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Facing the devastating impacts of global warming and energy insecurity, the US has mandated blending biofuels into its transportation fuel supply through the Renewable Fuels Standards (RFS) program. At current projected gasoline and diesel consumption rates, these standards mandate that 20% of US transportation fuels must be “renewable” by 2022 [1]. Although ethanol, the US’ most produced biofuel, is not suited to fulfill this mandate, recent breakthroughs in biofuels technologies are paving the way to potential solutions.

What’s wrong with ethanol?

Past biofuel mandates and tax benefits have successfully increased fuel-ethanol production in the US. Today, a full 40% of the US corn crop goes into producing fuel-ethanol. This is up from just 6% in 2000 [1]. Total ethanol production has risen from 1.6 to 13.3 billion gallons over this same time period [2]. So, what’s wrong with ethanol? Ethanol, as fuel, has two main problems: the feedstocks used for ethanol production and ethanol’s incompatibility with our liquid fuel infrastructure.

In the context of biofuel production, feedstocks are the plants or other materials used as starting materials in the production process. In the US, ethanol is most commonly produced by fermenting cornstarch. In this process, we extract sugars from corn kernels and feed them to yeast that, as a by-product of their growth, produce ethanol. We have gotten really good at this, and our best ethanol-producing yeast can turn ~95% of the sugar we feed them directly into ethanol [3].

Despite the efficiency of this conversion, the entire process is intrinsically limited by how much energy we have to put into producing the corn in the first place. When we grow corn to produce ethanol, we have to do a variety of things such as produce and apply fertilizer, till the land, and transport the ethanol product. The more energy we have to put into these processes, the less energy and green house gas (GHG) emissions we save from replacing gasoline with the ethanol produced. Although tallies of energy inputs into corn-ethanol production are somewhat dated, the US Department of Agriculture (USDA) estimates that corn-ethanol fuel only yields ~2.3 times the amount of energy that goes into producing it [4]. That is, if we add up all the energy stored in the ethanol and its co-products, energy obtained primarily from sunlight, this would only be 2.3 times the energy used to do things like produce fertilizer, run farm equipment and the ethanol distillation facility, and transport the final products. In addition, it is estimated that replacing a gallon of gasoline with an energy-equivalent amount ethanol only reduces our GHG emissions by ~20% [5].

Beyond these problems, it is estimated that there isn’t a lot of land left to increase corn production in the US; movement into previously unused lands could increase GHG emissions from corn-ethanol by releasing carbon stored in these unused soils. Finally, our increased use of corn for biofuel production will likely further increase corn and food prices [1,7].

Producing ethanol from feedstocks other than corn could require much less energy and further reduce GHG emissions. For instance, ethanol produced from sugarcane in Brazil yields ~8 times the energy required to produce it and lowers GHG emissions by >50% when used to replace gasoline [6]. However, unlike in Brazil, the US climate is not suitable for growing sugarcane.

Many feedstock problems may be overcome through what are called cellulosic biofuels. These are biofuels produced from the stems and woody portions of plants that aren’t normally used for food. Feedstocks for cellulosic fuels could come from crops that don’t have the same land and energy requirements as corn. Biotech researchers are currently engineering bacteria and yeast to eat cellulosic feedstocks, and others are developing non-biological processes to turn these materials into crude oil-like products.

It remains to be seen whether it is feasible to incorporate enough alternative feedstocks to meet the requirements of the renewable fuel standards and help overcome our dependence on petroleum-based fuels, but, even if we assume that we have a large supply of cellulosic feedstocks, and can develop bacteria and yeast that eat them readily, we cannot simply turn them into ethanol as we do with corn. The reason is that our current infrastructure cannot handle much more ethanol than it already receives.

This limitation is due, in large part, to ethanol’s chemical properties. Ethanol is hydrophilic or “water loving” whereas our current liquid fuels (gasoline, diesel, jet fuel) are hydrophobic or “water fearing.” Because ethanol is hydrophilic, unlike our current fuels, it interacts well with water. When it is blended into gasoline at small percentages, this is not a problem. At higher percentages, however, this interaction with water can cause ethanol to separate out of the fuel mixture, corrode the pipes used to transport it, and damage the engines it powers [8]. In fact, for vehicles up to model year 2000, the EPA only allows 10% ethanol mixtures and only 15% for vehicles model year 2001 and above. Clearly, neither of these reaches the 20% biofuel mixture that will be required by the renewable fuel standards in 2022 and comes no where near replacing all liquid fuels [1,9].

