Biochemical and molecular characterization of an Azotobacter vinelandii strain with respect to its ability to grow and fix nitrogen in olive mill wastewater

The bacterial strain A belongs to a collection of nitrogen fixing bacteria isolated from soil treated with olive mill wastewater (OMW). This strain can grow in OMW showing significant nitrogen fixing capacity. The study of growth and nitrogenase activity of the above strain during its growth into the waste showed that the maximum value of total acetylene reduction activity (expressed in nmol Ethyl/24 h/ml of culture) was obtained after 24 h of incubation as well as the maximum value of bacterial population. When the above nitrogen fixing capacity was expressed in reference to the bacterial population (nmol Ethyl/24 h/μg bacterial protein) its maximum value was observed earlier, since the first 7 h of incubation. Western blot analysis of total bacterial proteins, extracted at specific time intervals showed that nitrogenase activity was induced 30 mins after the inoculation of the waste with the strain A. The respective time of the enzymeʹs induction in chemical media (N-free) was 1 h. Southern blot analysis of total genomic DNA of strain A using as probes the three structural genes (nifH, nifD, nifK) encoding nitrogenase-1 in Azotobacter vinelandii gave hybridization patterns which are conserved between the above two bacteria. These results strongly support parallel biochemical taxonomy data indicating that strain A may belong to Azotobacter vinelandii species.