Endolysin LysMP as an Antibiotic Replacement to Control Lactobacillus Contamination in Small-Scale Corn Mash Fermentation

Authors: PATEL, MAULIK - Orise Fellow; Lu, Shao; Skory, Christopher

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 6/16/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Traditional bioethanol fermentation industries are not operated under strict sterile conditions and are prone to microbial contamination. Lactic acid bacteria (LAB) are often pervasive in fermentation tanks, competing for nutrients and producing inhibitory acids that have a negative impact on ethanol-producing yeast, resulting in decreased yields and stuck fermentations. Antibiotics are frequently used to combat contamination, but antibiotic stewardship has resulted in a shift to alternative antimicrobials. We demonstrate that LysMP endolysin, a bacteriophage-encoded peptidoglycan hydrolase, is an effective method for controlling growth of LAB. The LysMP gene was synthesized based on the prophage genome of Limosilactobacillus fermentum KGL7. Analysis of the recombinant enzyme expressed in E. coli and purified by IMAC showed an optimal activity at pH 6, with stability from pH 4 to pH 8 and from 20°C to 40°C up to 48h. Moreover, it retains more than 80% of its activity at 10% ethanol (v/v) for up to 48h. When LysMP was added at 250 µg/mL, it reduced bacterial load by at least 4-log fold compared to the untreated controls containing L. fermentum and prevented stuck fermentation in model corn mash fermentation. In comparison, untreated controls with contamination increased from an initial bacterial load of 1.50x10^7 CFU/mL to 2.25x10^9 CFU/mL and 1.89x10^9 CFU/mL after 24h and 48h, respectively. Glucose in the treated samples was fully utilized, while untreated controls with contamination had more than 4% (w/v) remaining at 48h. Furthermore, there was at least a 5-fold reduction in lactic acid (0.085 M untreated contamination controls vs. 0.016 M treated), and a 4-fold reduction in acetic acid (0.027 M untreated contamination controls vs. 0.007 M treated), when LysMP was used to treat contaminated corn mash fermentations. Most importantly, final ethanol yields increased from 6.3% (w/v)(contamination controls)to 9.3% (w/v)(treated), an approximate 50% increase and were comparable to uncontaminated controls 9.3% (w/v). These findings contribute to an overall alternative strategy to mitigate LAB contamination in biofuel refineries.