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Diesel from sugar cane

Fuel to be produced by genetic transformations in yeast

AMYRISSaccharomyces cerevisiae, the rod-shaped yeast, in a microscope photo, in the fermentation phase AMYRIS

Sugar cane no longer needs to be identified only by the name of the traditional sweetener. With the burning of cane bagasse to generate electricity this sweet grass can also be called “ethanol cane” and “electrical energy cane”, and “cachaça (Brazil’s national spirit) cane” should never be forgotten. Soon it may also be recognized as “diesel cane”. The news comes from Amyris-Crystalsev, a partnership in the form of a joint venture between Amyris, a North American biotechnology company, and Crystalsev, one of the biggest ethanol and sugar trading companies in Brazil, which belongs to the same group as the Santa Elisa Mill in Sertãozinho in Sao Paulo state. Also taking part in the venture is Votorantim Novos Negócios, a risk capital company that has started investing in the new company. The technology comes from Amyris, which has developed genetic modifications of commercial strains of the yeast, Saccharomyces cerevisiae, which is responsible for turning cane juice into ethanol during the fermentation process in the mills. The transformation makes the microorganism secrete a substance called farnesene, instead of ethanol, which can be used in any diesel engine, particularly in trucks, buses and tractors.

To make the technology viable on a large scale Amyris needed partners that had a lot of cheap raw material as a source of sugar and carbon. “In Brazil we’re improving the process for the industrial scale”, says Roel Collier, CEO of Amyris-Crystalsev. “The basic research, with development of the microorganism and even a pilot plant, was done in the United States.” For biologist, Fernando Reinach, executive director of Votorantim Novos Negócios, Amyris’ decision to produce diesel from sugar cane in Brazil is also due to the large scale fermentation knowledge mastered by Brazilian mills – Crystalsev, in this particular case – as well as the climate and production to be carried out using a cheap source of sucrose and carbon. “The production of biofuels from sugar cane has to be done here because it’s expensive to export it and transform the sugar cane juice somewhere else”, says Reinach, who in October joined the board of Amyris in the United States. For the new diesel to be competitive the price of a barrel of oil must be close to US$ 60. The new diesel is not biodiesel because it does not undergo the same processes as this biofuel. Reinach believes that he will find a good market abroad because in addition to being a commodity, the fuel is very pure. “It’s better than the best diesel available today, mainly because it doesn’t have sulfur (one of the substances that is most responsible for air pollution) like diesel made from oil.” This characteristic has strong environmental appeal, as well as the fact that the carbon dioxide (CO2) cycle is favorable to sugar cane because plantations, in order to photosynthesize and grow, absorb this gas to once more produce the sucrose that will result in the diesel.

Preparation of the new fuel requires few modifications in the process or the traditional machinery used for producing ethanol. Still without revealing all details of the process, Amyris-Crystalsev produces a graphic production scheme that shows that after fermentation, when the cane juice receives the microorganism that has been genetically modified by Amyris, there comes a separation phase followed by another chemical finalization stage, when the product is ready for market. Two stages substitute the ethanol distillation and dehydration phases.

The biotechnology used by Amyris was metabolism re-engineering. “This is modifying the genes that codify the enzymes responsible for transforming sugar not into ethanol, but into another product”, says Reinach. To do this the work was almost like reverse re-engineering, in which starting with a known product it can be discovered how it was made. The farnesene molecule, which forms a colorless liquid and is a component of fossil diesel, was already known from the chemical catalogues and has the same properties as diesel, such as far as combustion is concerned, although it is an expensive product extracted from other plants like citronella. Therefore, genetic modifications were directed towards the secretion of farnesene by Saccharomyces. Studies began with researchers from the company, which has its headquarters in Emeryville in California, producing the genome sequence of the yeast. They looked at all Saccharomyces genes that produce ethanol to also understand the differences there are between it and other strains of the same microorganism used in the laboratory (the same yeast is also used to produce bread, beer and cachaça, for example)”, says Reinach. “We did some one-off ‘micro-surgeries’ on the genetic material of the yeast which modified the metabolic route of the microorganism”, says Collier. “Genetic sequences were introduced that encouraged the production of diesel instead of ethanol.” Some 15 genes were modified. Those responsible for the new technique say that Saccharomyces is a genetically modified organism but do not disclose the origin of the genes, nor if they are from other organisms. This is because the process is still in the patent preparation phase.

