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

Authors: Roach, Dwayne; Khatibi, Piyum; Bischoff, Kenneth; Hughes, Stephen; Donovan, David

Submitted to: Biotechnology for Biofuels and Bioproducts
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/11/2013
Publication Date: 2/7/2013
Citation: Roach, D.R., Khatibi, P.A., Bischoff, K.M., Hughes, S.R., Donovan, D.M. 2013. Bacteriophage-encoded lytic enzymes control growth of contaminating Lactobacillus found in fuel ethanol fermentations. Biotechnology for Biofuels. 6(1):20.

Interpretive Summary: There is a need to treat bacteria-contaminated ethanolic biofuel fermentations to remove bacterial contaminants as they reduce the nutrients and conditions necessary for optimal ethanol yield. A major contaminants of concern are Lactobacillus bacteria that tolerate high acidic conditions. It is preferred to avoid the continued use of conventional antibiotics to remove these contaminants as they make the mash by-product less attractive as an alternative animal feed. Also, antibiotic resistant strains of lactobacilli are starting to show up in fermentations. Bacteriophage are bacterial viruses that produce enzymes that kill bacteria. This work identifies four bacteriophage enzymes that kill the major lactobacilli contaminant of ethanolic fermentations (L. fermentum) and demonstrates these enzymes can kill these contaminants at the pH, and ethanol content of ethanolic fermentations as well as in mock fermentations (small, experimental scale lab fermentations); one is derived from a Streptococcus bacteriphage (LambdaSa2 endolysin) and three from Lactobacillus bacteriophage (LysA, LysA2 and LysgaY). These findings are the first report to describe a non-antibiotic source of antimicrobials that can be used to ‘cure’ infected fermentations. The fuel ethanol industry has experienced rapid growth in recent years, with 10.6 billion gallons (40.2 billion liters) produced in 2009 with future need estimated to be 90 billion gallons (340.69 billion liters) by 2030 in the United States alone. The yeast used in the ethanol production can be engineered to produce the bacteriophage enzymes identified in this work and thus could eliminate the need for classical antibiotic use; this would be a tremendous savings to the producer that could readily be translated to reduced fuel ethanol costs to the consumer. Also, the use of antibiotics is shunned by EPA because of the eventual use of the mash by-product as animal feed. Thus, these enzymes could help improve the animal feed mash by-product by reducing the amount of antibiotic used and increase the acceptance of these alternative antimicrobials by the EPA and the farmer. Currently this work is still a work in progress and is primarily used currently by scientists in the field. Background: Reduced yields of ethanol due to bacterial contamination in fermentation.

Technical Abstract: Background: Reduced yields of ethanol due to bacterial contamination in fermentation cultures weakens 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 atimicrobials that are highly refractory to resistance development. Results: The streptococcal phage LambdaSa2 ('SA2) endolysin demonstrated strong lytic activity towards 17 of 22 strains of lactobacilli, staphylococci or streptococci and maintained an optimal specific activity at pH of 6.5 and in the presence of = 5% ethanol (fermentation conditions) toward L. fermentum substrates. 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 affect the lytic activity. Lysins were able to reduce both L. fermentum (0315-1) ('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.