Comparing Study Of Facultative Anaerobic Nitrogen-fixing Bacteria From Cow Rumen, Soil And Feed

With the depth and expansion on biological nitrogen fixation, the study of nitrogen-fixing bacteria(NFB) is become more and more popular. This study is mainly isolated facultative anaerobic NFB from soil, feed, cow rumen residue and rumen fluid, and then identified physical and chemical properties and species. The objective of this study was conducted to species relationship between strains from other sources and strains from rumen fluid and the characteristic, so as to provide relevant reference for the study of nitrogen-fixing bacteria in future.In this study, the tested strains were isolated from soil, feed, cow rumen residue and rumen fluid, and incubated and purified on N-free solid medium for 24-48 h at 39 °C under anaerobic and aerobic conditions. Nitrogenase activities of the isolated strains were assayed by gas chromatography based on the acetylene reduction test. The NFB were identified based on physiological–biochemical identification and 16 S rDNA gene analysis, and subsequently analyzed and compared homology.The results showed that 30 facultative anaerobe NFB were isolated from four sources, 9 NFB of isolated from rumen fluid(LY-1 to LY-9), 7 NFB of isolated from rumen residues(LC-1 to LC-7), 7 NFB of isolated from soil(TR-1 to TR-7) and 7 NFB of isolated from feed(SL-1 to SL-7), respectively.By comparison of nitrogenase activity, nitrogenase activity of strains from soil was the highest in four sources, and nitrogenase activity of TR-2 was 97.0523 nmol·mg-1·h-1. Nitrogenase activity of strains from rumen residues was higher than from rumen fluid, but the highest nitrogenase activity in two sources was from rumen fluid, and nitrogenase activity of LY-1 was 40.7075 nmol·mg-1·h-1.By analysis of molecule sequences, 9 NFB of isolated from rumen fluid were including 3 Bacillus sonorensis, 1 Bacillu methylotrophicus, 3 Acinetobacter baumannii, 1 Staphylococcus caprae and 1 Bacillus aerius; 4 NFB of isolated from rumen residues were including 2 Bacillus sonorensis, 1 Acinetobacter calcoaceticus and 1 Virgibacillus marismortui; 6 NFB of isolated from feed were including 5 Bacillus sonorensis and 1 Bacillus aerius; 6 NFB of isolated from rumen fluid were including 3 Bacillus sonorensis, 1 Bacillu methylotrophicus, 1 Staphylococcus caprae and 1 Bacillus aerius. LC-4 and LC-7 of isolated from rumen residues, TR-4, TR-5 and TR-6 of isolated from soil and SL-1, SL-2, SL-3, SL-6 and SL-7 of isolated from feed are the same specie as LY-3, LY-8 and LY-9 of isolated from rumen fluid. TR-1 and TR-2 of isolated from soil are the same specie as LY-1 and LY-4 of isolated from rumen fluid, respectively. Although the most similar specie of TR-3, SL-5 and LY-3 are all Bacillus aerius, the similarity of Strains is about 50% and cannot explain for the same specie of strains.In summary, the results indicated that there were a amount of facultative anaerobe NFB in rumen, and it may be likely to exist in rumen residues and make a great role. the results of species relationship indicated that some strains of isolated from rumen residues, soil and feed were the same species as some of isolated from rumen fluid. These NFB are not only existed in rumen and may be from the outside.