Contamination by lactic acid bacteria is a major source of inefficiency in the bioethanol industry. Contaminated fermentations exhibit lowered ethanol yields and contamination events often require shutdown of the entire process for cleaning and decontamination. The first objective of this study was to develop fermentation yeast that secrete bacteriocins, proteins that kill lactic acid bacteria, to counteract this contamination. A strain that secretes leucocin A was successfully developed; however, the strain did not exhibit secretion levels that were industrially relevant. Two reasons for poor secretion: the interaction of the bacteriocin with the cell membrane and the prevention of diffusion by the cell wall, ... More
Contamination by lactic acid bacteria is a major source of inefficiency in the bioethanol industry. Contaminated fermentations exhibit lowered ethanol yields and contamination events often require shutdown of the entire process for cleaning and decontamination. The first objective of this study was to develop fermentation yeast that secrete bacteriocins, proteins that kill lactic acid bacteria, to counteract this contamination. A strain that secretes leucocin A was successfully developed; however, the strain did not exhibit secretion levels that were industrially relevant. Two reasons for poor secretion: the interaction of the bacteriocin with the cell membrane and the prevention of diffusion by the cell wall, were established. The second objective of this study was to produce the circular bacteriocin, carnocyclin A, heterologous in E. coli, with the ultimate goal of producing industrial levels of the bacteriocin for use in the food and biorefining industries. The bacteriocin was produced but was not cyclized at detectable levels. The third objective of the study was to screen for additive and synergistic properties of antimicrobials used in the bioethanol industry against lactic acid bacteria isolated from industrial ethanol production facilities. The antimicrobials screened included a conventional antibiotic, a hop compound, and two bacteriocins. The antimicrobials exhibited additive properties, with leucocin A potentially showing synergistic properties with other inhibitors including nisin. The development of new biological fermentation pathways for yeast, as well as engineering yeast to produce proteins like bacteriocins, could be expedited by improving cloning processes. The fourth objective of this study was to develop a ligation independent cloning methodology to improve the workflow of the cloning process and the percentage of positive clones produced. This methodology had similar percentage positive clones to other cloning processes.
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