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The artisans of ethanol

Discoveries on yeast genetics encourage research into expanding alcohol productivity

Eduardo CesarTime to rest: yeasts recover in diluted sulfuric acid before being put back into the fermentation vatsEduardo Cesar

from Nova Europa

In less than five minutes, a crane hoists a cart; the cart tilts and lowers 30 tons of sugarcane onto a conveyor belt at the head of a sugar and ethanol production line at the Usina Santa Fé mill in the town of Nova Europa, São Paulo State. The cart moves away and a tractor brings in another cart, and then another, non-stop, day and night. The sugarcane travels on the conveyor belt, is put through mills that crush the cane and extract its green juice that turns into sugar after purification and evaporation. Further ahead, the sugar goes into fermentation vats – 25-meter high tanks – to be turned into ethanol. This transformation depends on specific strains of the Saccharomyces cerevisiae yeast, the same single-cell microorganism used to make bread, beer and wine.

“Until a while ago, we had no idea of what happened inside,” says Cláudio Câmara, the mill’s process manager, pointing at the vats. “All we knew was that the fermentation was OK.” After resorting to various strains of Saccharomyces cerevisiae suitable for smaller production volumes, Santa Fé adopted a combination of four yeast varieties to enable the production of one million liters of ethanol a day, which fills up 30 trucks. “That was the best result we managed to achieve,” he says. He would like to use fewer yeast varieties or less yeast – fermentation starts when 600 kilos of yeast are put into a vat filled with 80 thousand liters of juice, the diluted sugar. “Given this production volume, we cannot afford to run any risks.” Previously known only for their ability to transform cane sugar into fuel ethanol, these yeast strains are now better known and more respected.

Two studies – one conducted at the State University of Campinas (Unicamp) and another at the Federal University of Santa Catarina (UFSC) – examined the set of genes (genome) of the yeast strains used to produce ethanol. These studies describe the mechanism that enables the yeast to produce ethanol quickly and efficiently. With this information, the researchers are now trying to search for – or create – varieties better adapted to the fermentation vats. “Perhaps the performance of industrial yeasts will improve if we can remove or deactivate some genes,” says Gonçalo Pereira, coordinator of the team from Unicamp.

One of the arguments that supports this possibility is that ethanol production is still below its theoretical peak. At present, the yeasts produce 0.46 grams of ethanol per gram of sugar, says Sílvio Andrietta, a researcher from Unicamp. “The theoretical maximum is 0.51,” he says. “We have achieved 90% of this.” The financial impact can be huge if the efforts are successful. “If the efficiency of the process increases by 5%, we will achieve extraordinary gains, given the high production volumes,” says chemical engineer Saul Gonçalves D’Ávila, professor emeritus of Unicamp, who is involved with one of the teams. This year, 350 mills are expected to produce 27.5 billion liters of ethanol.

The specific set of genes – already familiar to researchers – of the yeasts used for ethanol production explains how these varieties have become as robust as a jeep, able to survive in strong heat and to compete successfully with the wild yeasts and other microorganisms that come with the sugar cane, all of them eager to feed on the vats’ abundant sugar. “The ethanol production process in Brazil is quite susceptible to contamination by microorganisms, which reduce productivity,” says fungi genome expert Gustavo Goldman, a professor at the University of São Paulo (USP) in Ribeirão Preto.

Resistance Genes
The said research studies have shown that the ethanol producing strains have acquired their own genetic features, which are quite different from those of the S288c, the reference strain. Maintained in the comfort of laboratories, this strain has been analyzed and manipulated for more than 10 years: its genome was the first from a microorganism with a nucleus to undergo sequencing, the result of which was described in a scientific paper published in 1996. The varieties used to produce wine and beer have also been widely studied, whereas the strains used in the production of ethanol have been focused on more heavily in the last few years.

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In a paper published in 2008, a team from USP, led by Luiz Basso, showed that the strains that start digesting the cane juice sugars were usually not the same as those that lasted until the end of the process; only the more robust strains survived the high temperatures and the growing concentration of ethanol, whereas the weaker strains – one of which is used to make bread – were replaced. Now these new studies clarify how the first strains manage to survive.

In one of the papers, published last month in Genome Research, experts from three São Paulo universities (Unicamp, USP and the Federal University of São Carlos) and from two American universities (North Carolina and Duke) analyzed the PE-2 variety of yeast, commonly used to produce ethanol and the one employed by the Santa Fé mill. The researchers compared the PE-2 with the S288c reference strain. Both have 16 chromosomes and about 6 thousand genes, yet each strain appeared to have gained or lost genes versus the other. Juan Lucas Argueso, a Brazilian researcher who currently works at Duke and who coordinated the genetic analysis, says that the PE-2 variety has 16 genes not found in the lab strain and which probably help this strain to survive in the fermentation vats. Two of these genes provide resistance to the toxicity of ethanol, whose concentration rises as fermentation progresses. “These genes were identified some 10 years ago in the yeast strains used to produce sake and survive in more toxic environments, where the concentration of ethanol is even higher,” says Argueso. Strains without these genes are more sensitive to ethanol and die more easily.” Two other PE-2 genes are new and are unlike any other identified gene. The researchers are yet to learn what their function is.

The central part of the two strains’ chromosomes is identical. The subtelomeric – or peripheral – parts of the chromosomes of the industrial yeast strain, however, are different and contain many genes that provide tolerance to environmental stress such as high temperatures and probably ethanol production efficiency. The tips of the chromosomes contain most of the genes specific to this strain; these genes are often repeated. “Gene repetition makes it easy for genetic material to be exchanged among the chromosomes that can recombine quickly at each new generation, creating distinct forms,” says Argueso. “This genome flexibility probably explains how this and other industrial strains survive competition with other microorganisms.” According to this study, this variety produces 50% more ethanol 30% faster than the lab strain. “The PE-2 does what it can and not what the industries would like,” says Pereira.

