SPP 2170: Subproject "CaproMix – Development of new and defined co-cultures for bioproduction of caproic acid from carbon dioxide"
This project brings together well complementary expertise of two academic groups from microbiology and molecular biology (Dr. FR Bengelsdorf) and bioprocess engineering/systems biology (Prof. AP Zeng). The objective is to gain quantitative and fundamental knowledge of synthetic co-culture(s) producing caproic acid (CA) via lactic acid (LA) from CO2 and H2. CA is an appealing C6-compound with a wide range of important applications. In the anaerobic co-culture to be studied an Acetobacterium woodii mutant should produce LA autotrophically and a selected strain from Clostridium kluyveri, Megasphera cerevisiae, and mutants of a newly isolated Caprobacter fermentas should convert LA into CA. The compatibility of different combinations (co-cultures) of these microbes will be first examined in terms of growth physiology (optimal pH, T, nutrient requirement and growth inhibition or enhancement). Metabolic interactions of the selected co-culture will then be studied more quantitatively and in more detail, e.g. regarding kinetics of cell growth, substrate consumption and product formation. For the quantification of cell population a metabolic engineering approach will be applied to generate A. woodii mutants expressing a special fluorogenic protein tag that causes a distinct fluorescence pattern to distinguish cells of different species, along with use of fluorescence-based flow cytometry to quantify the other microbe in the co-culture. A special two-chamber bioreactor system will be used to better study metabolic exchanges and interactions of the two microbes, among others for metabolic flux analysis. Similarly as with the characterization of the co-culture integrated biomolecular and bioprocess engineering approaches will be collaboratively applied to improve the performance of the co-culture for bioprocess development. In addition to extensive genetic and metabolic engineering work planned to improve LA production in A. woodii an adaptive laboratory evolution approach will be implemented to generate a hyper LA producer by suppressing cells that do not produce LA via the use of a LA induced selection pressure. A novel cultivation strategy using a bioelectrochemical system will be applied that allows tuning the in situ electrolytic H2 generation and consequently the metabolism of A. woodii according to the requirement of the LA-consuming and CA-producing strain in the co-culture. Accumulation of toxic LA concentration can be prevented via online measurement of LA which serves as a feedback control parameter for the in situ electrolysis. In summary, this project deals with a number of key scientific questions for developing efficient co-culture technology for the production of value-added products such as lactic and caproic acids from CO2 and regenerative electricity. It bears several methodological and technological innovations which not only help to realize the special goal of this project, but could be also applied to related products.