New advanced biofuel as an alternative to diesel fuel

September 27, 2011
This diagram shows the steps for synthesizing bisabolane, an alternative to D2 diesel, from the chemical hydrogenation of bisabolene, which is metabolized in microbes via an engineered mevalonate pathway. Credit: Image from Taek Soon Lee

Researchers with the DOE's Joint BioEnergy Institute (JBEI) have identified a potential new advanced biofuel that could replace today's standard fuel for diesel engines but would be clean, green, renewable and produced in the United States. Using the tools of synthetic biology, a JBEI research team engineered strains of two microbes, a bacteria and a yeast, to produce a precursor to bisabolane, a member of the terpene class of chemical compounds that are found in plants and used in fragrances and flavorings. Preliminary tests by the team showed that bisabolane's properties make it a promising biosynthetic alternative to Number 2 (D2) diesel fuel.

"This is the first report of bisabolane as a biosynthetic alternative to D2 diesel, and the first microbial overproduction of bisabolene in Escherichia coli and Saccharomyces cerevisiae," says Taek Soon Lee, who directs JBEI's program and is a project scientist with Lawrence Berkeley National Laboratory (Berkeley Lab)'s Physical Biosciences Division. "This work is also a proof-of-principle for advanced biofuels research in that we've shown that we can design a biofuel target, evaluate this fuel target, and produce the fuel with microbes that we've engineered."

Lee is the corresponding author of a paper reporting this research in the journal Nature Communications entitled "Identification and microbial production of a terpene-based advanced biofuel." Co-authoring this paper were Pamela Peralta-Yahya, Mario Ouellet, Rossana Chan, Aindrila Mukhopadhyay and Jay Keasling.

The rising costs and growing dependence upon foreign sources of petroleum-based fuels, coupled with scientific fears over how the burning of these fuels impacts global climate, are driving the search for carbon-neutral renewable alternatives. Advanced biofuels – liquid transportation fuels derived from the cellulosic biomass of perennial grasses and other non-food plants, as well as from agricultural waste – are highly touted for their potential to replace gasoline, diesel and jet fuels. Unlike ethanol, which can only be used in limited amounts in gasoline engines and can't be used at all in diesel or jet engines, plus would corrode existing oil pipelines and tanks, advanced biofuels are drop-in fuels compatible with today's engines, and delivery and storage infrastructures.

"We desperately need drop-in, renewable biofuels that can directly replace petroleum-derived fuels, particularly for vehicles that cannot be electrified," says co-author Keasling, CEO of JBEI and a leading authority on advanced biofuels. "The technology we describe in our Nature Communications paper is a significant advance in that direction."

JBEI is one of three Research Centers established by the DOE's Office of Science in 2007. Researchers at JBEI are pursuing the fundamental science needed to make production of advanced biofuels cost-effective on a national scale. One of the avenues being explored is sesquiterpenes, terpene compounds that contain 15 carbon atoms (diesel fuel typically contains 10 to 24 carbon atoms).

"Sesquiterpenes have a high-energy content and physicochemical properties similar to diesel and jet fuels," Lee says. "Although plants are the natural source of terpene compounds, engineered microbial platforms would be the most convenient and cost-effective approach for large-scale production of advanced biofuels."

In earlier work, Lee and his group engineered a new mevalonate pathway (a metabolic reaction critical to biosynthesis) in both E. coli and S. cerevisiae that resulted in these two microorganisms over-producing a chemical compound called farnesyl diphosphate (FPP), which can be treated with enzymes to synthesize a desired terpene. In this latest work, Lee and his group used that mevalonate pathway to create bisabolene, which is a precursor to bisabolane.

"We proposed that the generality of the microbial FPP overproduction platforms would allow for the biosynthesis of sesquiterpenes," Lee says. "Through multiple rounds of large-scale preparation in shake flasks, we were able to prepare approximately 20 milliliters of biosynthetic bisabolene, which we then hydrogenated to produce bisabolane."

When they began this work, Lee and his colleagues did not know whether bisabolane could be used as a biofuel, but they targeted it on the basis of its chemical structure. Their first step was to perform fuel property tests on commercially available bisabolene, which comes as part of a mixture of compounds. Convinced they were onto something, the researchers then used biosynthesis to extract pure biosynthetic bisabolene from microbial cultures for hydrogenation into bisabolane. Subsequent fuel property tests on the bisabolane were again promising.

"Bisabolane has properties almost identical to D2 diesel but its branched and cyclic chemical structure gives it much lower freezing and cloud points, which should be advantageous for use as a fuel," Lee says. "Once we confirmed that bisabolane could be a good fuel, we designed a mevalonate pathway to produce the precursor, bisabolene. This was basically the same platform used to produce the anti-malarial drug artemisinin except that we introduced a terpene synthase and further engineered the pathway to improve the bisabolene yield both in E. coli and yeast."

Lee and his colleagues are now preparing to make gallons of bisabolane for tests in actual diesel engines, using the new fermentation facilities at Berkeley Lab's Advanced Biofuels Process Demonstration Unit. The ABPDU is a 15,000 square-foot state-of-the art facility, located in Emeryville, California, designed to help expedite the commercialization of advanced next-generation biofuels by providing industry-scale test beds for discoveries made in the laboratory.