“In our search for more productive strains, we realized that one can change the genomes of the yeasts used to produce ethanol,” says Boris Stambuk, the professor from UFSC who coordinated the other study, to be published in Genome Research. Stambuk, Basso and a team from Stanford University in the United States had already sequenced the genome of CAT-1, a widely used strain of yeast. Recently, Stambuk and the Stanford team coordinated the genome analysis of the five yeasts most widely used in Brazil to produce ethanol. According to this analysis, the industrial strains (or varieties), as compared with the S288c, have more copies of the genes involved in the synthesis of vitamins B1 (thiamine) and B6 (pyridoxine). This peculiarity – which can kill microorganisms that are more delicate – makes the transformation of sugars into ethanol easier. “In sugar-rich environments, such as the industrial production of ethanol,” says Goldman, “these strains might provide an adaptive advantage over the others.”

Goldman firmly believes that “better adapted strains might be developed through genetic engineering or cross-breeding.” The Unicamp and UFSC teams are already exploring ways to improve the yeasts that produce Brazilian ethanol, but they know that this is no easy or quick task. Neither can they guarantee the outcome, because of the microorganisms’ robust nature. The external membrane of the industrial yeasts is one of the barriers to be overcome, as it prevents the entry of ethanol, which is harmful to yeasts. The membrane must be breached to introduce modifications in the genes. “Part of the resistance of these strains to genetic transformation is precisely due to their ability to keep harmful elements from entering,” says Stambuk.

Even if the researchers come up with new yeast strains, they know that they should not celebrate ahead of time: genetically modified organisms that work well in labs can be a disaster in bigger fermentation vats like the ones in mills. Pereira has already been disappointed once. In 2003, he presented a genetically modified yeast strain, which settled at the bottom of the fermentation vats after it had produced the ethanol. “In the lab it was beautiful, it functioned like clockwork,” he recalls. This could have been a way of simplifying ethanol production and reducing costs. In bigger equipment, however, these yeasts turned out to be less productive than the standard ones.

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This time, Pereira surrounded himself with people who keep track of what he and his team are doing and remind him that lab results must also be feasible from the technical, economic and environmental point of view. “Maybe we will find better strains by studying the genome and understanding yeasts’ behavior,” says Andrietta, a chemical engineer and co-author of the article published in the October issue of Genome Research.

The work of Pereira’s and Andrietta’s teams is part of a plan for producing plastic resins from ethanol. This project is being coordinated by Braskem, one of Brazil’s biggest petrochemical companies and part of the Odebrecht conglomerate. At present, this project involves a team set up in 2007 and involving three Unicamp units. That year, Antonio Queiroz, Braskem’s competitiveness and innovation director, concluded that the production of green polymers underwent biotechnological processes that the company’s researchers still barely understood. Shortly thereafter, he contacted Saul D’Ávila, one of his former chemical engineering professors, and set up “a group of people who trust each other and enjoy working together,” as he describes it. “We knew right from the start that we would be unable to do everything on our own and that we would have to partner with other teams.” Queiroz goes to Unicamp at least once a month to plan the next steps with the teams. In his opinion, the work is going smoothly, but it will need other partnerships soon. “I can’t do everything with the university. I know how far I can go.”

Queiroz also knows that a factory that manufactures plastic from sugarcane will not materialize in less than 10 years, even if things go smoothly. ETH Bioenergia – another company owned by the Odebrecht group – might be able to benefit from yeasts adapted to produce more ethanol in less time. ETH’s five mills are being expanded and are expected to double their crushing capacity to 10 million tons of sugarcane by the next harvest. “Medium- and long-term, we plan to use sugarcane biomass to make other products, such as special ethanols,” says Luis Felli, the company’s vice-president of agroindustrial operations.

The UFSC team is also involved with industry. Stambuk started studying yeasts in 2004 with Henrique Amorim, a former professor at USP in Piracicaba and owner of the company Fermentec. They joined forces with a sugar mill from the State of São Paulo willing to test the lab-modified yeasts in bigger equipment. “The results that Boris and Henrique achieved are the outcome of the basic research conducted in our labs,” says Pedro Soares de Araujo, from USP’s Chemistry Institute, who is part of the group and was Stambuk’s PhD thesis advisor. “The noteworthy aspect of our work is that basic research provided us with the elements needed to conduct applied research with people that have a sound scientific background.” Stambuk adds that: “By 2012, we will know whether we have been successful or not in the industrial environment.” People such as Câmara, from the Santa Fé mill, who deal with ethanol production every day, are eagerly awaiting the outcome.

The projects
Green routes for propylene (nº 07/58336-3); Type Partnered Research into Technological Innovation (Pite); Coordinator Gonçalo Amarante Guimarães Pereira – IB/Unicamp; Investment R$ 3,805,396.60 (FAPESP).
2. Bioethanol: development of Brazilian industrial yeasts for efficient fermentation of sugars in biomass; Type Research Aid (MCT public call); Coordinator Boris Ugarte Stambuk – UFSC; Investment R$ 3,500,000.00 (Braskem) and R$ 648,717.64 (CNPq)

Scientific articles
ARGUESO, J. L. et al. Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production. Genome Research. Oct. 2009.
STAMBUK, B. U. et al. Industrial fuel ethanol yeasts contain adaptive copy number changes in genes involved in vitamin B1 and B6 biosynthesis. Genome Research. In press.