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Researchers show mixotrophic fermentation process improves carbon conversion, boosting yields and reducing CO2

A team from White Dog Labs, a startup commercializing a mixotrophy-based fermentation process, and the University of Delaware have shown that anaerobic, non-photosynthetic mixotrophy—the concurrent utilization of organic (for example, sugars) and inorganic (CO2) substrates in a single organism—can overcome the loss of carbon to CO2 during fermentation to increase product yields and reduce overall CO2 emissions.

In an open-access paper published in Nature Communications, the researchers report on their engineering of the bacterium Clostridium ljungdahlii to produce acetone with a mass yield 138% of the previous theoretical maximum using a high cell density continuous fermentation process. In addition, when enough reductant (i.e., H2) was provided, the fermentation emitted no CO2. They further showed that mixotrophy is a general trait among acetogens.

The production costs for most chemicals via microbial fermentation are currently high compared to oil-derived products primarily because of operating costs associated with feedstock and feedstock processing. Consequently, first and second generation bioproduct manufacturing processes are economically challenged, particularly in light of recent low oil prices. One way to mitigate high feedstock cost is to maximize conversion into the bioproduct of interest. This maximization, though, is limited because of the production of CO2 during the conversion of sugar into acetyl-CoA in traditional fermentation processes.

Acetyl-CoA is a central building block and a link between glycolysis and almost all downstream metabolic pathways and serves as a focal point for the production of biofuels and industrial chemicals by microbial fermentations. However, the ability to achieve metabolically efficient production of acetyl-CoA is hindered by energetic requirements and biochemical pathway constraints, requiring the production of CO2 for every acetyl-CoA produced from glycolysis. Thus, one-third of all carbon in the feedstock is lost to CO2, resulting in maximum carbon conversion of 67% at best, and lower in actuality due to cell mass creation, cell maintenance needs and other constraints.

… [an] alternative approach that stoichiometrically converts sugar to acetyl-CoA is anaerobic, non-photosynthetic mixotrophic fermentation4 (here referred to as mixotrophy). … The amount of CO2 re-assimilated with mixotrophy depends upon the degree of reduction of the desired metabolite (product). The more reduced the product, the less CO2 can be re-assimilated, because NAD(P)H is directed towards product formation rather than CO2 fixation. However, this reducing equivalent deficiency can be overcome through H2-enhanced mixotrophy, whereby sufficient H2 is exogenously provided to fully recapture the CO2 lost in glycolysis. To avoid CO2 emissions associated with H2-production, electrolysis of water powered by solar, wind or hydroelectricity would be a preferred source and has achieved a level of maturity and success. Alternatively, syngas can be added to sugar fermentation to provide the necessary reducing power and carbon.

… In this study, we demonstrate the ability of a broad range of acetogenic organisms to conduct mixotrophy and H2- or syngas-enhanced mixotrophy without carbon catabolite repression (CCR), thus enabling sugar to metabolite yields that are not theoretically possible through heterotrophic (that is, traditional) fermentation.

—Jones et al.

The work was done in two labs at UD—those of Eleftherios (Terry) Papoutsakis, the Unidel Eugene du Pont Chair of Chemical Engineering, and Maciek Antoniewicz, the Centennial Junior Professor of Chemical and Biomolecular Engineering—in partnership with White Dog Labs.

The new quest started when Papoutsakis asked another doctoral student, Alan Fast, to try to calculate how well bacteria known as acetogens would do making biofuels.

Acetogens are anaerobic bacteria, which cannot grow in oxygenated environments. They can take carbon dioxide and hydrogen gas and turn them into chemicals such as acetone, butanol or ethanol.

In this study, researchers tested how C. ljungdahlii would do with two sources of fuel—sugars and synthesis gas—in the fermentation process.

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. Jones et al. Click to enlarge.

Before this, it was all hypothetical. This is the first demonstration that it takes place. We get both the increase in yield and consumption of all the carbon. There is a whole host of strains we could use, depending on the characteristics of the different strains.

—Shawn Jones, director of molecular and microbiology at White Dog Labs

Earlier this year, White Dog Labs was awarded a grant from the Department of Energy (DOE) to extend its MixoFerm mixotrophy technology from first- to second-generation biofuels and chemicals.

The company first pursued Acetone Butanol Ethanol (ABE) technology when it was founded in 2012. ABE was invented in the UK during WWI to provide acetone for gunpowder manufacturing. According to WDL Chairman, Dr. Sass Somekh, falling oil prices forced the company back to the lab to invent a new technology that would be more resistant to low fossil fuel prices.


  • Shawn W. Jones, Alan G. Fast, Ellinor D. Carlson, Carrissa A. Wiedel, Jennifer Au, Maciek R. Antoniewicz, Eleftherios T. Papoutsakis & Bryan P. Tracy (2016) “CO2 fixation by anaerobic non-photosynthetic mixotrophy for improved carbon conversion” Nature Communications 7, Article number: 12800 doi: 10.1038/ncomms12800



All of this relies on hydrogen.  Hydrogen is key to the biofuels, and it has to be cheap.

Of all the adjectives to describe hydrogen from RE, "cheap" is not one that I've seen.

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