"Once the complete fuel properties of hydrogenated biosynthetic bisabolene can be obtained, we'll be able to do an economic analysis that takes into consideration production variables such as the cost and type of feedstock, biomass depolymerization method, and the microbial yield of ," Lee says. "We will also be able to estimate the impact of byproducts present in the hydrogenated commercial bisabolene, such as farnesane and aromatized bisabolene."

Ultimately, Lee and his colleagues would like to replace the chemical processing step of bisabolene hydrogenation with an alkene reductase enzyme engineered into the E.coli and yeast so that all of the chemistry is performed within the microbes.

"Enzymatic hydrogenation of this type of molecule is a very challenging project and will be a long term goal," Lee says. "Our near-term goal is to develop strains of E.coli and yeast for use in commercial-scale fermenters. Also, we will be investigating the use of sugars from biomass as a source of carbon for producing bisabolene."

Explore further: Striking the right balance: Researchers counteract biofuel toxicity in microbes

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5 / 5 (2) Sep 27, 2011
There are a few different flavors of this type of research, but I would be willing to bet that we will see one or more of them move to production in the next 20 years. The US military is pushing really hard in this direction and has already stated that they are willing to be an early adopter if someone can produce it in large enough quantities. That almost makes it a sure thing. It seems like a perfect fit for military use here in the US, since we can make it here and it's a plug and play replacement for D2. They don't say in this article, but I wouldn't be surprised if this group is getting DARPA funding.

I have to admit that I chuckled a little when I got to the end and they said they are looking into "sugars from biomass" as a food for the microbes. So we're back to corn and sugar beets again, but making an oil based fuel rather than alcohol based ethanol. It's still a positive step I think.
5 / 5 (1) Sep 27, 2011
If the Bacteria feeds our cars, who feeds the bacteria? Hopefully these bacteria can live off water and sunlight. Sure beats the crap out of ethanol!
2.5 / 5 (2) Sep 27, 2011
I would recommend that ethanol be scrapped in favor of butanol. see: and http://www.techno...y/18443/
These exist for gasoline already and much work, including pilot plants, is in progress. This new replacement for D2 puts yet another iron in the fire. Stearing away from food sugars is key to resolving bio-fuels. Ethanol is/was a stepping stone and it is time we move on.
not rated yet Sep 27, 2011
Here is the latest on Cobalt Technologies:

not rated yet Sep 27, 2011
I know this research is in preliminary stages, but I think they could list the currently used feedstock, and their ideal feedstock that they'd like to get it to.

We also don't know how much time or energy it takes...etc.

They have already used this process to create a small amount, so they already know how the fed the original microbes, and apparently the initial properties of bisabolane were good enough for them to pursue it.

Then again, they are short on facts, while avoiding the excessive hype. Perhaps they are holding their cards close to their chest until they have an idea what it will take to produce this on an ideal basis.
1 / 5 (1) Sep 27, 2011
Butanol is okay, but it is almost exactly the same as ethanol. It is still an alcohol based fuel that's hard on storage tanks and motor parts compared to oily fuels like the one above. The difference between making ethanol and butanol is just a little different fermentation. As I understand it, butanol is a little more energy dense but takes a little more energy to produce, so it's basically a wash between ethanol and butanol.

All of these fuels still need something like corn or beets.

Algea fuels are probably better in the long term, but they need more work than the sugar based fuels. It's just a matter of time though.

not rated yet Sep 27, 2011
Interesting to be sure, but lacking in key areas such as cost, feedstocks, scalability. Theory and laboratory action is one thing, practicality can be quite another. I certainly hope this pans out.
When fuel can be EASILY be produced from organic waste, why is this not brought forward? Sure methane is a greenhouse gas, but is this true AFTER it's burned? Electroturbine hybrids should be able to run on nearly any fuel.
1 / 5 (1) Sep 28, 2011
Why not wastewater microbes=fuel
not rated yet Sep 28, 2011
Renewable yes, but misleading to say "green".
not rated yet Sep 28, 2011
a replacement for oil; to misquote Henry VIII "will no one rid me of these troublesome cars?" I was secretly hoping for fewer cars. There, my secret's out.
5 / 5 (1) Sep 28, 2011
This kind of biofuel research has its own special Catch 22. The use of waste feedstocks is certainly a viable source for biofuels, however the logistics and availability of such wastes represent a very small fraction of our actual energy needs. Inevitably, the assumption is made that primary biofuel feedstock production will be needed. All primary biofuel stocks are dependent on NPK fertilizers (which are dependent on peak petro and peak phosphate) and compete directly with human food production - inevitably raising the cost of that food.

In 2008 scientist estimated we had a 300 year supply of mineable phosphate reserves. By 2011 they estimated those reserves will peak in less than 30 years and be completely depleted in as little as 50 years.

The "Green Revolution" that took the human population from 2 B to 7 B was totally a function of cheap petro - which in turn allowed for cheap mined phosphate. Our future is critically dependent on phosphate.
not rated yet Sep 28, 2011
What we need is.... dun-dun-duuunnnnn!
Mr. Fusion-usion-sion-ion-on-n!

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