88-91_Biodiesel_153Campinas and Sertãozinho
In the present phase of the Amyris-Crystalsev Project the industrial plant that is likely to be installed in the Santa Elisa Mill in Sertãozinho in June 2010 is being engineered. A pilot plant will be ready before this, in 2009, at the company’s research center in Technopark in Campinas. Production will begin with 10 million liters of diesel a year. In 2011 it will go to 50 to 60 million liters at Santa Elisa. From then on the joint venture intends to offer the technology to other sugar and alcohol groups. Diesel consumption in Brazil is likely to reach 45 billion liters in 2008, but the expectation, as disclosed by the company itself, based on information from sector analysts, is for 80 billion liters by 2020.

However, the Amyris technology should not be limited to the new diesel. “We can already say that we’ll be able to make aviation kerosene, gasoline and also move towards the petro-chemical industry”, says Reinach. With microorganisms re-engineered by biotechnology and fed with sugar the company’s directors say that it is possible to produce all these fuels as well as inputs for the plastics industry. This whole technological process from Amyris was started with basic research carried out in the Department of Chemical Engineering and Bioengineering at the University of California, in Berkeley, by Professor Jay Keasling, founding partner of the company with three other post-doctoral researchers from the same university, Neil Renninger, current technology director, and Kinkead Reiling and Jack Newman, vice presidents. Keasling takes no part in the day-to-day activities of Amyris, but is on the company’s scientific board. He is currently the executive director of the Joint BioEnergy Institute (JBEI), a new North American scientific center also situated in Emeryville, the mission of which is to move towards developing new biofuels. Set up in June 2007, the institute was created by the Energy Department of the United States in partnership with the Lawrence Berkeley National Laboratory, the Sandia National Laboratory and the Lawrence Livermore National Laboratory, as well as the University of California in Berkeley and Davis.

Amyris Biotechnologies was founded in 2003 to develop technology platforms. In addition to the recent participation of Votorantim Novos Negócios, the company has already received investments from four other risk capital companies, like Kleiner Perkins Caufield & Byers that was involved with Google, Amazon and America Online from the outset, which come to more than US$ 100 million. It has also received investments from Khosla Ventures, TPG Ventures and eDAG Ventures.

The company’s first product was the laboratory development of the synthesis of artemisinin, the active principle of an anti-malarial drug used in Africa and Asia. Until then it had been extracted from sweet wormwood (Artemisia annual), in an expensive process that requires large amounts of the plant. The team from Amyris managed to produce artemisinin by the genetic reengineering of a bacteria that is widely used in laboratories, Escherichia coli. New genes, enzymes and sugar in a fermentation process make the modified bacteria produce the medication. As a result the product became 90% cheaper, according to the company. The project, which was started in 2004, lasted three and a half years and was carried out in partnership with the University of California in Berkeley and had an investment of US$ 42.6 million from the OneWorld Health Institute, of the Bill & Melinda Gates Foundation. Amyris is transferring its artemisinin production technology to Sanofi-Aventis, a French pharmaceutical industry that is going to produce the drug as from 2010.

In its headquarters in Emeryville Amyris is likely to have 200 employees by the end of 2008. In  Brazil, Amyris-Crystalsev already has 20 researchers, 50% of whom have PhDs. “Between January and March 2009 we’re going to hire more researchers to set up the pilot plant and the future plant”, says Collier, from Amyris-Crystalsev.