Studies have been conducted upon certain nitrogen-fixing clostridia isolated from Tama silt loam and Grundy silt loam, two acid soils widely distributed in Iowa. Tests have been made of their reaction requirements, capacity to fix gaseous nitrogen and production of carbon dioxide under varying controlled conditions in the laboratory. The results appear to warrant the following conclusions.

1. Nitrogen-fixing anaerobes were present in rather large numbers in the acid soils studied.

2. Varying applications of lime had little or no effect upon the numbers of anaerobic nitrogen-fixers or upon their ability to fix nitrogen in soil-solution cultures in the presence of nitrogen gas.

3. An incubation period of 3 weeks seemed to be suitable for obtaining maximum amounts of nitrogen fixed anaerobically. Pure cultures of Clostridium pasteurianum and Clostridium butyricum fixed only very small amounts of nitrogen in solution cultures in an atmosphere of nitrogen.

4. The initial pH of the medium apparently had very little effect upon the amounts of nitrogen fixed over a range of pH values from 5.0 to 9.5. In all the cultures the final pH after 3 weeks was near 5.0. The amounts of acid produced at various intervals over a 133-hour period in mixed cultures of soil anaerobes appeared to be closely related to the amounts of glucose utilized. No relationship was observed, however, between the initial reaction of the medium and the nitrogen fixed over a 20-day period. Apparently the optimum pH for growth and nitrogen fixation by the soil anaerobes extends over a wide range, and the organisms are somewhat tolerant of acidity.

5. The nitrogen metabolism of mixed cultures of soil anaerobes was studied in Winogradsky's nitrogen-free medium containing 0.2 gram of calcium chloride per liter or 15 grams of calcium carbonate per liter. Comparatively large amounts of nitrogen were fixed in both media, with a tendency for the amount to be larger in the calcium carbonate medium. In the calcium chloride medium larger amounts of ammonia and amino nitrogen were produced than in the carbonate medium. Larger amounts of acid were present in the cultures containing the calcium chloride.

6. The metabolism of the two pure cultures of soil anaerobes, No.4, not identified, and No.5, Clostridium butyricum, was also studied in these two media. In the calcium chloride medium culture 5 was somewhat more efficient in fixing nitrogen. Glucose utilization was greatly stimulated by the presence of calcium carbonate, the effect being somewhat more pronounced with culture 5. The carbonate also had a beneficial effect upon the amounts of nitrogen fixed in both cultures. There was some relation between the rate of nitrogen fixation and the glucose utilization and also between the rate of acid production and the glucose utilization. Nitrites and nitrates were produced in both cultures in the calcium carbonate medium, but none were found in the presence of calcium chloride.

7. Results obtained with Clostridium butyricum growing in media containing varying nitrogen sources and in a nitrogen-free medium, all with calcium carbonate, showed that the presence of combined nitrogen in the medium greatly stimulated the utilization of glucose during the first part of the incubation period. The entire amount of glucose present in all the cultures was utilized, however, by the end of 25 days. The most rapid utilization occurred when sodium nitrate was used as a nitrogen source. Practically negligible amounts of nitrogen were fixed in the media containing combined nitrogen, while comparatively large amounts were fixed in the nitrogen-free medium. Apparently the calcium carbonate did not immediately neutralize the acids as they were formed, but the reaction proceeded slowly over the entire incubation period.

8. In experiments on carbon dioxide production with a pure culture of Clostridium butyricum in various media containing calcium carbonate, it was found that large amounts of carbon dioxide were produced under anaerobic conditions. The largest amounts were produced in the peptone medium. Production was about the same in sodium nitrate and nitrogen-free media. Only very small amounts of carbon dioxide were produced in the similar media containing calcium chloride instead of calcium carbonate. The presence of air apparently stimulated the production of carbon dioxide by Clostridium butyricum in the peptone medium, but exerted a detrimental effect when the sodium nitrate and nitrogen-free media were used. The retarding effect of calcium chloride was less pronounced in the peptone medium when air was present.

9. The source of the carbon dioxide produced in the peptone medium containing calcium carbonate was presumably in the glucose molecule. A small portion of the carbon dioxide was produced as a result of the interaction of the calcium carbonate with the acids formed.

10. Throughout all the experiments marked beneficial effects upon the nitrogen-fixing Clostridia were observed owing to the presence of calcium carbonate in the medium. This effect was not only noted upon the growth of the organisms in the various cultures and the utilization of glucose but also upon the fixation of nitrogen.