Optimizing Corn Steep Liquor as Fermentation Media for the Production of Recombinant Antifreeze Proteins

Abstract

Corn processing byproducts light corn steep liquor (CSL) and thin stillage were utilized as fermentation growth media for higher growths of wild type and recombinant Lactococcus lactis modified to produce antifreeze proteins (AFPs). A combination of additives and trace elements- disodium-β-glycerophosphate (DG), tryptone (T), ascorbic acid (AA), iron (Fe), zinc (Zn), and magnesium (Mg) were evaluated in media at 100 mL volume in a 96-well Bioscreen plate with three replicates. The addition of additives and trace elements increased the optical density OD600 by more than 40%, indicating the growth of wild type as well as recombinant L. lactis strains when compared to control without any additives in growth media. The CSL media at 50% (v/v) in water with 5 g/L DG, 5 g/L T, 0.5 g/L AA, and 4 mg/L Zn was selected as the best-optimized media for the further fermentation process. The specific growth rates and cell doubling times for the bacteria were compared for both wild-type and recombinant-type strains. Compared to the wild type, the recombinant type had a lower growth rates and longer doubling times. The highest growth rate (0.52 ± 0.00 h-1) and smallest doubling time (1.32 ± 0.01 h) were observed for M17 media, which is a well-defined sugar-rich commercial media for L. lactis. The lowest growth rate (0.39 ± 0.01 h-1) and highest doubling time (1.77 ± 0.06 h) were observed for thin stillage-based media. Results indicate that AFPs can be produced comparatively well in corn processing coproduct-based media; such AFP would have the potential for use in many food and non-food applications.