Although Clostridium pasteurianum was the first free-living nitrogenfixing bacterium to be isolated in pure culture (WI~OG~A])SKY, 1895), studies on the biochemistry of asymbiotic nitrogen fixation for many years were limited almost exclusively to species of the aerobic Azotobacter. This preference likely arose from the relative ease of culturing and the much greater rate, extent and efficiency of the N-fixing process of the aerobe. In the early 1950's investigations on the physiology of the elostridia provided methods that overcame this technical handicap. Nevertheless, it was somewhat unexpected that the first demonstration of cell-flee fixation was obtained with a preparation from C. pasteurianum (CA~N~mtN et at., 1960). This discovery stimulated research in many laboratories resulting in notable advances in our knowledge of the biochemistry of the nitrogen fixation process. As might be expected, most if not all of this research has been done with one strain, W-5, since the biochemist usually prefers for the biological component of his system one possessing an extensive backlog of research information. The bacteriologist, however, may choose less intensive investigation on more strains. With this in mind, we have undertaken a comparative survey of nitrogen fixation by strains of three species belonging to the Bacillaceae--C. pasteurianum, Bacillus polymyxa and B. macerans, the results of which are reported here.
Material and Methods
Organisms. Six strains of Clostridium pasteurianum were isolated from soils and their physiological characteristics, including ability to fix nitrogen, compared with stock strains isolated by SPIEGET.BE~G (1940) (ATCC 7040, ATCC 7041) and Wisconsin W-5 (ATCC 6013). The new isolates with the source of soil are: Lake (Lake Mendota mud), BB (Wisconsin field soil), Z-15 (Texas loam), Delta (Indiana field), Lambda (Georgia field), Sigma (Wisconsin field). Since Clostridium pasteurianum is tolerant of high concentrations of sucrose, a nitrogen-free medium containing 15~ sucrose was used for isolation (SPIEGELBERG, 1940). After further passage on N-free medium and heat shocking at 80 ~ C for 10 rain, the cultures were streaked on glucose yeast-extract agar and incubated anaerobically. Cultures were allowed to sporulate and then transferred to sterile soil for storage.