An experimental study was carried out to examine the auxotropic nature of a newly developed mutant Micrococcus glutamicus AB 200. Yeast extract from the minimal salt medium was replaced by 0.1 μg/mL of biotin, to investigate the role of biotin on growth as well as production of L-lysine by this strain. Biotin (0.1 μg/mL) was proved to be an essential element for growth and production, however in excess amount a significant increase in cell growth lead to an anaerobic condition, which adversely affected L-lysine production.