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| Fig. 1: Types of biomass. (Courtesy of the DOE) |
Increased fossil fuel dependency paired with consequent reserve depletion necessitates a serious exploration of alternative energy sources. One possible alternative is biomass. Biomass refers to any renewable organic material derived from animals and plants. Different types of biomass are delineated in Fig. 1. They include but are not limited to animal waste, wood, crops and alcohol fuels, and while animal waste and wood comprise the majority of biofuel resources, genetically engineered Escherichia coli (E. coli) has recently garnered attention around the world. [1] E. coli is a gram negative bacteria that is found in the gut of all humans and can be genetically engineered to be a promoter of alcohol fuels.
Contrary to public belief, most E. coli strains are harmless and comprise the microbiota of the human gut. [2] E. coli is also well studied - in fact it is the most widely studied organism when it comes to gene regulation and expression, and its genome has been sequenced and engineered in depth. Furthermore, E. coli can utilize both pentose and hexose sugars, unlike other studied organisms which can only use one of the two, making E. coli the optimal candidate for genetic engineering for biofuel production. [3]
Bioethanol is the most widely used biofuel and has a long and global history of usage. In 1876, German inventor Nicholaus Otto developed the first modern internal combustion engine fueled by ethyl alcohol. [4] By 1908 in the United States, the Ford T Model was designed such that it could run on corn-based ethanol. [5] As seen in Fig. 2, ethanol production has been continually increasing, with the majority of the world's ethanol being produced in the United States and Brazil. [6]
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| Fig. 2: Global ethanol production and trends. [6] (Courtesy of the USDA) |
It must be noted, however, that more widespread use of ethanol falters when considering its low energy density, high vapor pressure, and high hygroscopicity. Alternative biofuels such as butanol and isopropanol thus may be a more viable and economic alternative to bioethanol due to their higher energy densities and lower vapor pressures, compared to ethanol. [3] In addition to these biofuels, engineered E. coli can produce two jet fuel precursor monoterpenoids and three precursor sesquiterpenes - all of which have been identified as promising sustainable alternatives to current aviation fuels. [7,8]
All of the aforementioned biofuels can be produced by E. coli. Broadly speaking, the bacteria intake sugar as their food source, convert it to adenosine triphosphate (ATP), and through a fermentation process, convert this energy carrier into a biofuel. More specifically, in the absence of oxygen, E. coli can endogenously produce ethanol through a process where "one mole of glucose is metabolized into two moles of formate, two moles of acetate, and one of ethanol". [3] But, while wild type E. coli can produce ethanol, the native process falls short with a yield of just 0.26 g ethanol/g of glucose compared to the theoretical yield of 0.51 g ethanol/g of glucose. Through genetic engineering, however, biofuel yields can be optimized. To produce more ethanol, the genes pdc and adhB are inserted into the E. coli genome. These genes are found in operon from a plasmid that expresses a promoter of ethanol production. This, in conjunction with deletion of a gene that encodes succinate production, gives rise to E. coli that yields ethanol at 46 g/L minimal media, which is a medium containing only water, salts, and a carbon source. Through similar manipulations, E. coli can produce n-butanol at 6.1 g/L and isopropanol at 5 g/L. [3] To understand the significance of these values, the reported yields must be placed in the context of their respective energy densities. E. coli yields a net ethanol energy density of
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| Table 1: Energy densities of different fuels. |
For butanol,
and for isopropanol,
It is important to note that the volume in these values refers to the volume of minimal media the bacteria are grown in. While ethanol's energy density is lower than other biofuels, the net energy density is 6-8 times greater than that of butanol and isopropanol, making it presently the best candidate for microbial fuel. While there may be concern about unintended environmental and health effects of engineered E. coli, ethanol-producing strains have already been successfully engineered to block recombination, and it is a goal of bioengineers to delete metabolic pathways such that the E. coli cannot survive in the wild. [9,10] Thus, environmental impacts are not a concern and E. coli-derived ethanol proves to be a promising candidate among different types of biofuels. However, certain strains of S. cerevisiae - also known as brewer's yeast - have yields as high as 96.9 g/L, whereas E. coli can tolerate ethanol up to 50 g/L and thus is limited in its efficiency, compared to other microorganisms. [11,12] While E. coli is a viable microorganism for ethanol production, whether it is the one best suited for mass production therefore requires further exploration and consideration of many factors, such as glucose uptake efficiency.
Biofuels are promising energy sources that are cleaner than fossil fuels. Burning ethanol results in reduced CO2 emission, but the extent of this reduction is not yet confirmed. Engineers are now working to genetically modify E. coli so that it consumes CO2. Thus the CO2 released through burning ethanol could be offset by this strain of E. coli. [13] Furthermore, while ethanol is the most fruitful biofuel produced by the engineered E. coli, further examination of butanol and isopropanol could shift the current biofuel landscape. However, while biofuels are cleaner than fossil fuels, they are not clean - at least not yet. During times where the threat of irreversible environmental damage looms, cleaner energy may simply not be enough, and so the future of biofuels remains uncertain.
© Rochelle Radzyminski. The author warrants that the work is the author's own and that Stanford University provided no input other than typesetting and referencing guidelines. The author grants permission to copy, distribute and display this work in unaltered form, with attribution to the author, for noncommercial purposes only. All other rights, including commercial rights, are reserved to the author.
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[3] V. Koppolu and V. K. R. Vasigala, "Role Of Escherichia coli in Biofuel Production," Microbiol. Insights 9, 29 (2016).
[4] E. C. S, "Nikolaus August Otto, 1832-1891," Nature 129, 892 (1932).
[5] W. A. Payne, "Are Biofuels Antithetic to Long-Term Sustainability of Soil and Water Resources?" in Advances in Agronomy, Vol. 105, ed. by D. L. Sparks (Academic Press, 2010), pp. 2-43.
[6] J. Beckman and G. Nigatu, "Global Ethanol Mandates: Opportunities for U.S. Exports of Ethanol and DDGS," U.S. Department of Agriculture, BIO-05, October 2017.
[7] D. Mendez-Perez et al., "Production of Jet Fuel Precursor Monoterpenoids from Engineered Escherichia coli," Biotechnol. Bioeng. 114, 1703 (2017).
[8] C.-L. Liu et al., "Renewable Production of High Density Jet Fuel Precursor Sesquiterpenes from Escherichia coli," Biotechnol. Biofuels 11, 285 (2018).
[9] K. Ohta et al., "Genetic Improvement of Escherichia coli for Ethanol Production: Chromosomal Integration of Zymomonas mobilis Genes Encoding Pyruvate Decarboxylase and Alcohol Dehydrogenase II," Appl. Environ. Microb. 57, 893 (1991).
[10] D. Biello, "Bacteria Transformed into Biofuel Refineries," Scientific American, 27 Jan 10.
[11] M. A. Cotta, "Ethanol Production from Lignocellulosic Biomass by Recombinant Escherichia coli Strain FBR5," Bioengineered 3, 197 (2012).
[12] M. Parapouli et al., "Saccharomyces cerevisiae and Its Industrial Applications," AIMS Microbiol. 6, 1 (2020.
[13] S. Gleizer et al., "Conversion of Escherichia Coli to Generate All Biomass Carbon from CO2," Cell 179, 1255 (2019).