194 



THE ACTINOMYCETES, Vol. I 



found capable of producing this vitamin 

 (Ganguly et al.). 



According to Hall et al. (1953), S. olivaceus 

 when grown under submerged aerobic con- 

 ditions is capable of producing large amounts 

 of vitamin B12 . The yields of the vitamin 

 were influenced by the composition of the 

 medium. In the presence of proteinaceous 

 material such as distillers' solubles, glucose, 

 CaCOs , and cobaltous ion, about 1.5 /ig of 

 vitamin B12 was produced per millilter in 

 deep tank fermentors, but as much as 3 ng 

 of the \-itamin was obtained in some media. 

 Appreciable amounts of the B-complex vita- 

 mins, niacin, pantothenic acid, biotin, py- 

 ridoxine, thiamine, and riboflavin were pro- 

 duced. 



By selective cultivation, including mutant 

 formation by ultraviolet light and x-rays, 

 it is possible to increase considerably the 

 yields of vitamin B12 by a given culture. 



The effect of cobalt as a limiting factor in 

 the biosynthesis of the acti^T vitamin B12 

 by S. griseus was immediately recognized 

 (Hendlin and Ruger, 1950). Shull and Rou- 

 tien (1951), Pridham et al. (1951), Saunders 

 et al. (1951), and Garey et al. (1951) made a 

 sur\'ey of \'itamin B12 production by different 

 actinomycetes. Particular attention was paid 

 to *S. griseus (Rickes et al., 1948) and »S'. 

 fradiae (Jackson et al., 1951). 



Chemical investigations have brought out 

 the fact that there are several forms of 

 vitamin B12 . Possibly not all of them occur 

 naturally but are formed during the isolation 

 process. The incorporation of cobalt into 

 the actinomycete metabolite has been estab- 

 lished l)y the use of radioactive cobalt in 

 fermentation media and the isolation of the 

 vitamin containing the ial)eled isotope. 



Some of the vitamin B12 produced by ac- 

 tinomycetes is ])ound on the cells, and ma}'- 

 be released by treatment with acid, alkali, 

 ionizal)ie salts, sonic energj', or heat. Sub- 

 seciuent to release, the cell-free li(iui(l may 

 lie treated with cyanide to convert the vita- 

 min present to the more stable cyanide form. 



In addition to vitamin B12 , various other 

 biologically active compounds, designated as 

 B121, , Bi2c , and Bi2d , are produced by S. 

 griseus (Anslow et al.). The formation of 

 vitamin Bi2b by S. aureofaciens was demon- 

 strated by Lichtman et al. (1949). The B^b 

 was effective, by parenteral administration, 

 in the treatment of Addisonian pernicious 

 anemia. 



In a study of the production and purifica- 

 tion of vitamin B12 by \'arious actinomycetes, 

 Tarr has shown that Bi2a was formed by S. 

 griseus and by *S'. aureofaciens in aerated 

 herring press water containing 2 mg per 

 milliliter of added cobalt (as cobaltous ni- 

 trate). Highest recovery of vitamin Bi2a in 

 these products (about 1.1 mg per milliliter 

 with S. aureofaciens and 0.8 mg per milli- 

 liter with S. griseus) was obtained by chro- 

 matography on filter paper strips treated 

 with potassium dihydrogen phosphate, elu- 

 tion of the vitamin, and aseptic addition of 

 the eluates to previously sterilized Lactobacil- 

 lus leichmannii assay medium. Treatment of 

 the crude fermentation products with po- 

 tassium cyanide (2.5 mg. per milliliter) 

 caused partial resolution of Bi2a to B12 . 



Letunova (1958) made a comprehensive 

 study of cultures isolated from the lime of 

 stagnant reservoirs of the biogeochemical 

 province enriched in cobalt. Nearly 86 per 

 cent of these cultures, as well as 90 per cent 

 of strains isolated from lime deposits of the 

 Co-impoverished pro\'ince, were able to form 

 vitamin B12 . 



Certain nocardias are also capable of pro- 

 ducing B12 . Bardi et al. (1958) studied a 

 strain of .¥. rugosa that was found to release 

 free porphyrins into the medium under suit- 

 able aeration conditions. By ultraviolet ir- 

 radiation, mutants were obtained that gave 

 a much higher yield of porphyrins. The rela- 

 tion l)etween vitamin B12 and porphyrin 

 ])r()du('ti()ii was demonstrated. The free por- 

 l)hyrins consisted mainly of coproporphyrin 

 III, and of uroporphyrin, probably III, in 



