A technical overview of C1 gas fermentation

Metabolomics

Carbon dioxide (CO2) and methane (CH4) are abundant one-carbon (C1) components of greenhouse gases and their atmospheric concentrations have seen a drastic increase since the industrial revolution. Besides industrial activities, agricultural practice also causes substantial emissions of these two kinds of gases. Nowadays it is an urgent demand to reduce emissions of greenhouse gases for controlling global warming. Biological conversion of C1 gases to industrial high-value hydrocarbon-based chemicals (such as the ABE — acetone, butanol and ethanol1) via fermentation has been proven to be an effective approach to meet the demand without completing for photosynthetic resources (e.g., food) or land2. Through converting waste C1 gases into biofuels, we can reduce our reliance and demand on fossil fuels, which in turn reduces our total carbon emission. Because of this great environmental benefit, here I outline technologies that recycle waste C1 gases for industrial and environmental purposes.

1. Bacterial chassis for industrial C1 gas recycling

By far, a large proportion of bacterial fermentation chassis that produces biofuels and industrially high-value chemicals from C1 gas feedstock are restricted to acetogenic Clostridium spp., such as C. autoethanogenum, C. acetobutylicum, C. saccharoperbutylacetonicum, C. ljungdahlii and C. aceticum 1–3, 7. Bacteria of the genus Clostridium are spindle-shaped, Gram-positive and spore-forming and most of them are obligate anaerobic4. C. autoethanogenum and its closely related species C.ljungdahlii produce ethanol from carbon monoxide (CO) and CO2 through the Wood-Ljungdahl pathway2, 3.

Bacteria of a soil-living aerobic species Cupriavidus necator (previously known as Ralstonia eutropha) constitute a second kind of chassis used in industry for capturing its waste C1 gases. The ability of C. necator in biosynthesis of poly[(R)-3-hydroxybutyrate] (PHB) in the presence of CO2 and H2 has been exploited for decades by industry producing biodegradable plastic5. Moreover, an engineered C. necator strain converts formate to isobutanol and 3-methyl-1-butanol (3MB) when linked to an electrochemical device that synthesising formic acid with CO2 and H2O and protecting the bacterium from inhibitory by-products, making it a promising chassis for industrial production of biofuels6.

2. C1 gas fixation: the Wood-Ljungdahl pathway

The Wood-Ljungdahl (WL) pathway (Figure 1), also known as the acetyl-CoA pathway11, is the most ancient and efficient CO2-fixation pathway in nature12, 16, with a possible existence in the last common ancestor of all cells (LUCA)8. The pathway reduces CO2 with H2 to produce acetyl-CoA, which is then converted into biomass via a complete/incomplete reductive tricarboxylic acid (TCA) cycle (Figure 2)7 and acetate (Figure 3)9.

Wood-Ljungdahl pathway
Figure 1. Wood-Ljungdahl pathway. This diagram was created by S. Ragsdale and E. Pierce7. CoA: acetyl-coenzyme A (H3C-CO-SCoA).

As illustrated in Figure 1, the WL pathway consists of a methyl branch (Eastern branch) and a carbonyl branch (Western branch)7. Homologues of the carbonyl branch is shared between archaea and bacteria, while the methyl branch differs between these two taxonomical domains in C1-carriers, cofactors, electron transporters and enzymes13. Furthermore, the end product of the WL pathway differs between most archaea and bacteria when the pathway is used for energy production and carbon fixation: the archaea produce methane (hence these archaea are called CO2-reducing methanogens) while the bacteria produce acetate (acetogens)13.

Incomplete TCA cycle
Figure 2. An incomplete reductive TCA cycle that enables converstion of acetyl-CoA into cellular intermediates. Dashed arrows denote enzymes that are not found in the genome of Moorella thermoacetica (formerly known as Clostridium thermoaceticum)7, 10. Acetogens utilises this incomplete reverse TCA cycle to produce biomass from acetyl-CoA7. This diagram was created by S. Ragsdale and E. Pierce7.

In regards to the generation of pyruvate in the TCA cycle, it has been shown that C. necator mutants defective in PHB production secrete a large amount of pyruvate into the medium when cultured chemolithoautotropically5.

Acetate generation
Figure 3. Four pathways leading to the generation of acetate in prokaryotes and eukaryotes. This diagram was created by van Grinsven et al9.

3. Bioengineering for C1 gas recycling

The development of bioreactors for recycling industrial waste C1 gases requires a multidisciplinary collaboration of mechanical engineering, material science, process control (automation), biochemistry, bioengineering, microbial metabolism and genomics, and so forth. Here, I summarise two recent advances in creating these bioreactors to show common methodologies of this field. For more details of this field, see a profound technical review of C1 gas fermentation published by Liew et al17.

