More hydrous alcohol

Another potential alternative could lie in genetic improvement of the microorganisms utilized in the conventional industrial production process for the biofuel through fermentation.

By Elton Alisson

Agência FAPESP – The sugar alcohol sector is facing a greater challenge to increase ethanol production in order to meet demand from the domestic and export market.

For this reason, several alternatives are being studied to increase the sugarcane planting area, to increase agricultural yield (produce per hectare), to implement improvements in the industrial process or to produce the fuel in new ways, using the cellulose found in sugarcane bagasse or other vegetable raw materials – the so-called second generation ethanol.

A project conducted by researchers at the Polytechnic School at Universidade de São Paulo (USP) in collaboration with research groups at the Universidade Federal de Santa Catarina (UFSC) and Delft University of Technology (Holland) revealed that another potential alternative could lie in genetic improvement of the microorganisms utilized in the conventional industrial production process for the biofuel through fermentation. In this process, yeast of the Saccharomyces cerevisiae species convert saccharose (sugar) into ethanol.

Through metabolic engineering strategies combined with further evolution in laboratories in bench scale testing, the group managed to increase ethanol production yield over saccharose by 11% utilizing genetically modified yeast.

“This experiment still has not been tested in the industrial environment. But taking into consideration the large production volume at the moment, even a 3% increase in the alcoholic fermentation yield would afford a 1 billion liter increase per year in Brazil alone, using the same amount of sugarcane. That would already be an extraordinary gain,” comments Andreas Karoly Gombert, professor at USP Polytechnic School, to Agência FAPESP.

The project emerged as part of an initiative by professor Boris Ugarte Stambuk at Universidade Federal de Santa Catarina (UFSC), who developed and patented a metabolic engineering strategy that alters the topology and the energetic potential of saccharose’s metabolism in Sacchromyces cerevisiae yeast.

In order to verify to what extent genetically modified yeast was better than conventional yeast, Stambuk sought out Gombert with a view to initiating collaboration.

Through the research project entitled “Yeast improvement by metabolic and evolutionary engineering,” financed by FAPESP under the FAPESP Bioenergy Research Program (BIOEN), Gombert and colleagues cultivated the genetically modified yeast using a process called long-term chemostat cultures, limited by saccharrose, at the Polytechnic School’s Biochemistry Laboratory and also at Delft University of Technology’s biotechnology department.

Through this process, the yeast is submitted to selective pressure over several generations, during which genetic alterations occur and the individuals better adapted to the growing conditions are selected.
In this manner, the researchers managed to select a clone with greatly multiplied capacity to transport saccharose, compared to the strain provided by Stambuk from UFSC. The strain increased the ethanol yield over saccharose by 11%, compared to the wild variety.

“This result is unprecedented and surpasses our quantitative forecasts based on the theoretical model of the Sacchromyces cerevisiae’s consumption of saccharose. The next step will be to introduce the same strategies in industrial yeast and finally test it under industrial conditions,” says Gombert.

In laboratory studies, the researchers tried to verify which modifications occurred in the yeast genome during the process of evolutionary engineering through transcriptome analysis, PCR and genetic sequencing of some genes. This way, they managed to identify which genes of the microorganism were duplicated.

However, according to Gombert, this duplication of genes is only part of the explanation for the improved yield, since it is not possible for the time being to reproduce the phenotype obtained by evolution in laboratory through directed genetic modification.

According to him, the increased yield of Brazil’s alcohol fermentation industry in the last few decades was obtained basically through improvement in process conditions. But in order for this yield – which has held steadily at 90% to 92% of the stoichimetric value – to increase further, new real technological advances will be necessary. Among them, the use of genetically modified yeasts is a strong candidate.

“There is already at least one foreign company that is introducing genetically modified yeast for large scale production of another molecule, which is not ethanol, based on sugarcane saccharose. If this experiment proves successful, we will have proof that it is possible to grow these microorganisms in a large-scale process, with asepsis and cellular recycling during the several months of the sugarcane harvest, for production of a low value compound. Which, to my knowledge, is very rare and even unprecedented,” he said.