Making “drop-in biofuels” through bioengineering

How, then, are we supposed to replace our liquid fossil fuels with biofuels? As indicated above, companies such as Kior Inc. from Texas are developing non-biological means to directly convert cellulosic feedstocks into crude oil substitutes [10]. While this might be the way of the future, there are, as yet, very few commercial entities doing this. In the meantime, researchers are finding ways to alter the biology of commercially used microorganisms like E. coli and yeast to produce what are known as “drop-in biofuels.” These are biofuels that are, theoretically, similar enough to gasoline, diesel, or jet fuel that they can be mixed directly into these fuels at high percentages without modifications to engines or the distribution infrastructure.

As you can see in figure 1, our current liquid fuels are actually complex mixtures, rather than being composed of one type of molecule, like ethanol. Furthermore, different types of vehicles require different mixtures. Through what’s known as metabolic engineering researchers are developing microorganisms that produce the compounds founds in these mixtures.

Figure 1. Compounds Found in Liquid Fossil Fuels A) This is a small smattering of the compounds found in liquid fossil fuels. In each molecule, the black lines are bonds connecting carbon atoms, which can be found at the ends or corners between lines. Carbon must form four bonds with other atoms. Where 4 bonds are not indicated, carbon is bound to hydrogen. Fossil liquid fuels primarily contain carbon and hydrogen. B) Some biological molecules. Notice that nucleic acids, amino acids, and sugars all have large amounts of oxygen (O), nitrogen (N), or both. Fatty acids, however, are more similar to liquid fossil fuels in that they are composed of mostly carbons and hydrogens.

Metabolic engineering

Living things make many different types of chemicals. These are responsible for a vast variety of functions that keep us alive. As already mentioned, yeast and other microorganisms produce ethanol as a normal part of their growth. Interestingly, many organisms, and even humans, do produce compounds that are much more similar to the chemicals in liquid fuels than ethanol (Figure 2B). The problem is that we generally do not produce these chemicals in quantities great enough to fuel our cars. Through metabolic engineering, researchers are striving to change this.

Many of the chemicals found in living things are produced in reactions catalyzed by a particular class of proteins known as enzymes. Like all proteins, enzymes are produced using the information encoded in genes. Scientists have known for a long time that they can prevent organisms from making certain chemicals by getting rid of genes for enzymes that catalyze certain reactions. This is useful for producing biofuels; when we prevent an organism from creating a particular chemical, the resources that would have been used to produce that chemical have to go somewhere else. If we can get rid of enzymes that make things other than biofuels, we can funnel the resources these organisms get from food into the production of fuels and not other chemicals (Figure 2).

Figure 2. Metabolic Engineering A) When feedstocks such as sugars are fed to microorganisms, they are first broken down into a variety of chemical precursors which are turned into all of the different compounds (proteins, nucleic acids, carbohydrates, and lipids) that make up the microorganism. B) Using metabolic engineering, researchers direct chemical precursors to the production of useful compounds by getting rid of genes encoding enzymes that catalyze reactions making unwanted compounds. In this theoretical example, researchers would shunt chemical precursors to lipids, which are similar in structure to a variety of fuels.

Thanks to decades of research on enzymes and the genes that encode them, scientists have mapped out the pathways that foods can take to different chemicals in many different organisms. In fact, there are now computer programs that can be used to predict how getting rid of certain genes might lead to increased or decreased production of a certain compound in a certain organism. While these programs are incomplete and require development, they have already been used successfully to help produce different types of chemicals.

Beyond simply getting rid of enzymes that catalyze unwanted reactions, researchers have found many ways to take genes (and the enzymes they encode) from one organism and put them into another. Even though an organism may not produce a certain chemical, we can give it the enzyme or series of enzymes it needs to produce this chemical and, if we’re lucky, make it produce that chemical.

