Building Better Biofuels

If you were going to create the perfect biofuel, what would you make? When the founders of San Carlos, Calif.-based LS9 asked the question, their answer was quite simple, says Gregory Pal, senior director for corporate development. "You would make petroleum."
By Diane Greer
Like ethanol, the bio-petroleum would be produced from renewable feedstocks using a fermentation process. But LS9's renewable petroleum would overcome many of ethanol's shortcomings. The fuel would contain more energy than ethanol, be supported by the existing infrastructure of pipelines, refineries and fueling stations and run in a wide range of engines. "You would make a fuel that was dropiin compatible with existing fuel systems," Pal says.

The question wasn't spurred by fanciful musing but by new techniques enabling scientists to customize an organism's biological processes to produce novel substances. LS9's founders saw an opportunity to leverage this emerging technology, called synthetic biology, to create "designer" microbes producing biofuels that are chemically equivalent to petroleum and diesel.

LS9 is not the only company developing renewable fuel using synthetic biology. Gevo, in Pasadena, Calif., is manipulating organisms to make butanol, Emeryville, Calif.-based Amyris Biotechnology is turning microbes into miniature factories generating diesel and jet fuel substitutes and Synthetic Genomics, in La Jolla, Calif., is developing genomes from scratch, tailored to biofuel production.

Beyond Genetic Engineering
Synthetic biology aims to modify existing biological systems or build new systems to perform novel tasks. The technology extends well beyond genetic engineering, which typically attempts to alter a few characteristics of an organism by inserting genes from other organisms.

"Synthetic biology applies more of a systems or engineering approach," Pal explains. Working from the bottom up, scientists define the biological processes they wish to build and identify the genes or sets of genes needed to produce the intermediate chemicals and control the biochemical reactions within an organism to execute the process. They then rewire the organism's genetic coding, by inserting, removing and disabling genes, to meet the design specification.

New gene synthesis technology facilitates the process, allowing scientists to decipher natural DNA sequences and then replicate and modify the genes in the lab. These artificial genes are then inserted into existing organisms.

Advances in our understanding of how genes interact and simple organisms function are making the science more targeted and effective, allowing practitioners to reengineer an organism's genetic map so that it works like a fine-tuned machine, says Kinkead Reiling, senior vice president at Amyris.

Retooling Microbes
LS9 is using synthetic biology to rewire the metabolic processes of yeast and e.coli to make its biofuels.

When microbes break down (metabolize) food into energy, excess energy produced by the process is stored. Many organisms, including humans, store excess energy by
converting fatty acids produced during metabolism into lipids (i.e. fats), what we humans know as the love handles around our abdomens.

LS9 is tapping into this storage mechanism by modifying the organism's metabolic process to divert fatty acids into biofuel production, Pal explains.

Fatty acids are molecularly similar to hydrocarbons, which are the building blocks of gasoline, diesel and jet fuel, Pal says. By re-engineering the genetic coding of e.coli and yeast, LS9 creates a miniature assembly line (metabolic pathway) to synthesize the biofuel.

Genes missing from the microbes and required to produce intermediate substances and enzymes (which produce biochemical reactions) are inserted into the organisms. Genes producing unwanted substances or diverting energy from the biofuel production process are silenced.

To make diesel LS9's microbes ferment (metabolize) sugars into fatty acids. But instead of converting the fatty acids into lipids, the cell's modified metabolic process produces enzymes that combine the fatty acids with alcohol (also produced by the cell) to generate diesel, which the cell excretes.

The process consumes 65 percent less energy than ethanol production since the energy-intensive distillation process is eliminated. Unlike ethanol, diesel is not water-soluble and floats to the surface of the fermentation mixture, facilitating its removal.

LS9's goal is to create fuel that is cost competitive with oil at $40 to $50 per barrel, Pal says. A small-scale pilot facility, planned for this year, will generate the performance and economic data to support investment in a large-scale commercial facility. Pal expects to have a product to market in three to four years.

Building Better Bugs
Modifying an organism's genetic circuitry is only the first step in the process. The next major challenge is re-engineering the microbe to produce fuels more efficiently and in commercial quantities.

Gevo, founded in 2005, initially focused on redesigning the metabolic processes of microbes to convert waste methane gas into methanol. This work led to technology to re-engineer an organism's metabolic pathways to increase its tolerance to toxic environments, explains Pat Gruber, company chief executive officer.

Fuel produced by microbes during fermentation accumulates and eventually attains concentrations that are toxic to the organisms. Constant intervention is required to remove the fuel, which adds costs to the process. Increasing an organism's tolerances improves process efficiency and facilitates scaling.

Discussions in the company soon turned to the best means to employ the new technology. "Butanol is a more interesting fuel than methanol, both economically and from a performance standpoint, so we shifted directions," Gruber explains.

Butanol contains more energy than methanol or ethanol, it can be blended with gasoline without retrofitting engines and it can be distributed in existing pipelines. It is also used as a chemical intermediate, creating numerous market opportunities, Gruber says.

