Vitamin B 12 from Propinonibacterium Shermanii

7.4.1. Vitamin B 12 from Propinonibacterium Shermanii

There are, in fact, three different types of medium that are skillfully used in the production of vitamin B 12 from P. shermanii, such as : (a) maintenance medium ; (b) seed-culture medium ; and (c) main-culture medium. All these media shall now be treated individually in the sections that follows :

(a) Maintenance Medium : The maintenance medium for P. shermanii essentially includes per 1L the various ingredients as : tryptone 10g ; yeast extract – 10 g ; filtered tomato juice – 200 g ; and agar 10 g. The pH of the prepared medium is adjusted to 7.2. The inoculated media is duly incubated for a duration of 96 hours at 30°C.

(b) Seed-Culture Medium : The seed-culture medium is usually of different types which are prepared according to the following two stages, namely :

(1) First-Stage Medium : It is very much identical in composition to the maintenance me- dium and is precisely devoid of agar. It is normally incubated for a duration of 48 hours at 30°C without any agitation whatsoever.

(2) Second-Stage Medium : The exert composition of the second stage medium 1L is as follows : cornsteep liquor solids — 20 g ; glucose — 90 g ; and the pH is maintained at 6.5. In general, the medium is duly incubated for 24 hours at 30°C devoid of any aeration, and pH is adjusted to 6.5

(c) Main-Culture Method : The main-culture (i.e., production) media essentially comprise of the following ingredients in 1L : cornsteep liquor solids — 40 g ; glucose — 100 g ; cobalt chloride [CoCl 2 . 6H 2 O] — 0.02 g ; and the pH adjusted to 7.0. It is usually incubated at 30°C. Nevertheless, the first phase of 80 hours is allowed to carry on without aeration, but with slight introduction of N 2 with agitation. Later on, a slight aeration to the tune of 0.1 v/v/m is sustained, and pH is adjusted to 7.0.

It has been observed that ‘propionibateria’ are invariably grown/cultivated upon carbohydrate- based media specifically and that too in an unaerated environment. However, the cobalt supplment is an absolute necessity for vitamin B 12 production. Besides, it also solely depends upon either the internal generation or external supply of 5, 6-dimethyl benzimidazole (or 5, 6-DBI). Importantly, the mutant strainsof P. shermanii are capable of synthesizing their own 5, 6-DBI, which ultimately enhances the

yield of vitamin B 12 to an extent of 65 mg . L –1 in a pilot scale. It is, however, pertinent to state here that the ‘aeration’ definitely augments the formation of 5,

6-DBI, whereas it distinctly lowers the vitamin B 12 biosynthesis at one of its various steps. Therefore, it is quite necessary and equally vital that the very first stage (80 hours) the fermentative process in the bioreactor must be carried out predominantly in an anaerobic environment, but a little agitation is still necessary until the main bulk of the carbohydrate present in the media is fully consumed for the growth and the ultimate formation of cobamide. Of course this kind of sequential steps will exert hardly any undue effect. The subsequent follow up stage (next 88 hours) is supplemented with moderate agitation and slight aeration. The aeration afforded at this stage essentially induces the biosynthetic pathway of

5, 6-DBI, whereby the resulting cobinamide gets converted ultimately to cobalamin.