This is exactly what’s being done in biofuels production. Recent papers have shown that we can engineer E. coli, a bacterium commonly used in research and industrial applications, to produce long and medium-size alkanes, chemicals that are key components of fossil liquid fuels [11-13]. These researchers realized that alkanes are similar in structure to fatty acids, the compounds that make up the membranes of all cells (Figure 1B). Working from the knowledge that there are organisms that naturally produce a small amount of alkanes, these researchers hypothesized that they could find enzymes that catalyzed reactions that turn these fatty acids into alkanes. Once they found enzymes that could catalyze this conversion for long fatty acids, researchers were then able to look for similar enzymes. They found enzymes in plants and microorganisms that allowed E. coli to produce alkanes of different lengths. Similar techniques have enabled researchers to produce a variety of compounds with properties similar to those of our liquid fuels [14].

While running a car off of any one of these compounds alone is likely to have its own problems, using the right mixture, we should be able to make biofuels to suit all of our transportations needs. It’s very important to note however, that even though these studies have demonstrated that we can get organisms to produce compounds contained in fuels, we still have a long way to go in improving how efficiently we produce them. Remember, we have yeast that can produce ethanol at ~95% efficiency. We produce some of these compounds at such low yields that efficiencies aren’t even reported. Don’t dismay just yet though, with years of metabolism research behind them, it’s likely that researchers will come up with the right tweaks to increase these yields in the near future. After all, we’ve been using yeast to produce ethanol in alcoholc beverages for centuries. If our thirst for biofuels matches our thirst for alcohol, we’re sure to find ways to produce them efficiently!

Tyler J. Ford is a Graduate Student in the Biological and Biomedical Sciences Program at Harvard Medical School.

References:

[1] Schnempf, R., and Yacobucci, B. D. (2013) Renewable Fuel Standard (RFS): Overview and Issues, Congressional Research Service.

[2] Statistics: Historic U.S. fuel Ethanol Production, Renewable Fuels Association 2013.

[3] Gulati, M., Kohlmann, K., Ladisch, M. R., Hespell, R., and Bothast, R. J. (1996) Assessment of ethanol production options for corn products, Bioresource Technology 58, 253-264.

[4] Shapouri, H., Gallagher, P. W., Nefstead, W., Schwartz, R., Noe, S., and Conway, R. (2010) 2008 Energy Balance for the Corn-Ethanol Industry, United States Department of Agriculture.

[5] (2013) Ethanol Myths and Facts, US Departmnet of Energy: Bioenergy Technologies Office. http://www1.eere.energy.gov/bioenergy/printable_versions/ethanol_myths_facts.html

[6] (2013) Biofuels Compared, National Geographic. http://ngm.nationalgeographic.com/2007/10/biofuels/biofuels-interactive

[7] Wise, T. A. (2012) US corn ethanol fuels food crisis in developing countries, Aljazeera.http://www.aljazeera.com/indepth/opinion/2012/10/201210993632838545.html

[8] Allen, M. (2010) Can E15 Gasoline Really Damage Your Engine, in Popular Mechanics.http://www.popularmechanics.com/cars/alternative-fuel/biofuels/e15-gasoline-damage-engine

[9] Wald, M. L. (2013) For First Time, E.P.A. Proposes Reducing Ethanol Requirement for Gas Mix, in The New York Times, New York.

[10] Kior. http://www.kior.com/

[11] Howard, T. P., Middelhaufe, S., Moore, K., Edner, C., Kolak, D. M., Taylor, G. N., Parker, D. A., Lee, R., Smirnoff, N., Aves, S. J., and Love, J. (2013) Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli, Proceedings of the National Academy of Sciences of the United States of America 110, 7636-7641.

[12] Schirmer, A., Rude, M. A., Li, X., Popova, E., and del Cardayre, S. B. (2010) Microbial biosynthesis of alkanes, Science 329, 559-562.

[13] Choi, Y. J., and Lee, S. Y. (2013) Microbial production of short-chain alkanes, Nature 502, 571-574.
[14] Peralta-Yahya, P. P., Zhang, F., del Cardayre, S. B., and Keasling, J. D. (2012) Microbial engineering for the production of advanced biofuels, Nature 488, 320-328.

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