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SUMMARY: Glucose utilization by a strain of Streptomyces griseus was studied. Under highly aerobic conditions, glucose was converted mainly to structural material and CO,, but under restricted aeration, lactic acid was formed. Pyruvic acid was also formed during the stages of most rapid growth. The metabolism of glucose was dependent upon the presence of phosphate, and the optimal hydrogenion concentration for both glucose oxidation and the rate of disappearance of inorganic phosphate was about pH 7. Phosphate esters, tentatively identified as glucose-1-phosphate and glucose-6-phosphate, were obtained in fluoride-inhibited systems. Glucose oxidation was depressed by M-sodium iodoacetate and Msodium arsenite but was stimulated by 10-2M-sodium arsenate ; 1 0 -3~-2 : 4-dinitrophenol and 10-sM-sodium azide had no effect. Streptomycin production was decreased by 3 x lO-*M-sodium arsenate but not by 10-2M-sodium fluoride or ~O-*Msodium iodoacetate. S. griseus metabolized members of the tricarboxylic acid cycle, although citrate and a-ketoglutarate gave much lower values of Qo, a t pH 7.3 than pyruvate, acetate, succinate, fumarate or malate. Ketoacids were produced in presence of arsenite from fumarate, malate, glucose, lactate, acetate, succinate, glutamate and citrate in descending order of yield. Except from fumarate, which yielded some material behaving like a-ketoglutarate, the product was chiefly p p v a t e .

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Banks has supervised the maintenance of our radioactivity measuring apparatus, and we are grateful for his unfailing help in this respect. We are also pleased to acknowledge our indebtedness to Miss M. E. Lake, and Miss M. McKerrow who have assisted us in a few of these experiments, and to Mr M. P. Curwen for help and advice on statistical matters.

These attempts failed because the livers contained negligible amounts of vitamin B12. We recognized at the time that it would have been preferable to use ruminants, but such animals were not available to us. Subsequent work suggests that we ought to have extracted the faeces rather than the livers of our animals.About the same time we succeeded in preparing very small amounts of radioactive vitamin B12 concentrates by fermentations with Aerobacter aerogenes isolated from hen faeces. More recently we have returned to this problem, using Streptomyces gri8eu; in the meantime the preparation by this method of [6OCo] vitamin B12 with low or moderately high specific activity has been reported (Chaiet, Rosenblum & Woodbury, 1950;. RESULTS Preparation of vitamin B12 containing 60CoTo avoid diminishing the specific activity of the added radioactive cobalt, we used a semi-synthetic medium (Dulaney, 1948) shown to be nearly free from cobalt. We added only about 0-2 p.p.m. of radio-cobalt, thus minimizing any radiation hazard and securing efficient conversion ofcobalt to vitamin B12. The fermentation liquors were worked up to yield a few mg. of crystalline vitamin B12 by a simple procedure described in the Experimental section. The latest fermentation with 60Co at 3-5 mc./mg. has yielded vitamin B12 with a specific activity of 120 ,uc./mg., which is considerably higher than any previously recorded (Rosenblum & Woodbury, 1951). Preparation of vitamin B12 containing 32pIt was of interest to attempt the preparation of vitamin B12 labelled with radioactive phosphorus, since this would be safer for projected human metabolism experiments than vitamin B12 containing radioactive cobalt with its much longer half-life and penetrating gamma radiation. It was foreseen that it would be difficult to prepare material with adequate specific activity because preliminary experiments had shown that Streptomyce8 gri8eus needs a moderately high level of phosphate in the medium, growth and vitamin B12 production being impaired when the total phosphorus level is reduced to about 0-01 %.It was decided to use 10 mc. Of 32p and to aim at preparing about 1 mg. of the radioactive vitamin. It was then almost immaterial what volume of medium was used for the fermentation; the smaller the volume, the higher the specific activity of the resulting vitamin B12 would be, but the greater the proportion of ordinary vitamin B12 that would have to be added.The radioactive phosphate was added aseptically 24 hr. after fermentation started. This was done in the hope that part of the phosphorus in the medium would already have been taken up by the mould and converted into organic phosphorus compounds that would not exchange with the added 32p. This hope was not realized; the specific activity of the product was the same as that from a pilot run with

DEHYDROASCORBIC ACID AND BACTERIA 605 factor is not replaceable by methylene blue, pyocyanine, glutathione, coenzyme ii, yeast extract, or boiled or unboiled whole yeast. 3. The effect of hydrogen-donator concentration on the rate of DHA reduction, as compared with the rate of methylene-blue reduction, indicates that the rate of hydrogen transfer may be greatly reduced in the DHA-reducing system. 4. Growth on lactate or formate in place of glucose increases the hydrogenase activity, but decreases the DHA-reducing activity. 5. When more than one hydrogen donator is present, the rates ofDHA reduction are not additive. The rate of reduction when formate, lactate and hydrogen are present together is not as great as when glucose is present alone.

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