Most efforts to ferment sugars into butanol rely upon bacteria, Clostridium acetobutylicum. But even with genetic modification, the bacterium doesn't produce enough butanol to be economically viable.

Gevo's approach is to concentrate on organisms, such as e.coli and yeasts, that serve as outstanding platforms for biofuel production, explains Matthew Peters, Gevo vice president and chief scientific officer.

The company recently licensed technology from James Liao, a chemical engineer at the University of California, Los Angeles, which re-engineers e.coli to make butanol. Liao rewired e.coli's genetic circuitry by adding genes to convert keto acids, produced during metabolism, into butanol.

Once the new genetic machinery is in the cell, the next step is optimizing the organism's metabolic processes to increase biofuel yields and throughput rates, Gruber explains. During metabolism some operations are essential to producing fuel molecules, others are not. The goal is to enhance those processes making fuel while eliminating processes that generate undesired coproducts.

Liao removed genes producing nonessential substances and enhanced the productivity of others. These modifications increased keto acid production, boosting butanol production.
Gruber, who previously worked at Cargill developing large-scale fermentation technologies, expects Gevo's technology will continue to evolve. "I've seen plants double an organism's productivity after they have been built," Gruber says. "It is a different paradigm than what people are used to thinking about in the chemical world."

Gevo's goal is to produce fuel at an unsubsidized price that is less than gasoline, says Tom Dries, vice president of business development. To keep costs down, the company will retrofit existing ethanol plants to run its processes, at a cost of about $20 million per facility. Dries expects to produce its first product sometime in 2009.

Scaling Up
John Melo, chief executive officer at Amyris Biotechnologies is also focused on scale. His goal is to produce 338 million gallons of diesel from his synthetically modified microbes by 2011.

Amyris originally started to commercialize an inexpensive version of Artemisinin, an anti-malarial drug, created using synthetic biology techniques. Work on the project was bolstered by a $42-million grant from the Gates Foundation.

Using some of the technology developed for synthesizing Artemisinin, the company is now producing diesel, jet fuel and gasoline substitutes. "The Artemisinin project taught us a good bit about how a microbe would tolerate the type of chemicals we were trying to put into it and we learned how to take a plant enzyme and move it into a microbe effectively," Reiling explains.

The Amyris team is using computation tools to identify the suites of genes to assemble within an organism to produce its biofuels, along with tools to optimize the genes for use in the system. "Dozens of genes are affected, inserted and changed in the process," Reiling says.

The company is initially focusing its efforts on commercializing its diesel product. "Diesel is growing at two to three times the rate of gasoline," Melo says. "There is not a scaleable renewable fuel today servicing the diesel market."

Melo breaks the challenge of scaling his process into three components: cost, feedstock and infrastructure. On the cost side, the company is working on increasing the productivity of its process to reach parity with oil at $55 to $60 per barrel. To achieve scales in feedstock and infrastructure, Amyris is forming partnerships.

In April, Amyris announced a joint venture with Crystalsev, one of Brazil's largest ethanol producers, to commercialize its diesel technology in Brazil. Crystalsev will provide 2 million tons of sugarcane crushing capacity and will convert two of its ethanol plants to produce Amyris' renewable diesel from cane juice, Melo explains. Production is slated to begin by 2010.

Melo expects to sign a second major deal in Brazil in the July to October timeframe. "By the end of the year we will have geographic expansion beyond Brazil."

Producing Genomes from Scratch
Most companies employing synthetic biology to produce biofuels are modifying small segments of an organism's genetic materials using existing and man-made genes. At Synthetic Genomics, research efforts are also focused on creating all the genetic material for an organism (its genome) from scratch (de novo), tailored to biofuel production. "Most of these organisms have other priorities in life producing substances for their own particular needs," explains Ari Patrinos, the company's president. "There is a limit to how much you can tweak them to do what you want."

"If you can design the genome de novo, you only include those processes and activities of interest to you," Patrinos says. As a result, the biological processes will be more efficient and productive and include built in tolerances.

To date no one has produced a functioning genome from scratch. In January Synthetic Genomics progressed toward that goal when it announced the successful assembly of the entire genome of the bacteria Mycoplasma genitalium, the largest man-made DNA structure ever produced. The next step is to insert the synthetic genome into an existing cell, essentially "booting it up", to produce a functioning organism, Patrinos says.

"Once you have demonstrated that you can do the genome, you can add the appropriate promoters that turn on and off genes," Patrinos says. He envisions inserting sets of genes into the genome, observing the outcomes and then optimizing the final combination of genes that produces the best product at the highest efficiencies.

Patrinos believes Synthetic Genomes will begin producing biofuels in the next few years. "I think we have a leg up on scaling up because the organisms can be tailored for the scaling process."

Diane Greer is a New York-based writer and researcher specializing in renewable energy, clean technologies and sustainable business.