Climate change is escalating into a global climate disaster. Rising greenhouse gas emissions (e.g. CO₂) are fuelling this trend. However, the use of fossil fuels such as crude oil is still increasing (Ripple et al., 2024) and to reverse this trend climate-friendly alternatives are needed. Microbial gas fermentation is investigated to produce platform chemicals such as hexanoate and butyrate. Our approach is using the acetogens A. woodii and C. drakei in a synthetic co-culture.
A. woodii is capable of autotrophic growth utilising efficiently H₂ + CO₂, while producing natively acetate. By plasmid-borne expression of a D-lactate dehydrogenase (ldhD) from Leuconostoc mesenteroides and knocking out A. woodii´s native genes encoding the bifurcating lactate dehydrogenase complex, a strain was constructed, capable of producing lactate as a second product (Mook et al., 2022). C. drakei is capable of producing butyrate and hexanoate via reverse-β-oxidation using H₂ + CO₂ as well as lactate as a substrate. One challenge during co-cultivation is the monitoring of two different species over the cultivation time. Therefor quantitative PCR (qPCR) is an option to quantify the respective cells ampilification of unique genes.
The idea is to define a correlation between respective c_q values and cell counts or OD₆₀₀ values. Primers were designed to amplify the gene encoding the formate dehydrogenase from A. woodii and the gene encoding the phosphate butyryltransferase from C. drakei. To determine a strong correlation between cell counts and OD600 values several measurements were done over the course of heterotrophic growth experiments. Furthermore, dilution series of each specie obtained at the highest OD₆₀₀ were also prepared and subsequently analysed. A correlation (R²=0.96) was found between OD₆₀₀ values and the logarhithm of the cell counts for C. drakei during growth. qPCR has been performed with this isolated genomic DNA from the dilution row and the results are currently evaluated. Another method to determine the number of cells in this co-culture is by labelling the cells with species specific fluorescent labelled DNA probes. Therefor probes binding in the V4 region of 16S rDNA of A. woodii and C. drakei have been designed. The probe binding in A. woodii´s DNA is labelled with the fluorophore ATTO495, while the probe binding in C. drakei´s DNA is labelled with Pacific Blue. The fixed and labelled cells will be analysed according to an established hybridization strategy.