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FAPESP history of VII

The rise of ethanol

Joint efforts of researchers from São Paulo is helping to multiply the use of bioenergy

Delfim MartinsExperimental sugar cane field at the Barra Mill in Barra Bonita (SP), in 2000Delfim Martins

In 2011, researchers from the University of São Paulo (USP) and the State University of Campinas (Unicamp) unveiled some 10.8 gigapairs of sugar cane DNA bases, 33 times more than was produced in the two years of the Cane Genome Project, which ended in 2001 and which mapped out the genes expressed from the plant. The result is part of two thematic projects coordinated by molecular biologist, Glaucia Souza, and geneticist, Marie-Anne Van Sluys, professors from USP, who are looking to map sugar cane genes. The projects are forecast to conclude in 2013. Given the complexity of the genome, 300 regions are already organized into strands that have more than 100,000 bases containing between 5 and 14 contiguous sugar cane genes. The researchers want to go further than the Sugar Cane Genome program, both in terms of the quantity of data as well as in questions about how the plant’s genome, which has become synonymous with renewable energy, functions. Studies of grasses, like sorghum and rice, have shown that to improve the productivity of plants it is necessary to know how gene activity is controlled, the function of DNA strands known as promoters.

The research is an example of how knowledge about sugar cane and ethanol has advanced over the last 15 years, with support from FAPESP. From the Sugar Cane Genome project, which mapped the genes expressed from sugar cane between 1998 and 2001, to the FAPESP Research Program in Bioenergy (Bioen), which started in 2008, of which Glaucia is the coordinator, the Foundation has supported a large investigation, linking researchers from various areas of knowledge, directed at improving the productivity of Brazilian ethanol and advancing basic science and technology related to the generation of energy from biomass.

After three years the results of Bioen are both palpable and varied. An innovative process for producing kerosene from various types of vegetable oil, which may make the fuel used in airplanes less polluting and cheaper, was developed at the Faculty of Chemical Engineering (FEQ) at the State University of Campinas (Unicamp). After extracting and refining the oil, it is placed in a reactor along with a specific quantity of ethanol and a catalyst, which is responsible for accelerating the chemical reactions. “The greatest contributor to the process of obtaining bio-kerosene is the high level of purity of the end product,” said Rubens Maciel Filho, a professor at FEQ and the study coordinator.

Another contribution from Maciel is a project that seeks to create compounds with high economic value from cane substrate. The project has obtained good results in producing acrylic acid and propionic acid from lactic acid. “It’s possible to develop products that are worth 190,000 times more than sugar,” Maciel says.

The experience in genomics of geneticist, Maria-Anne Van Sluys from USP, led to her heading up a project whose objective is to generate a partial sequencing of two cane cultivars (R570 and SP80-3280) and provide information for developing molecular tools capable of helping to understand this genome. One of the targets is the study of the so-called transposition elements, regions of DNA that can be transferred from one region to another in the genome, leaving a copy in the place they were originally located, or not. “Improvement programs can also benefit by having access to molecular information with the potential for developing markers,” says Marie-Anne.

A project led by Ricardo Zorzetto Vêncio, from USP’s Ribeirão Preto School of Medicine, has developed a pilot version of software for characterizing the gene functions of sugarcane. The approach is innovative because it is not limited to attributing to an organism’s gene sequence the functions already observed in a similar sequence in another living being. The idea is to use algorithms that consider the uncertainty contained in this association. “Instead of simply saying that a gene has a specific function we want to say what the probability of it having this function is and in this calculation take into account different evidence, such as the evolutionary relationship with other genes or if there is some experiment that confirms the function,” says Vêncio. Augusto Garcia, a professor at USP’s Higher School of Agriculture Luiz de Queiroz (Esalq), is developing software for using genetic markers in improvement programs, by exploring the genetics and physiology of sugar cane. “This is one of the great expectations to obtain cultivars more quickly,” says Glaucia Souza. Every year the Sugar Cane Technology Center (CTC) tests 1 million cuttings in their search for more productive plants. It takes twelve years for two or three promising varieties to appear.

