Submerged Fermentation

6.5.2. Submerged Fermentation

The submerged fermentation process essentially makes use of bioreactors which are very much identical both in design and function to those employed in the antibiotic production. It is mostly made of stainless steel having a capacity ranging between 10 to 15 m 3 and adequately provided with such devices as : mechanical internal agitator — for mixing the contents of the bioreactor ; external pumps — for circulation, loading and evacuation ; bubble columns — for aeration of the medium ; and

ANTIBIOTICS

the air-lift loop. Cultivation progessively involves the suspension growth of microorganisms in the liquid environment. Nevertheless, the sterilization as well as the process control in the submerged fermentation are relatively easy and convenient to accomplish.

In general, the pH of the prepared culture medium is first optimized and then pumped right into the previously steam-sterilized bioreactor via HTST* sterilization devices. The thermobavile additives are carefully introduced individually into the sterile medium at an ambient temperature. Adequately propogated inoculum must be transferred into the production-stage vessels through sterilized SS pipes/ pumps with utmost care and precaution. At this particular stage sterile-compressed air is introduced into the fermentation broth via a strategically positioned sparger** at the bottom of the bioreactor. In com- mercial bioreactors the agitation is done by the aid of multiple flat-bladed disc turbines located onto a vertical shaft. Besides, other suitable devices, such as : counter-current stirrers, and axial-flow propellars, may also be employed as per the necessary requirements. It is, however, absolutely necessary to main- tain the contents of the bioreactors (or fermentors) to an ambient temperature by two commonly used devices, namely : (a) external heat-exchangers ; and (b) internal cooling-coils.

Note : The evolution of CO 2 , as a byproduct, in fermentative procedures essentially gener- ate adequate heat energy, which if not taken care of properly would cause serious unwanted microbial processes to triger off thereby lowering the yield of desired products.

Modern bioreactors are frequently provided with in situ sophisticated probes and sensitive in- strumental gadgets to monitor as well as stringently control various physical parameters during devel- opment of the inoculum in the course of various production stages. It is, however, pertinent to mention

here that the different physical parameters like : dissolved O 2 , evolved CO 2 , redox potential, and pH value must be monitored and simultaneously controlled adequately.

Factors Governing Submerged Culture : In fact, there are three major factors that essentially govern the submerged culture, namely : (i) temperature ; (ii) pH ; and (iii) dissolved oxygen tension (DOT). These factors would be treated individually in the sections that follows :

(a) Temperature : Each fermentative process leading to a particular bioproduct essentially requires optimum temperature for the specific production of an enzyme, and hence the sub- sequent growth of the microorganism, which varies accordingly with the progressive stages of the entire process. Interestingly, the activity of an enzyme for an already utilized substrate gets remarkably influenced by temperature. It is, therefore, almost mandatory to closely monitor the range of temperature during fermentation in such a manner which would en- courage cell growth appropriately.

(b) pH : The stability of an ‘enzyme’ is exclusively guided by the pH optima. Therefore, it is quite evident to sustain and maintain pH optima so as to accomplish the maximum growth rate of the specific organism in question. In other words, it is absolutely vital and necessary that not only the ‘pH profile in the course of fermentation alone’, but also the ‘enzyme production phase’ must be imposed as well as monitored in such a manner so as to achieve the maximum growth and enzyme production simultaneously.

(c) Dissolved Oxygen Tension (DOT) : In actual practice, the dissolved oxygen tension (DOT) may be maintained stringently by controlling three most important physical conditions, such

* HTST : High temperature short time pasteurizers. ** A device for inlet of sterile compressed air viz., single, multiple inlet type or ring type.

PHARMACEUTICAL BIOTECHNOLOGY

as : adequate rate of aeration ; agitation ratio ; and gas-phase pressure. It has been amply demonstrated that the ‘level of O 2 ’ just lower than its critical concentration does affect the growth of microorganisms adversely. Therefore, it is very essential to maintain ‘appropriate

aerobic condition’ in the growth culture medium. Interestingly, the desired ‘level of O 2 ’

predominantly in the submerged microbial cultures may be obtained by adopting either of the two following methods, namely :

(i) Enhancing the mass of O 2 being provided to the bioreactor per unit time by increasing the overpressure in the head-space of the bioreactor, and

(ii) Increasing the ensuing rate of air supply to the bioreactor. CAUTION : Oxygen-enriched air could attribute to higher oxygen transfer rates.