Biofuels Production projects
Controlling Fermentation Hygiene via Chlorate Addition and Expression of Chlorate Dismutase in Process Organisms
Our objective is to demonstrate proof-of-concept for a novel bioprocess hygiene strategy. Industrial fermentation facilities are vulnerable to contamination with unwanted eukaryotes, bacteria, and bacteriophage. Growth of contaminating organisms can reduce product yields or lead to bioreactor collapse, entailing financial losses. In Saccharomyces cerevisiae-based fuel ethanol fermentations, lactic acid bacteria and wild yeasts are common contaminants (5, 41). Some facilities use antibiotics to treat or prevent bacterial infections in fuel bioreactors, but antibiotics are not universally effective against bacteria, and they do not prevent contamination by wild yeasts. In the case of bacterial fermentations, antibiotics could be used to selectively inhibit or spare certain species, but they do not protect process organisms from bacteriophage infection. We propose a strategy in which process organisms are engineered to produce the enzyme chlorite dismutase (Cld), which detoxifies the disinfectant compound chlorite by converting it to chloride and molecular oxygen (43). Chlorite added to the fermentation system would then selectively damage non-Cld-expressing contaminants, including eukaryotes, bacteria, and bacteriophage. We will demonstrate that Cld expressed in S. cerevisiae will protect this organism from concentrations of chlorite that kill or inhibit the growth of known contaminants of fuel ethanol fermentations. These studies pave the way for the development of a widely applicable technique to address fermentation hygiene concerns in a variety of industrial fermentations.
Our long-term goal is to protect large-scale continuous fermentation systems, such as those used in biofuel production, from contamination by unwanted microorganisms. Our strategy, initiated in 2014, is to engineer process organisms, such as yeast and bacteria, to express the enzyme chlorite dismutase (Cld). This enzyme detoxifies the oxidant chlorite into chloride and oxygen. Cld-expressing process organisms should be protected from low concentrations of chlorite added to the fermentation systems, while contaminating yeast, bacteria, and bacteriophage will be rendered non-growing or non-infective. In the first year of the project, as proof of concept, we have demonstrated that Cld expression simultaneously protects a native chlorate-reducing bacterium from chlorite and from bacteriophage-mediated killing. We have expressed functional Cld in a heterologous bacterium, and we have demonstrated that this strain outcompetes the non-Cld-expressing parent strain when both are co-cultured in the presence of chlorite. We are currently completing a set of plasmids to express Cld at different levels in the cytoplasm of Saccharomyces cerevisiae, to determine if we can increase the native chlorite resistance of this organism. In the future, we propose to express Cld in Clostridium species that perform acetone-butanol-ethanol (ABE) fermentation for the production of biodiesel.