This Small Business Innovation Research Phase I project will develop a process to increase the productivity and yield of biobutanol from cultures of solvent-forming Clostridium by treating cultures with recently discovered Clostridium quorum-sensing molecules. The biobutanol fermentation can be difficult to operate due to changes in the producing bacteria driven by unknown biological mechanisms, which result in ?stuck? or ?degenerate? cultures that don?t produce butanol. As the proportion of unproductive bacteria increases biobutanol productivity drops, yield from starting materials decreases and process economics suffer. Previously undescribed biological mechanisms in Clostridium that are involved in controlling the ability to make biobutanol have recently been discovered. An application of that discovery can prevent stuck or degenerated biobutanol fermentations and increase productivity and yield. Consequently, investment and operating costs for a biobutanol fermentation facility will decrease, and annual revenues will increase. Phase 1 research will focus on optimizing culture treatment levels with quorum-sensing molecules, and obtaining biobutanol yield and productivity data for batch and continuous cultures.
The broader/commercial impacts of this research are that as a second generation biofuel, biobutanol produced by Clostridium can use a broader range of substrates than bioethanol production, and has significant advantages over bioethanol as a fuel and industrial chemical for which demand is increasing. New companies are engaged in improving biobutanol fermentation processes and in genetic engineering to improve biobutanol formation. A new understanding of biological mechanisms that regulate Clostridium biobutanol biosynthesis, however, has the potential to generate transformative technology for the biobutanol fermentation industry.
. Award Number: IIP-1046592 The goal of this project is to develop a process to increase and extend butanol production by treating solvent-forming Clostridium cultures with recently discovered Clostridium quorum-sensing molecules. The molecular mechanisms by which butanol-producing Clostridium control the biosynthesis of butanol are not completely understood. Specifically, cultures exhibit reduced butanol formation through the course of repeated subculturing and in continuous culture. Current methods used to extend butanol production by Clostridium rely on process stages that separate the acid- and solvent-producing cell forms, resulting in increased capital and operating costs. Increasing butanol formation by manipulation of intercellular communication will result in new and enhanced butanol fermentation processes. As a result, capital investment for a butanol fermentation facility will be reduced as will operating costs. The quorum sensing technology being developed by Butrolix, LLC has the potential to be transformative for the biobutanol industry. The production of butanol and acetone using Clostridium was one of the first large-scale industrial fermentation processes ever developed. Increased oil production and lower oil prices from the 1950s and on were ultimately the demise of the biobutanol industry. More recently, the continuing climb in the cost of oil and recognition of the need to reduce greenhouse gas emissions have renewed interest in the fermentation process, and a new round of innovation in biobutanol production has begun. Butanol is a feedstock chemical for the paint and plastics industries where current annual sales are about $10 billion. Production costs for petroleum-based butanol are at the point that biobutanol from fermentation can be competitive, and innovation in the butanol fermentation process could create ‘shut-down’ economics for petro-butanol. Also, as a biofuel, butanol produced by fermentation can use a broader range of substrates than can be used for bioethanol, and has significant advantages over bioethanol as a fuel extender. New companies are engaged in improving classical butanol fermentation processes and in genetic engineering to improve butanol formation. A new understanding of biological mechanisms that regulate Clostridium butanol biosynthesis, being developed by Butrolix, LLC, has the potential to generate transformative technology for the butanol fermentation industry. Three different types of experiments were conducted in order to achieve the technical goals of this project. First, small batch cultures were treated with different levels of chemically synthesized peptides and were then transferred sequentially, continuing the peptide treatment with each transfer. Each transfer was subsequently analyzed for butanol content and the results used to determine a peptide treatment level that, in comparison to untreated cultures, produced the greatest amount of butanol. The results showed that peptide-treated cultures produced more butanol than untreated cultures, and that the optimal treatment level was very low. Second, time-course studies were conducted using the optimum peptide treatment level that was determined by the initial experiments. Replicate samples were taken from treated and untreated cultures at regular intervals during growth of the cultures and were analyzed for butanol in order to calculate yield and productivity of the cultures. In this set of experiments, peptide-treated cultures yielded 50% more butanol than untreated and had peak productivity that was double that of untreated cultures. Finally, continuous culture studies were conducted for 20 days using the optimum peptide treatment level that was determined by the initial experiments. Replicate samples taken from treated and untreated continuous cultures were analyzed for butanol in order to calculate yield and productivity of the cultures. Similar to the results from the first two sets of experiments, peptide-treated continuous cultures produced more butanol than the untreated cultures. The outcomes of the experiments conducted during this Phase I SBIR project confirm the potential utility of using quorum sensing peptides to increase butanol production by Clostridium. This technology, being developed by Butrolix, LLC, has the ability to positively impact the economics of biobutanol production to an extent that could be transformative for the industry.
