Mixed Culture Of Nitrogen - Fixing Bacteria And Cellulose - Decomposing Bacteria In Termites And Cloning Of Nitrogenase Gene

Termite is a typical creature that can usually thrive on nitrogen-poor food and mainly feeds on wood materials with low nitrogen content. Nitrogen-fixing microorganisms living in termites make nitrogen content of termites reach110g/kg and cellulose-decomposing microorganisms digesting lignocellulose in wood materials also exist in termites, which are of significance for termites’life.This work used Odontotermes formosanus obtained from Luo Jia Mountain in Wuhan as the test material and cellulose-decomposing microorganisms with higher cellulase activity were isolated from O. formosanus’s gut in the isolation medium containing the only carbon source CMC-Na by the three division. And the taxonomic placements of cellulose-decomposing microorganisms were identified by morphology, physiological and biochemical test and molecular biology. The results showed that19cellulose-decomposing bacteria and27cellulose-decomposing fungi were isolated. By identification, the strain HUB-III-621with the highest cellulase activity was Bacillus subtilis. And the isolated cellulose-decomposing microorganisms were co-cultured with Azotobacter in this work. The result showed that when Azotobacter was co-cultured with the isolated cellulose-decomposing microorganism, compared with single culture of each strain, the colony number, cellulase activity and nitrogenase activity had significant improvement (p<0.05) and every indice reached the highest values when inoculation amount of Azotobacter was6%,2%and6%respectively. When3strains were co-cultured at the most optimum condition, compared with single culture of each strain, the colony number, cellulase activity and nitrogenase activity had significant improvement (p<0.05); compared with co-culture of the2strains, the colony number and cellulase activity were higher, but had no significant difference (p>0.05). Otherwise, ni/D gene (1451bp) and ni/K gene (1563bp) were cloned from the gut of termites. The sequences, encoding regions and taxonomic placements of the two genes were analyzed; at the same time, the secondary structure and the third structure of encoded proteins were predicted, which provided good basis for the research of nitrogenase from proteomics and the important theoretical meaning for the research of nitrogen fixation mechanism of termites.