Respiration Rates of Rhizobium; Their Estimation and Significance

FRANKLIN E. ALLISON AND SAM R. HoovER RESPIRATION studies with Rhizobium meliloti and Rhizobium trijolii, carried out both according to the resting cell technique and with actively growing cultures, are reported. Organisms that were not assimilating (storing) carbohydrate or undergoing cell division usually gave Qc>2 values (cmm. Oa consumed per mgm, dry wt. of organisms per hour) on glucose of 5 to 8 at 28°C, the values being highest with the youngest organisms. Where substrate was not present (endogenous respiration) the Qo2 values were 2 to 5 or even lower, depending upon the initial carbohydrate reserves in the cells. If the organisms were maintained in a complete medium, and under conditions suitable for growth, the observed Qos was usually considerably above the resting cell value of about 8. In the presence of glucose, asparagin, and yeast extract, a Qoa of 25 to 35, or occasionally of 40, was observed. Studies of organisms of various ages showed a close correlation between the rate of respiration and the growth rates, as measured by increase in dry weight of the organisms. The Qo2 was highest during the period of logarithmic growth and decreased rather rapidly to a value of near 10 when growth had nearly stopped. Ordinarily anything that increases the growth rate of Rhizobium also increases the Qc>2; likewise, an increase in Qc>2 of organisms maintained in a complete medium commonly indicates increased growth except where special respiration stimulants are added. A value covering the range above approximately 8 seems to be characteristic of growth just as lower values indicate truly resting cells. The resting cell technique was found to be much less satisfactory for use with Rhizobium than with Azotobacter and various other organisms that have been used previously by a number of workers. Ordinarily this method has been used chiefly in studies with organisms that have resting Qo2 values on glucose of 20 to 50, or in the case of Azotobacter, near 1,000. With such organisms the Qo2 values without subtrate are seldom more than 20% as high as with substrate, and may be as low as i% with Azotobacter. In contrast, strains of Rhizobium usually show an endogenous respiration amounting to 30 to 60% of the respiration rate with substrate, which is also low. The result is that the oxidation of any given substrate being studied is partially complicated, or at least may be, by the oxidation of cell material, and this makes the interpretation of the results difficult. Wilson attempted to overcome the difficulty by using young cultures of Rhizobium grown on a yeast medium without other substrate. These organisms were high in nitrogen and gave Qoo values of 30 to 40 at 34°C (equivalent to 18 to 24 at 28°C). Unfortunately he did not prove that they were non-assimilating and non-growing cells, and our general observations, as well as those of numerous other workers using various organisms, would indicate strongly that they were not truly resting cells. Any results obtained with this type of cell do not, therefore, lend themselves to a simple interpretation. Certain generalizations regarding the resting cell technique that apply to most microorganisms can be made. ( I ) A resting cell should be defined as a nonproliferating and non-assimilating cell, and not merely as a non-proliferating cell as has previously been done. (2) In using resting cells the experimenter should be prepared to prove the condition of the cells by either showing that the added substrate is completely oxidized, or that the dry weight has remained constant during the experiment. It may be difficult to comply with this requirement where the experimental period is short. (3) There would seem to be no need in most types of respiration experiments to 'Contribution from Bureau of Chemistry and Soils U. S. Dept, of Agriculture, Washington, D. C. Published originally in Trans. Third Com. Int. Soc. Soil Sci., A:32~38. 1939. Senior Chemist in charge of biochemical investigations and Assistant Chemist, respectively.