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Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)


Biology and Microbiology

First Advisor

William Gibbons


The initial impetus to evaluate nisin as a contaminant control agent came from Minnesota Corn Processors (MCP), who recognized that the ethanol fuel industry needed a means of controlling bacterial contamination during fermentation. Bacterial contamination is a significant problem in this industry --reducing product concentrations and yields, limiting productivity, and increasing production costs. In light of the problems associated with contamination, and the limited options available for successfully controlling and/or preventing contamination, MCP chose to investigate nisin as an alternative biocontrol agent. Nisin is a nontoxic, digestible polypeptide bacteriocin produced by Lactococcus lactis subsp. lactis. It is a biocontrol agent used internationally as a food preservative in dairy products and canned foods. Nisin inhibits the growth of many Gram-positive spoilage bacteria, but has little effect on most Gram-negative bacteria and fungi. This is important since many industrial processes utilize Gram-negative bacteria or fungi. Unfortunately the high cost of nisin has limited its application to food products. MCP realized that before nisin could be commercialized as a biocontrol agent for bulk fermentation processes, the costs of producing and utilizing nisin must first be reduced. This would involve the development, optimization, and scale-up of new production and utilization technologies. The most critical factors relating to nisin production involve increasing nisin yields, while minimizing raw material and processing costs to produce a high potency, low cost biocontrol agent. Therefore, the main objective of this project was to develop and commercialize a technically and economically feasible system for the production of nisin, using alternative low-cost media and developing a system to maintain proper pH. Creation of a value-added nisin production industry would improve the efficiency and profitability of the ethanol fermentation process by providing an effective tool for controlling contamination. In the first phase of this project, the MCP process was characterized in order to isolate and identify bacterial contaminants found to be detrimental to ethanol fermentation. The most significant bacterial reservoir (with levels of 8.6 x 1010 Gram-positive bacteria/ml) was the cooler supply tank that fed our batch fermentation process. This was also where the most detrimental organism tested -- MCP #3 -- was isolated. When this bacterium (genus Lactobacillus) was inoculated into mash at either a 2:1 or 1:1 ratio of bacteria to yeast, only 76-83% of the expected ethanol yield was produced. The next phase of the project sought to develop a means of producing nisin - more economically using low-cost agricultural by-products. Different complex media were compared for nisin and cell mass yields. Thin stillage, a fuel ethanol production by-product, was shown to be the most efficient, low-cost medium for large cell mass yields (3.6 x 1010CFU/ml) and high nisin titres (2600 IU/ml). These yields were obtained from filtered thin stillage, buffered with 0.5% dipotassium phosphate and 0.5% sodium carbonate, with 4% glucose also added, and the pH maintained at 6.5-6.8 with periodic additions of 6N NH4OH. The maximum yields of nisin were generally obtained by the seventh hour of fermentation. Compared to other agricultural by-products (steep water or whey) thin stillage looks very promising due to the fact that it is an abundant, inexpensive substrate which produces high titres of nisin with the above additions. In the final phase of this project, fermentationderived nisin and commercial nisin (Nisaplin) were compared for their effectiveness in controlling bacterial contamination. This was done to determine the purity and concentration of nisin required for efficacy at various stages of the ethanol production process. It was found that nisin is effective at relatively low concentrations (100 IU/ml) both in the purified and unpurified state, when introduced early in the fermentation process (12 h prior to yeast addition). Nisin has no adverse affects on yeast and is very thermostable under acidic conditions. During the various phases of this project the detrimental effects of bacterial infections on ethanol yields were documented. With the utilization of nisin, levels of bacterial contaminants could be reduced, thus improving ethanol productivity' s. This work also showed that nisin can be produced from a less expensive agricultural by-product and that this fermentation-derived nisin is nearly as effective as purified nisin. This should reduce the cost of producing nisin and make it more economical for use in the ethanol fuel industry. Nisin presents a unique opportunity to solve MCP's occasional contamination problems, and create a new industry solely directed at supplying nisin to other companies. Development of nisin as an inexpensive, yet effective means of controlling bacterial contamination in ethanol plants would greatly increase productivity, thereby reducing processing costs, making ethanol a more economical product.

Library of Congress Subject Headings

Alcohol as fuel
Contamination (Technology)




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