This project was funded under the Bioenergy Technologies Incubator 2 program with the aim of developing and demonstrating a novel biochemical conversion technology called Anaerobic, Non-Photosynthetic (ANP) Mixotrophy. The goal was to show production of acetone from a cellulosic hydrolysate at commercial production metrics (43wt% yield, 40 g/L titer, and 3 g/L/hr productivity). The key innovation was the use of ANP mixotrophy to improve the yields of acetone beyond theoretical limits of traditional fermentation. Produced through traditional fermentation, the maximum possible yield for acetone is 32wt% (one mole acetone per mole of glucose), but with ANP mixotrophy, this can be increased to 45wt%. This increase is possible by using specific bacteria called acetogens. These bacteria use the excess reducing equivalents from glycolysis, normally used to generate H<
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produced from glycolysis back into acetyl-CoA via the Wood-Ljungdahl pathway. The specific tasks for this project were to: 1) improve the ability of the host strain (Clostridium ljungdahlii) to grow on both C6 and C5 cellulosic hydrolysates, 2) optimize the strain for acetone production, and 3) determine bioprocessing parameters to achieve the production metrics. Over the two-year project, significant progress was made in both adapting the strain to grow on hydrolysate feedstocks and adapting the fermentation platform (a continuous, cell-retention system) to tolerate the residual solids in the commercial hydrolysates. Substantial knowledge was also gain on genetically engineering the strain for acetone production, though a final production host capable of meeting all metrics was not achieved. Based on the data and skills generated through this project, White Dog Labs was able to secure a Joint Development Agreement with Air Liquide and another industrial partner to continue developing the process with the aim of commercial deployment.