Use of Cheese Whey for Vitamin B12 Production: I. Whey Solids and Yeast Extract Levels1

The suitability of cheese whey as a substrate for vitamin B(12) production by Propionibacterium shermanii was studied. It was found that with a given level of whey solids a definite amount of yeast extract was required to give maximal yields of vitamin B(12). Of the levels of materials studied, 10% whey solids and 1.5% yeast extract gave the best yields of vitamin B(12). Most of the lactose of the whey had been utilized in all flask cultures after 168 hr at 29 C.     

Use of Cheese Whey for Vitamin B12 Production: II. Cobalt, Precursor, and Aeration Levels1

Within the limits of this study, it was found that 5 ppm of cobalt was adequate to give good levels of vitamin B(12). The vitamin B(12) precursor 5,6-dimethylbenzimidazole was found to be adequate at 10 ppm in the absence of aeration. In the presence of aeration, a zero level of precursor was found to be most desirable. The analysis of variance showed aeration to be highly significant, and the aeration and precursor interaction to be significant. No other significant effects were observed.     

Utilization of Non-Sugar Sources for Vitamin B12 Production

Assimilation of non-sugar carbon sources for vitamin B₁₂ production was studied.     

Production of Vitamin B12 by Micromonospora

Various species of Micromonospora produced yields of vitamin B(12) activity as high as 11 mug/ml under conditions of shaken flask fermentation.     

Transport of Vitamin B12 in Escherichia coli: Genetic Studies

The chromosomal location of two genetic loci involved in the transport of cyanocobalamin (B(12)) in Escherichia coli K-12 was determined. One gene, btuA, is believed to code for the transport protein in the cytoplasmic membrane, because a mutant with an alteration in this gene has lost the ability to accumulate B(12) within the cell although normal levels of the surface receptors for B(12) are present. The other locus, btuB, apparently codes for the surface receptor on the outer membrane. These mutants have lost the ability to bind B(12) and have greatly reduced transport activity, although growth experiments have shown that they can utilize B(12) for growth, but with decreased efficiency. This surface receptor for B(12) also appears to function as the receptor for the E colicins, because btuB mutants are resistant to the E colicins, and mutants selected for resistance to colicin E1 are defective in B(12) binding and transport. The gene order was determined by transduction analysis to be cyc-argH-btuA-btuB-rif-purD. In addition, mutations in metH, the gene for the B(12)-dependent homocysteine methylating enzyme, were obtained in this study. This gene was localized between metA and malB.     

Utilization of Hydrocarbons and Vitamin B12 Production by Bacillus badius

The accumulation of vitamin B₁₂ by Bacillus badins grown on hydrocarbon was investigated. The bacterium could assimilate n-alkanes of C₁₁–C₁₈, ethanol, fumarate, α-ketoglutarate and malate. n-Alkanes of C₁₆–C₁₈, were the best for vitamin B₁₂ production. The bacterium utilized well all of the nitrogen sources tested. Above all, ammonium dihydrogen phosphate was the best for the bacteria] growth and vitamin B₁₂ production. Addition of organic nutrients such as malt extract and meat extract, and addition of metal ions such as ferrous and cobalt promoted the growth and vitamin B₁₂ production. Interestingly, vitamin B₁₂ was produced mostly in the supernatant. The cyanoform of the corrinoid predominantly formed in the supernatant would confirm the identity with cobalamin.     

VITAMIN B12 AND THE MACROMOLECULAR COMPOSITION OF EUGLENA : II. Recovery from Unbalanced Growth Induced by Vitamin B12 Deficiency

When vitamin B₁₂ is added to B₁₂-deficient cultures of Euglena gracilis, the cells undergo two relatively synchronous cell divisions within a shorter than usual period of time, apparently as a result of a transitory shortening of the cell cycle. The first cell division pulse, occurring 4.5 h after addition of B₁₂, is preceded by the completion of DNA duplication, but appears to involve no net synthesis of RNA or protein. Before the second round of cell division at about 11 h, a significant amount of DNA synthesis is observed. This time it is accompanied by a minor increase in the RNA and protein content of the culture. The cellular contents of RNA and protein were observed to decrease steadily after the resumption of cell division in B₁₂-depleted cultures receiving the vitamin. Ultimately all three macromolecules returned to their nondeficient, plateau stage levels; by this time, cell division had ceased.     

Vitamin B12 Production and Depletion in a Naturally Occurring Eutrophic Lake

The distribution of vitamin B(12) within Upper Klamath Lake was surveyed at approximately monthly intervals during a period from September 1968 to November 1969. High concentrations (up to 1.8 mug/g of dry sediment) characteristically occurred at the water-sediment interface, with a sharp decline below this area. A heavy bloom of Aphanizomenon flos-aquae occurred from the latter part of May through October 1969. B(12) concentrations of the uppermost sediments, from all but one sampling site, increased gradually through the bloom, followed by a drastic increase during the die-off period. B(12) is probably not a limiting factor for primary productivity, since sufficient levels of this vitamin were found to occur throughout the year. Of 42 cultures isolated from Upper Klamath Lake water and sediments, 20 were found capable of producing 50 pg or more of B(12)/ml of medium. Phytoplankton samples were found to contain up to 5 mug of B(12)/g of dry material. Degradation of B(12) occurred in sterilized as well as fresh sediment samples.