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Developing an optimal medium for fermentation is a key concept to efficient optimization. One-factor-at-a-time (OFAT) is the preferential choice that researchers use for designing a medium composition. This method involves changing only one factor at a time while keeping the other concentrations constant. This method can be separated into multiple sub-groups that include removal, supplementation, and replacement experiments. In a removal experiment, all the components of the medium are removed one at a time and their effects on the medium are observed. Supplementation experiments involve evaluating the effects of nitrogen and carbon supplements on production. Replacement experiments are the final method and this involves replacing the nitrogen and carbon sources that show an enhancement effect on the intended production. The OFAT method has an advantage as well as a disadvantage when compared to other medium designing methods. The advantage is that the method is simple to conduct and the results can be easily analyzed. However, this method also provides the disadvantage of requiring a large number of experiments to isolate the optimum level. This can result in a large number of experiments needing to be conducted and resulting in a costly process.

An example of a supplementation experiment is its use on chitosan formation by the fungus Absidia Orchidis. The experiment done found that the use of manganese and ferrous ions gave the highest yield of chitosan. This type of medium allowed for the experimentation of the best supplements to promote cell growth

A Replacement medium experiment was conducted by Evrogen for photobleaching of fluorescent proteins. The use of replacement medium, in this case, was allowed for the medium to be flush with proteins without diminishing as many compounds in a normal media. This type of medium is more useful in long-term experiments.