Studies by André Meloni Nassar, managing director of the Institute for International Trade Negotiations (Icone), also made progress in the use of economic models for evaluating changes in the use of land caused by the large scale production of biofuels. In the search for ethanol from cellulose, one of the highlights is a project that assesses how possible it is to break down the resistance of the cell walls of lignified plants, like sugar cane, using enzymatic hydrolysis. Lignin is a macro-molecule found in plants and associated with cellulose in the cell wall, whose function is to confer rigidity and resistance. Breaking it down is one of the challenges to obtain ethanol from cellulose. “To understand how the removal of lignin may reduce the recalcitrance of cell walls, in addition to commercial varieties, hybrid varieties of sugar cane with contrasting concentrations of lignin have been evaluated,” says Adriane Milagres, a professor from USP’s Lorena School of Engineering, one of the coordinators of the project. “When materials are dealt with by selective methods, removal of 50% of the original lignin already raises the conversion level of cellulose to 85-90%.”

Since its early years, FAPESP has been supporting initiatives that have created critical mass for the recent effort. An example was the launch in 1968 of the Laboratory of Industrial Technology at USP’s Polytechnic School. The Poli had had a pilot plant for producing ethanol by fermentation since the 1940s, but lacked the small reactors and equipment that would allow more complete work to be carried out. Another contribution of the Foundation was the Bioq-FAPESP Program, launched in 1972 (see Pesquisa FAPESP 185). In forming human resources in the field of biotechnology it opened the way to sequencing the genome of various organisms in the 1990s and 2000s, among them the sugar cane genome. “Both Bioq-FAPESP, in the 1970s, and the Integrated Genetics Program of the CNPq in the 1980s were pillars of the current effort,” says Marie-Anne Van Sluys, a professor at the Institute of Biosciences at USP and one of the coordinators of Bioen.

A leap in interest in research into sugar cane and ethanol occurred in April 1999, with the advent of the Sugar Cane Genome project, whose official name was the FAPESP Sucest Program (Sugar cane Est). The project, which mapped 250,000 functional gene fragments of sugar cane, was characterized by interaction with the private sector which until today is making research efforts into bioenergy. Paulo Arruda, a professor at Unicamp, remembers he was invited to head up a project after the Sugar and Alcohol Producers Cooperative of the State of São Paulo (Copersucar) approached the scientific board at FAPESP and proposed a partnership between universities and industry to map out the sugar cane genome. “Professor José Fernando Perez, scientific director at the time, asked me what I thought. I observed that sugar cane has a very complex genome and suggested mapping out the functional fragments of the genome,” says Arruda, who today is one of the coordinators of FAPESP’s Research for Innovation area. Sugar cane is a polyploid organism: each chromosome has between 6 and 10 copies that are not always the same. This peculiarity means that the idea of sequencing the whole of the genome was discarded.

Challenges and talent
The Sugar Cane Genome Project lasted two and a half years, brought together 240 researchers and had funding of around US$ 4 million from FAPESP and a further US$ 400,000 from Copersucar. “The project was really innovative. Centered around very young people, who were better able to deal with the technology than more experienced researchers, the Sugar Cane Genome Project showed that it was possible to identify great challenges and bring together talent to resolve them,” says Arruda. Basically, it started the effort that is still ongoing today to develop in–depth knowledge about the metabolism of sugar cane in order to obtain more productive and more drought or poor soil-resistant varieties more quickly.

The conclusion of the Sugar Cane Genome Project did not cool the interest of researchers or of the industry in continuing to seek out knowledge about the plant. After 2003, Glaucia Souza assumed coordination of Sucest and started the Sucest-FUN Project, dedicated to analysis of sugar cane genes. The identification of 348 genes associated with sucrose levels was carried out in a project between the CTC, the Lucélia Central Alcohol Distillery and researchers from USP and Unicamp, in a project led by Glaucia. Another important project was identification of molecular markers from the Sucest sequences, under the leadership of researcher, Anete Pereira de Souza, from the Institute of Biology at Unicamp. “Glaucia’s and Anete’s projects were two milestones, because they showed there was a community prepared to invest in the theme. The progress they achieved made it feasible to map out the sugar cane genome, which was not possible at the time of Sucest,” says Marie-Anne.

