eduardo cesarFlocculent yeast grown in a liquideduardo cesar
Within a year, if all goes well, new technology for the production of ethanol should be available in the market, leading to a reduction in the production costs of the country’s sugar and alcohol refineries. Researchers from the Multidisciplinary Center for Chemical, Biological and Agricultural Research (CPQBA) at the State University of Campinas (Unicamp) managed to successfully select strains of the Saccharomyces cerevisiae yeast that have the capacity to flocculate (group together as they grow, forming flakes or clumps that are of varying sizes) for use in the column reactors used in industrial fermentation processes by Brazilian distillers. The advantage of these yeasts over those traditionally used is that they do away with the centrifuge stage in alcohol production that occurs immediately after fermentation. Researchers also developed fermentation vessels that are specific for this process and that have been working experimentally for two years in a pilot refinery in the Ribeirão Preto region in São Paulo State.
To have some idea of what an achievement the innovation developed by the Unicamp group is, one must understand the stages involved in ethanol production. After the sugarcane has been harvested it is sent to the refinery where it goes through a crushing process. The resulting liquid, consisting of cane juice or molasses mixed with water, goes to the fermentation vats. At this stage the Saccharomyces cerevisiae yeast fungi play a vital role, because they are responsible for transforming the sugar into ethyl alcohol. In the conventional processes used in practically all the 400 Brazilian refineries, the fermented wash is sent to centrifuges where the yeast is separated out. The centrifuged yeast, containing yeast cells, is treated with an acid and returns to the fermentation vats, while the wash is sent to the column distillery, the final step in the process for producing ethanol.
With the strains of flocculent yeasts selected by the CPQBA, the wash that leaves the vats contains no yeast, which is retained in the equipment itself. “Unlike conventional yeasts that are dissolved in the wash, flocculent yeasts are deposited on the bottom of the vat and so we have managed to eliminate the centrifugation stage, the only purpose of which is to separate the yeast from the fermented wash”, explains biologist Maria da Graça Stupiello Andrietta, coordinator of the Biotechnology and Processes Division of the CPQBA. “The big advantage of this new process is a reduction in the cost of refineries acquiring and maintaining equipment, since they will no longer need to have a centrifuge.”
From the group’s calculations the elimination of centrifugation and the acid treatment of conventional processes can lead to a saving in processing costs of R$ 0.02 to R$ 0.03 per liter of ethanol produced – the total processing cost is R$ 0.20 per liter, excluding the cost of the sugar-cane. Furthermore, the elimination of this machine facilitates the automation process since the centrifugation unit is the only aspect of the process that cannot be fully automated. Despite there being one stage less in the production cycle, the yield and the productivity of the process are still the same.
According to the researcher, almost 15 years ago some Brazilian refineries were pioneers in using processes that employed flocculent strains of Saccharomyces cerevisiae, but they abandoned them because they were unable to master the technology. “At the time we were already selecting strains and we saw that flocculent yeasts could perform efficiently in fermentation if suitable equipment was available. There was a gap in technology”, recalls Maria da Graça, who in the past worked at the Copersucar Technology Center, currently the Sugarcane Plantation Technology Center (CTC), in Piracicaba (SP), one of the main research institutions in the sugar and alcohol sector.
eduardo cesarFlocculent yeast grown in a gelatinous environmenteduardo cesar
Another major hurdle to the implementation of this process on an industrial scale was the formation of a stable bed of cells that would allow the system to be operated for long periods of time without major variations in the operating conditions. One of the factors with which the stability of the bed is strongly associated is the characteristic of the strains present in the reactors; hence the importance of the work being done at CPQBA. Of the nearly 300 strains of flocculent yeast isolated from industrial units in the country and stored in the Strain Bank of the center’s Biotechnology and Process Division, 12 were selected for the study that was funded by FAPESP.
One of the aspects that the group tried to understand was the flocculation profile of each of the strains, since the size of the clump formed is directly related to the microorganism’s fermentation performance. To select the most efficient, the researchers chose 12 strains and placed them in a reactor to see how they behaved: during fermentation the yeast strains compete with each other and the most efficient remain in the process. Identifying the 12 strains used in the study was done by electrophoresis, a method that isolates the yeasts’ DNA and produces a profile of their chromosomes. At the end of the studies two strains were selected for use in the pilot plant as an inoculant, the name given to the seed that starts the fermentation process.
