The Growth Metabolism of Rhizobium, With Evidence on the Interrelations Between Respiration and Synthesis

Numerouscultural studies with rhizobia, reported during the past 50 years, have established that these organisms have a comparatively simple metabolism.The quantity of sugars consumed in respiration in comparison with that consumed in growth is smaller than for most bacterial species.The formation of intermediate products of carbohydrate oxidation, such as organic acids and alcohols, is usually almost negligible.The main by-product ordinarily formed is a complex gum (7), the quantity varying markedly with the bacterial strain and growth conditions.This paper presents manometric and analytical data dealing with the respiration and growth metabolism of a strain of Rhizobium grown under conditions such that practically no by-products were formed.The carbohydrate consumed is accounted for quantitatively and its efficiency of utilization calculated.From these data some idea can be had of the interrelations existing between respiration and growth. MethodsThe general procedure was to culture the organisms in a thin layer in large Erlenmeyer flasks and aseptically to remove 4 to 10 cc.samples at various times, starting as soon as there was sufficient growth to study, and continuing until growth stopped.The samples were examined for oxygen consumption, CO2 production, respiratory quotient, dry weight of organisms, composition of organisms, composition of medium, and efficiency of growth.Analyses were made directly on the growing cultures, 181This is an Open Access article under the CC BY license. Growth Metabolism of Rhixobiumand not on "resting cells" centrifuged from the medium.The technique used was worked out in an effort to understand both the resting oxidation and growth metabolism, especially the use which the organism makes of the carbohydrate.Organism Studied-Rhizobium meliloti, No. 131, of the University of Wisconsin cultures, obtained through the courtesy of Dr. I. L. Baldwin, was used.This strain produces a negligible quantity of gum on our medium, is a rapidly growing organism of high infectiveness and effectiveness, and has been used in much of our previous work.Culture Conditions-The organisms were grown on a sucroseinorganic salts medium (1) containing iron and coenzyme R ( 6), with ammonia or nitrate nitrogen added as desired.The bacteria were grown at 28" in 2 liter Erlenmeyer flasks containing medium to a depth of about 1 cm.The initial pH was 6.8 but as growth proceeded the ammonia cultures tended to become slightly more acid and the nitrate ones more alkaline.The cultures were not aerated but were shaken well each day and before sampling.Respiration Measurements-The usual Warburg technique (3) was used at 28", the measurements being made in duplicate.The respiratory quotient was determined by the first method of Warburg, in which the COZ is absorbed in Ba(OH)2 as the 02 consumption is measured, and subsequently displaced by HCl tipped in from the side arm, Growth Measurements-The quantity of organisms present in a sample was determined by centrifuging 10 cc.aliquots, washing once with distilled water, and determining oxidizable material by the method of von Fellenberg (4).The sample is digested in a mixture of 0.1 N KzCrz07 and concentrated HzS04 and diluted, KI is added, and the iodine, set free by the excess chromic acid, titrated with 0.1 N NazSz03.Digestion for 1 hour on a steam bath gave better reproducibility than 15 minutes at room temperature as in the original method.An empirical factor of 1.25 cc. of 0.1 N KzCrzOi per mg. of organisms was obtained by titration versus dry weight analysis.Determination of Sugar and Extracellular Growth Products-The modified procedure of von Fellenberg as given above was also used for the determination of oxidizable materials left in the medium