180 PROVASOLI [chap. 8 



balamin being the growth-factor needed by the majority of the auxotrophic 

 algae. 



The data indicate that bacteria are the major producers of vitamin B12 in 

 the sea. Ericson and Lewis (1953) isolated 34 bacteria from the Baltic Sea and 

 epiphytic on seaweeds; 70% of them (mainly Pseudomonas and Achromobacter 

 spp.) are cobalamin producers (Escherichia coli bioassay). An equal number of 

 marine bacteria was isolated by Starr et al. (1957) from mud and waters from 

 the southeast and west coast of Texas, a lagoon, laboratory tanks, and mullet 

 intestine : 70% of them had activity for Escherichia coli and 30% for Euglena 

 gracilis. Twenty per cent of 60 bacteria isolated from muds of the Bahia 

 Fosforescente (Puerto Rico) had Escherichia coli activity (Burkholder and 

 Burkholder, 1958). Burkholder (1959) isolated from muds and water of Long 

 Island Sound 344 bacteria ; 24% of them produced cobalamins. Marine bacteria 

 collected near shore produce more non-Bi2 cobalamins than true vitamin B12 : 

 this is indicated by the following data. Chromatographic analysis of the filtrates 

 show more non-Bi2 cobalamins than true B12 (Ericson and Lewis, 1953) ; more 

 bacterial strains produce cobalamins active for Escherichia coli than for Euglena 

 gracilis (Starr et at., 1957). Most marine bacteria live epiphytically on particu- 

 late matter. Suspended solids and muds of Sapelo Island, Georgia coast, the 

 Bahia Fosforescente and Long Island Sound are relatively rich in Escherichia 

 coli-active cobalamins (Starr, 1956; Burkholder and Burkholder, 1956, 1958; 

 and in litt.). A differential assay with Ochromonas malhamensis and Escherichia 

 coli on the same bottom deposits of the Bahia Fosforescente shows that the 

 quantity of true B12 is 7-23% of the Escherichia coZi-active cobalamins (Burk- 

 holder and Burkholder, 1958). A similar situation should exist in sea-water but 

 data are lacking. Most determinations of "B12" in sea-water have been done 

 with the Euglena gracilis, which, as noted, is not specific for true B12. Cowey 

 (1956) did a few differential assays: in sea-water from Aberdeen Bay almost 

 half the total cobalamins was B12; in oceanic waters the non-Bi2 cobalamins 

 were present in far lower relative quantities. More work is needed to find out 

 whether this holds generally ; if it is so then we should explain the discrepancy 

 between what is actually found in the waters, what has been produced by 

 marine bacteria, and what is present in muds. In any case, differential assays 

 for sea-water are indispensable because the Bi2-requiring algae themselves 

 have different specificity patterns ; those of low specificity (Escherichia coli 

 pattern) utilize all cobalamins, but the others able to utilize only vitamin B12 

 may be discriminated against, depending on the ratio of non-Bi2 cobalamins 

 to vitamin B12. 



Many marine bacteria require vitamins for growth and they compete for 



cyanocobalamin (Ochromonas malhamensis and "vertebrate" pattern) ; Euglena gracilis 

 responds also to pseudo-Bi2 and factor A, and Escherichia coli to all cobalamins including 

 factor B (no nucleotide), and methionine. The Escherichia coli bioassay hence is equiva- 

 lent to "total cobalamins", the Ochromonas assay only to cyanocobalamin, and the 

 Euglena gracilis assay is intermediate between the Ochromonas and Escherichia coli 

 assays. 



