Bacteriophage-encoded lytic enzymes control growth of contaminating Lactobacillus found in fuel ethanol fermentations

Background: Reduced yields of ethanol due to bacterial contamination in fermentation cultures weaken the economics of biofuel production. Lactic acid bacteria are considered the most problematic, and surveys of commercial fuel ethanol facilities have found that species of Lactobacillus are predominant. Bacteriophage lytic enzymes are peptidoglycan hydrolases that can degrade the Gram positive cell wall when exposed externally and provide a novel source of antimicrobials that are highly refractory to resistance development. Results: The streptococcal phage LambdaSa2 (lambda Sa2) endolysin demonstrated strong lytic activity towards 17 of 22 strains of lactobacilli, staphylococci or streptococci and maintained an optimal specific activity at pH 5.5 and in the presence of <= 5% ethanol (fermentation conditions) toward L. fermentum. Lactobacillus bacteriophage endolysins LysA, LysA2 and LysgaY showed exolytic activity towards 60% of the lactobacilli tested including four L. fermentum isolates from fuel ethanol fermentations. In turbidity reduction assays LysA was able to reduce optical density >75% for 50% of the sensitive strains and >50% for the remaining strains. LysA2 and LysgaY were only able to decrease cellular turbidity by <50%. Optimal specific activities were achieved for LysA, LysA2, and LysgaY at pH 5.5. The presence of ethanol (<= 5%) did not reduce the lytic activity. Lysins were able to reduce both L. fermentum (BR0315-1) (lambda Sa2 endolysin) and L. reuteri (B-14171) (LysA) contaminants in mock fermentations of corn fiber hydrolysates. Conclusion: Bacteriophage lytic enzymes are strong candidates for application as antimicrobials to control lactic acid bacterial contamination in fuel ethanol fermentations.