An Electricity-enhanced Anaerobic Non-photosynthetic Mixotrophy Platform Based On Clostridium Carboxidivorans

Biofuels such as ethanol and butanol are important directions for the development of renewable energy.However,the cost of microbial fermentation is higher than that of fossil fuel.Increasing the substrate conversion rate and the yield of the target product can effectively reduce the cost of biofuels production.This paper adopts the method of anaerobic non-photosynthetic mixotrophy(ANPM),and uses Clostridium carboxidivorans to reabsorb one-third of the carbon lost in the process of producing acetyl-Co A from glycolysis,which improves the carbon recovery rate and establishes efficient biofuel production platform.Using methyl viologen(MV)and biochar as electron mediators,using wild bacteria C.carboxidivorans and modified bacteria C.carboxidivorans E2 for electricity-enhanced anaerobic non-photosynthetic mixotrophy(EE-ANPM)to further increase the carbon recovery rate and reduce the production cost of biofuels.This study uses C.carboxidivorans to fix CO₂ through the EE-ANPM method,which improves the yield of biofuels,fills the gaps in this research,and is of great significance for solving the energy crisis and environmental problems.MV has a lot of advantages as an electronic mediator.Using MV as an electronic mediator,six different fermentation conditions are designed using wild-type C.carboxidivorans,and the effects of MV,applied electricity,calcium carbonate and other conditions on the fermentation spectrum are compared and analyzed.It was found that MV can inhibit the growth of microorganisms while improving the efficiency of electron transport.After adding MV,the carbon recovery rate of EE-ANPM increased from 66.4%to 82.8%.In addition,whether it is serum bottle fermentation or current-enhanced fermentation,the alkyd ratio increases after adding MV,indicating that MV can cause the redistribution of intracellular reducing power,change the metabolic pathway,and make more reducing power available for alcohol production.Both the applied electricity and calcium carbonate can improve the carbon recovery rate and the alkyd ratio of the product.Overexpression of the bifunctional aldehyde/alcohol dehydrogenase encoded by adh E2can significantly increase the production of alcohol.The modified strain C.carboxidivorans E2overexpressing adh E2 was used to perform EE-ANPM with MV as the electron mediator.In the fermentation of serum bottles without MV,the carbon recovery rate of the modified bacteria (55.3%)was higher than that of the wild bacteria(52.2%),indicating that overexpression of adh E2 can increase the product yield.The alkyd ratio(0.32 mol/mol)of the modified bacteria was much higher than that of the wild bacteria(0.07 mol/mol),indicating that overexpression of adh E2 greatly increased the yield of reduced products.The product carbon recovery rate(85.2%)and alkyd ratio(0.49 mol/mol)of the modified bacteria containing MV-containing EE-ANPM are significantly higher than the product carbon recovery rate(55.3%)and alkyd acid ratio(0.32 mol/mol)of the modified bacteria containing MV-free ANPM indicates that under the combined action of MV and electricity,overexpression of adh E2 can significantly increase the carbon recovery rate of the product and increase the level of reducing products.Biochar has redox groups and can be used as an electron mediator.Using wild-type C.carboxidivorans,biochar was used as the electronic mediator,four different fermentation conditions were designed,combined with the partial fermentation results using MV as the electronic mediator,and the effects of biochar,electricity,and calcium carbonate on the fermentation results were compared and analyzed.It is found that biochar can improve the efficiency of electron transport.After adding biochar,the carbon recovery rate of EE-ANPM increased from 66.4%to 82.8%,which was similar to the effect of adding MV.The alkyd ratio of adding biochar(0.17 mol/mol)is lower than that of adding MV(0.40 mol/mol),indicating that the ability of biochar to regulate intracellular reducing power and increase the production of reducing products is weaker than that of MV.