CO2 fixation by anaerobic non-photosynthetic mixotrophy for improved carbon conversion

Abstract

Maximizing the conversion of biogenic carbon feedstocks into chemicals and fuels is essential for fermentation processes as feedstock costs and processing is commonly the greatest operating expense. Unfortunately, for most fermentations, over one-third of sugar carbon is lost to CO₂ due to the decarboxylation of pyruvate to acetyl-CoA and limitations in the reducing power of the bio-feedstock. Here we show that anaerobic, non-photosynthetic mixotrophy, defined as the concurrent utilization of organic (for example, sugars) and inorganic (for example, CO₂) substrates in a single organism, can overcome these constraints to increase product yields and reduce overall CO₂ emissions. As a proof-of-concept, Clostridium ljungdahlii was engineered to produce acetone and achieved a mass yield 138% of the previous theoretical maximum using a high cell density continuous fermentation process. In addition, when enough reductant (that is, H₂) is provided, the fermentation emits no CO₂. Finally, we show that mixotrophy is a general trait among acetogens.

Figure 1. The concept of mixotrophy and its demonstration.

(a,b) Different modes of fermentation are shown as an abbreviated metabolic network (a) and block flow diagrams (b). Heterotrophy (case I): hexose is consumed and CO₂ and potentially H₂ are produced. Mixotrophy (case II): hexose is consumed and excess reducing equivalents are used to fix endogenously produced CO₂; any unconsumed CO₂ is released from the process. H₂-enhanced mixotrophy (case III): hexose along with H₂ are fed to the microorganism and no CO₂ is released. Syngas-enhanced mixotrophy (case IV): hexose and CO:CO₂:H₂ are fed to the microorganism. Depending on the composition of the syngas and the metabolite of interest, CO₂ may still be released from the process. Dashed lines indicate potential pathways or products. (c,d) ¹³C-labelling fermentation profiles of CLJ (c) and CAU (d) during syngas-enhanced mixotrophy. Fructose (black line) consumed and metabolites produced during fermentation in the presence of a syngas mixture (¹³CO, ¹³CO₂, H₂ and N₂). The percentage of acetate labelled with ¹³C is shown in light blue for each time point. The s.d. of two biological replicates is shown in black error bars.

Figure 2. Metabolic engineering of CLJ to demonstrate mixotrophic production of acetone.

(a) Metabolic pathways downstream of pyruvate for the native and engineered CLJ. Native metabolites and enzymes are shown in black, and heterologous enzymes along with non-native metabolites are shown in blue. Integration with the WLP is shown through light blue arrows. The gene deletion is shown in red. PFOR, pyruvate:ferredoxin oxidoreductase; ALS, acetolactate synthase; ALDC, acetolactate decarboxylase; 23BDH, 2,3-butanediol dehydrogenase; SADH, secondary alcohol dehydrogenase; AK, acetate kinase; PTA, phosphotransacetylase; AAD, alcohol:aldehyde dehydrogenase; THL, thiolase; CoAT, CoA-transferase; AADC, acetoacetate decarboxylase. (b,c) Product profiles of CLJ ΔSADH (pTCtA) under mixotrophy and H₂-enhanced mixotrophy. Total molar yields (b) and product distributions (c) are shown. The s.d. of three biological replicates is shown in black error bars.

Figure 3. High cell density continuous acetone fermentation.

Fermentation performance for 150 h of cell retention fermentation. At hour 96, a harvest was initiated to maintain a constant cell density. Titers and cell densities (a) are shown in addition to volumetric productivities and the total acetone pathway (acetone, 3-HB, and isopropanol) product yield (b). (a) Acetone pathway titers (blue diamond), acetate titers (orange triangle) and cell densities (grey circle with broken line). (b) Fructose volumetric consumption (black squares), acetone pathway volumetric productivity (blue diamonds), acetate volumetric productivity (orange triangles) and acetone pathway mass yield (%g g⁻¹) from consumed fructose (red circle with broken line). Individual product and fructose titers are shown in Supplementary Fig. 2.

Figure 4. Mixotrophy is a general trait of acetogens.

(a) Carbon molar yields (C_M C_S⁻¹; C_metabolites C_substrate⁻¹), defined as the carbon moles produced divided by sugar carbon moles consumed, of C. acetobutylicum (CAC), C. ljungdahlii (CLJ), C. autoethanogenum (CAU), M. thermoacetica (MTA) and E. limosum (ELM) grown under different conditions. Cultures were grown on fructose with either a N₂ headspace (heterotrophy and mixotrophy) or a syngas headspace (syngas-enhanced mixotrophy). The red dashed line indicates a yield of 67%. (b,c) Product profiles of CLJ, CAU, MTA and ELM under mixotrophy (b) or syngas-enhanced mixotrophy (c) are shown. Yields of CO₂ in b are calculated based on total carbon consumed for each strain. The s.d. of three biological replicates is shown in black error bars.

Figure 5. Theoretical yields for various metabolites to demonstrate the potential of mixotrophy.

(a) Calculated maximum biological mass yields for several metabolites of interest under heterotrophic, mixotrophic and H₂-enhanced mixotrophic conditions (where only enough H₂ is supplemented to consume all CO₂ evolved). Amounts of H₂ required to achieve these yields are shown in b. 2,3-BD, 2,3-butanediol; 1,4-BD, 1,4-butanediol; 3-HB, 3-hydroxybutyrate; 2-HIB, 2-hydroxyisobutyrate.