The group, which also included chemical engineer Sílvio Roberto Andrietta, faced another challenge: developing specific equipment for use with these flocculent yeasts. The pilot system comprises two cylindrical tower-type bio-reactors, which are taller and slimmer than conventional ones, which work in series and can ferment 4,500 liters of wash an hour, which means a daily production of 12,000 – 15,000 liters of ethanol. “Conventional vats would be unsuitable, because they would allow the clumps to move forward with the fermented wash, causing the distillation equipment to block”, explains Sílvio Andrietta. The pilot refinery, built with support from a major machinery manufacturer in the sugar and alcohol sector, which prefers to remain anonymous, has been operating for two years with promising results, according to the researchers. “We’re going to test it with one more crop and then it’ll be ready to be launched on the market. We’re already in the phase of licensing the equipment for an industry in the sector”, says Maria da Graça. “I think the process using flocculent yeasts will be an interesting alternative for alcohol producers.”
Database
The objective of another project developed by researchers from CPQBA’s Biotechnology and Process Division is to establish the cellular composition of different strains of Saccharomyces cerevisiae yeast – flocculent or not – that are used industrially. “With this information we’re going to know how the strains most used in industrial fermentation processes in Brazil are constituted”, says chemist Cláudia Steckelberg, coordinator of the research, which also receives financial support from FAPESP. The researcher collected at random 35 dominant yeasts used by sugar and alcohol refineries from different States, of which four parameters are being assessed: fermentation performance, ethanol tolerance, cariotyping (chromosome profile) and cellular composition (profile of fatty acids).
Cariotyping is done using the electrophoresis method, while cellular composition uses gas chromatography, which identifies fatty acids in micro-organisms; it is already known that the most ethanol-tolerant yeasts are those with high levels of fatty acids of the palmitoleic and oleic types. To assess the fermentation performance of the micro-organisms, the researchers create ideal fermentation conditions in the laboratory, put the 35 strains together and then observe those that ferment best. “The project is on-going. Its purpose is not to identify the best yeast strain but to get to understand them fully. Our idea is to create a large database that contains information about the kinetic characteristics (productivity and yield) and their cellular composition. This is a type of yeast- biome”, says Maria da Graça.
Thanks to the knowledge acquired over the last twenty years from research in the sugar and alcohol sector, the group has also worked on identifying other micro-organisms present in the sugarcane environment that are of industrial interest, but not directly linked to the production of ethyl alcohol. “The purpose of this work, the result of two masters’ degree dissertations, is to assess the potential of strains of bacteria isolated from sugarcane when it comes to synthesizing products from sucrose that are of commercial interest”, says Maria da Graça. The researchers have already managed to isolate a bacteria found in sugar-plantations, Leuconostoc sp., which produces a type of gum, called dextrane, which is much used by food, medication and cosmetics manufacturers. Gums are biopolymers with a microbial (dextrane and xantane gums, etc.) or vegetable (guar and carageen gums, etc.) origin that have wide industrial applications. Dextrane, for example, is used in food as a thickener or stabilizer, thereby guaranteeing consistency, and in the pharmaceutical industry, as a substitute for blood plasma.
Interest in this new line of research arose when it was realized that the bacteria that produce dextrane-type gums are frequently found in samples of decaying sugar-cane. “We have mapped three dextrane gums with properties that are different from those that already exist in the market and that can be used in the food industry”, says the researcher. The studies are still on-going and for the time being no contact has yet been made with potentially interested companies to transfer the knowledge generated in the laboratories of the CPQBA.
The projects
1. Selecting flocculent yeasts for use in reactors (column-type) in the production of ethanol (nº 01/02064-9); Modality Regular line of help for research; Coordinator Sílvio Roberto Andrietta – Unicamp; Investment R$ 116,567.13 (FAPESP)
2. The characterization of industrial yeasts to produce ethanol using their cellular composition and kinetic characteristics (nº 03/00177-6); Modality Regular line of help for research; Coordinator Cláudia Steckelberg – Unicamp; Investment R$ 82,720.37 (FAPESP)
3. Production of dextrane and oligosaccharides using new strains isolated from sugarcane (nº 03/07846-0); Modality Regular line of help for research; Coordinator Maria da Graça Stupiello Andrietta – Unicamp; Investment R$ 92,778.99 (FAPESP)