  • In 2017, Liew et al. revealed that aldehyde:ferredoxin oxidoreductase (AOR) plays a critical role in ethanol formation in acetogens and inactivation of a bifunctional aldehyde/alcohol dehydrogenase (AdhE) leads to a substantial increase in ethanol yield (up to 180%). For this study of metabolic engineering, the investigators used ClosTron and allelic exchange mutagenesis to create AdhE mutants14.

    Autotrophic product formation in C. autoethanogenum
    Figure 4. Autotrophic product formation in C. autoethanogenum. WLP: the WL pathway. This figure was produced by Liew et al14.

  • Li et al. (2012) built an integrated electromicrobial bioreactor that utilises C. necator to convert formate into 3-methyl-l-butanol (3MB) and isobutanol15. For this bioreactor, the investigators exploit an electrochemical reaction to produce formic acid from CO2 and H2O, which circumvent the safety issue of direct usage of H2. In order to prevent bacterial stress response resulted from the toxicity of O2- and NO, they shielded the anode with a porous ceramic cup.

4. Industrial applications

Industrial waste C1 gases, such as those from steel mills, oil refining and coal gasification, and synthetic gas (syngas for short) have been used in industry as sustainable feedstocks for the production of ethanol16, 18. Compared to starch-based feedstocks (the so-called first generation feedstocks), the waste gases (the second-generation feedstocks) have the lowest carbon cost18. Several companies emerged to commercialise relevant techniques and they launched collaborative projects with steel manufacturers. For example, LanzaTech operated demonstration projects in collaboration with BaoSteel (China Baowu Steel Group, headquartered in Shanghai, China) and Shougang Group (Beijing, China). The project with BaoSteel yields ethanol showing competitive price with that produced from the starch-based feedstocks18. Despite of this success, there are still challenges to be overcome. For instance, the creation of clostridia strains for commercialisation18.

References

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  2. Humphreys, C. M. et al. Whole genome sequence and manual annotation of Clostridium autoethanogenum, an industrially relevant bacterium. BMC Genomics 16, 1085 (2015).
  3. Huang, H. et al. CRISPR/Cas9-Based Efficient Genome Editing in Clostridium ljungdahlii, an Autotrophic Gas-Fermenting Bacterium. ACS Synth. Biol. 5, 1355–1361 (2016).
  4. Riley, T. V. Clostridium: Gas gangrene; tetanus; food poisoning; pseudomembranous colitis. in Medical Microbiology (eds. Greenwood, D., Barer, M., Slack, R. & Irving, W. B. T.-M. M. (Eighteenth E.) 245–255 (Churchill Livingstone, 2012). doi:https://doi.org/10.1016/B978-0-7020-4089-4.00037-8.
  5. Burbidge, A. & Minton, N. P. SBRC-Nottingham: sustainable routes to platform chemicals from C1 waste gases. Biochem. Soc. Trans. 44, 684 LP – 686 (2016).
  6. Li, H. et al. Integrated Electromicrobial Conversion of CO2 to Higher Alcohols. Science (80-. ). 335, 1596 LP – 1596 (2012).
  7. Ragsdale, S. W. & Pierce, E. Acetogenesis and the Wood–Ljungdahl pathway of CO2 fixation. Biochim. Biophys. Acta - Proteins Proteomics 1784, 1873–1898 (2008).
  8. Weiss, M. C. et al. The physiology and habitat of the last universal common ancestor. Nat. Microbiol. 1, 16116 (2016).
  9. van Grinsven, K. W. A. et al. Acetate:Succinate CoA-transferase in the Hydrogenosomes of Trichomonas vaginalis: IDENTIFICATION AND CHARACTERIZATION . J. Biol. Chem. 283, 1411–1418 (2008).
  10. Pierce, E. et al. The complete genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum). Environ. Microbiol. 10, 2550–2573 (2008).
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  12. Varma, S. J., Muchowska, K. B., Chatelain, P. & Moran, J. Native iron reduces CO2 to intermediates and end-products of the acetyl-CoA pathway. Nat. Ecol. Evol. 2, 1019–1024 (2018).
  13. Borrel, G., Adam, P. S. & Gribaldo, S. Methanogenesis and the Wood–Ljungdahl Pathway: An Ancient, Versatile, and Fragile Association. Genome Biol. Evol. 8, 1706–1711 (2016).
  14. Liew, F. et al. Metabolic engineering of Clostridium autoethanogenum for selective alcohol production. Metab. Eng. 40, 104–114 (2017).
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  17. Liew, F. et al. Gas Fermentation — A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks. Front. Microbiol. 7, 694 (2016).
  18. Jiang, Y., Liu, J., Jiang, W., Yang, Y. & Yang, S. Current status and prospects of industrial bio-production of n-butanol in China. Biotechnol. Adv. 33, 1493–1501 (2015).