At the same time interest from companies was growing in research into bioenergy. In 2006, FAPESP, in a partnership with the BNDES, signed an agreement with Oxiteno, of the Ultra Group, to develop cooperative projects in which everything, from the enzymatic hydrolysis process of cane bagasse for obtaining sugars to the production of ethanol from cellulose is investigated. The following year, Dedini Indústrias de Base entered into an agreement with FAPESP to fund projects relating to techniques for converting cane bagasse into ethanol. At the beginning of 2008, FAPESP and Braskem also established an agreement to develop biopolymers. Two biotechnology companies, formed largely by researchers linked to the FAPESP Genome Program, Alellyx and Canavialis, were acquired at the end of 2008 by multinational company, Monsanto, which transformed them into its global sugar cane research platform – Paulo Arruda, who headed up the Sugar Cane Genome project used to work for Alellyx.

Corn and subsidies
The growing economic importance of sugar cane has helped drive the interest of researchers. In the 2009 season Brazil harvested 569 million tons of sugar cane, almost double the harvest in 1999, according to data from the Sugar Cane Industry Union (Unica). Half of the production was transformed into ethanol, the equivalent of 27 billion liters, which makes Brazil the second biggest producer of the fuel in the world. First place goes to the United States, which extracts ethanol from corn on the basis of heavy subsidies. São Paulo accounted for 60% of Brazilian production. The gain in productivity has been bigger than 3% a year over the last 40 years, the result of genetic improvements in sugar cane. Ethanol makes Brazil a unique example of a country that has replaced the use of gasoline on a large scale. In the State of São Paulo, 56% of the energy comes from renewable sources, 38% of it from sugar cane.

To link the existing efforts and to drive research into areas that are still in their early stages, in July 2008 FAPESP launched the Bioen Program. One of its objectives is to overcome technological obstacles and expand even further the productivity of first generation ethanol made from fermenting sucrose. Another aspect is to take part in the international race for second generation ethanol, produced from cellulose. This program has five strands. The first is research into biomass, with a focus on improving sugar cane. The second is the process by which biofuel is made. The third is linked to ethanol applications for automotive and aviation engines. The fourth is linked to studies on bio-refineries, synthetic biology, sugar chemistry and alcohol chemistry. The fifth deals with the social and environmental impacts of the use of biofuels.

A development of Bioen was the creation in 2010 of the São Paulo Research Center in Bioenergy. This is an effort to encourage interdisciplinary research and expand the contingent of researchers involved with the theme, supported by FAPESP, the São Paulo state government and the three São Paulo state universities. According to the agreement, the government passes on funds to USP, Unicamp and Unesp, which will be used for building laboratories, refurbishment and the purchase of equipment. It is incumbent upon the universities to hire more researchers in various areas of bioenergy. FAPESP, on the other hand, has assumed the mission of selecting and financing projects linked to the center. “Currently the three universities are organizing official applications for the public examination to select the first 17 researchers for the center, 7 of whom will be in USP units, 5 in Unicamp and 5 at Unesp,” says Luis Cortez, a professor from Unicamp and the center´s coordinator. This number is likely to reach around 50 as new investments are made by the government. An example is the Center for Synthetic and Systemic Biomass Biology at USP, the 2008 brainchild of Glaucia Souza, Marie-Anne Van Sluys and Marcos Buckeridge. This center is going to bring together researchers from the Institutes of Chemistry, Mathematics, Statistics, Biosciences and Biomedical Sciences and the Polytechnic School. Synthetic biology combines biology and engineering for building new functions and biological systems. “The intention is to invest in an area in which Brazil still has no great expertise and involve researchers from various disciplines,” says Glaucia Souza.

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