Technical Abstract: Cyanophycin (CGP) is a bacterial bioproduct having a straight-chain poly(aspartic acid) as a backbone with arginine pendant groups attached to it. It has many potential industrial applications in the areas of water softerning, hydrogel, metal-ion chelation, and nutriceuticals. Biotechnological production of CGP employs as producing strains the recombinant organisms that express heterologous cyanophycin synthase (cph) gene. A systematic study of fermentation parameters influencing CGP synthesis by a recombinant E. coli expressing a cphA of Synechocystis sp. showed that high aeration conditions as provided by 400 rpm stirrer speed and 1.0 L/min air flow in a Sixfors vessel (450 mL culture working-volume) resulted in high yields of cell biomass and crude CGP product. Glycerol substrate was found to yield 1.8-times higher crude CGP than glucose did under similar conditions. With glycerol as substrate, we found that a simplified fermentation scheme consisting of a straight 48-h fermentation at 37 deg C (without a 30-to-37 deg C temperature-shifting induction step) yielded compatible or higher amounts of crude CGP as those obtained under various temperature-shifting conditions. By studying the effects of glycerol concentration on CGP yields and analyzing glycerol consumption patterns, we demonstrated that substrate-to-product conversion could be increased by at least 15% and that costly leftover of unused substrate could be alleviated. The results provide important bases to optimize fermentative production of CGP using glycerol that may be obtained as a coproduct from biodiesel production.