Bacteriophage application restores ethanol fermentation characteristics disrupted by Lactobacillus fermentum

Authors: LIU, MEI - Ecolyse Inc; Bischoff, Kenneth; GILL, JASON - Texas A&M University; MIRE-CRISCIONE, MIRANDA - Ecolyse Inc; BERRY, JOEL - Texas A&M University; YOUNG, RY - Texas A&M University; SUMMER, ELIZABETH - Ecolyse Inc

Submitted to: Biotechnology for Biofuels and Bioproducts
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/25/2015
Publication Date: 9/4/2015
Citation: Liu, M., Bischoff, K.M., Gill, J.J., Mire-Criscione, M.D., Berry, J.D., Young, R., Summer, E.J. 2015. Bacteriophage application restores ethanol fermentation characteristics disrupted by Lactobacillus fermentum. Biotechnology for Biofuels. 8:132.

Interpretive Summary: In this study, we discovered two new strains of viruses that kill bacteria and we demonstrated their use in reducing bacterial contamination of fuel ethanol fermentations. Antibiotics are frequently used to prevent and treat bacterial contamination of commercial fuel ethanol fermentations, but there is concern that their use contributes to the emergence of antibiotic resistant bacteria. Bacteriophage are viruses that infect and kill bacteria, and they may be used as alternatives to antibiotics. We isolated two new bacteriophage strains that have a broad spectrum of activity against strains of bacteria that infect commercial fuel ethanol facilities, and we showed that the bacteriophage will mitigate the effects of contamination in experimentally infected ethanol fermentations. Implementing antibiotic-free strategies to control fermentation contaminants is important to fuel ethanol producers, the animal feed industry, and regulatory agencies that seek to control bacterial contamination in commercial fermentation cultures while minimizing the selection for antibiotic resistance.

Technical Abstract: Background: Contamination of corn mash by lactic acid bacteria (LAB) reduces ethanol yields and the overall efficiency of the ethanol fermentation process, and the industry relies heavily on antibiotics for contamination control. There is a need to develop alternative methods for the control of contamination. The goals of this study were to determine the diversity and abundance of bacteria contaminating commercial ethanol fermentations, and to evaluate the potential of anti-LAB bacteriophages in controlling production losses. Results: Bacterial populations in 27 corn mash fermentation samples collected from nine different commercial plants were determined by pyrosequencing of 16S rRNA amplicons. The results showed that the most abundant bacteria (>50% of total population) in 24 of the 27 samples included LAB genera such as Lactobacillus, Streptococcus, Lactococcus, Weissella, Enterococcus, and Pediococcus. Lactobacillus was identified as the most prevalent genus at all fermentation stages in all plants, accounting for between 2.3% to 93.7% of each population and constituting the major genus (>50%) in 9 samples from five plants and the most abundant genus in five other samples. Lactobacillus species, including L. delbrueckii, L. fermentum, L. mucosae, and L. reuteri were the most well represented species. Two bacteriophages that target L. fermentum strains from ethanol plants, vB_LfeS_EcoSau and vB_LfeM_EcoInf (EcoSau and EcoInf), were isolated and characterized as a siphophage and myophage, respectively. Analysis of the 31,703 bp genome of EcoSau revealed its similarity to the P335-like phage group, and the 106,701 bp genome of phage EcoInf was determined to be a novel phage type despite its distant relationship to the SPO1-like phages. Addition of phages EcoSau and EcoInf to L. fermentum-contaminated corn mash in ethanol fermentation models mitigated L. fermentum infection and restored the yields of ethanol and levels of residual glucose, lactic acid, and acetic acid to levels comparable to the infection-free control. Conclusions: This study provides detailed insight into the microbiota contaminating commercial ethanol fermentations, and highlights the abundance of LAB, especially L. delbrueckii, L. fermentum, L. mucosae, and L. reuteri, in the fermentation process. This study suggests a model in which phage cocktails with broad coverage of major LAB species can be applied directly to corn mash as non-antibiotic contamination controls in the ethanol fermentation industry.