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: 6/22/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Most bioethanol fermentation facilities lack strict sterile conditions and are susceptible to microbial contamination. Lactic acid bacteria (LAB) are typically present in fermentation tanks, competing for nutrients and producing acids that inhibit ethanol-producing yeast. This leads to reduced productivity and lower yields. While antibiotics have traditionally been used to combat contamination, there is a growing emphasis on utilizing alternative antimicrobials. We discovered a bacteriophage-encoded peptidoglycan hydrolase, endolysin LysMP, that is effective in controlling LAB contamination. The LysMP gene, was synthesized based on the prophage genome of Limosilactobacillus fermentum KGL7. The recombinant enzyme expressed in E. coli displayed optimal activity at pH 6, with stability ranging from pH 4 to pH 8 and temperatures between 20°C and 40°C for up to 48 hours. Furthermore, it retained over 80% of its activity in the presence of 10% ethanol (v/v) for the same duration. Addition of LysMP at a concentration of 250 µg/ml reduced the bacterial load by at least 4-log fold compared to untreated controls containing L. fermentum, effectively preventing stuck fermentation in corn mash fermentations. Conversely, untreated controls experienced a significant increase in contamination from an initial bacterial load of 1.50x10^7 CFU/ml to 2.25x10^9 CFU/ml and 1.89x10^9 CFU/mL after 24 and 48 hours, respectively. The glucose was fully utilized in treated samples, while untreated contaminated controls still had over 4% (w/v) remaining at the 48-hour mark. Moreover, when LysMP was employed to treat contaminated corn mash fermentations, there was a minimum five-fold reduction in lactic acid (0.085 M in untreated contamination controls vs. 0.016 M in treated samples) and a four-fold reduction in acetic acid (0.027 M in untreated contamination controls vs. 0.007 M in treated samples). Importantly, the use of LysMP resulted in an approximate 50% increase in final ethanol yields, elevating them from 6.3% (w/v) in contamination controls to 9.3% (w/v) in treated samples, which were comparable to uncontaminated controls at 9.3% (w/v). These findings offer an alternative strategy to effectively manage LAB contamination in biofuel refineries.