A genetic modification developed by researchers at Unicamp, within the framework of an international research agreement with two institutions in the United States, could increase bioethanol production yield by up to 30%. The invention uses an alternative route to unlock the production of second-generation (2G) biofuels, based on the use of genetically modified bacteria. The technology has been protected by a patent application and licensed, through a strategy of the Inova Unicamp Innovation Agency , to the North American company Terragia Biofuel , which seeks to advance the testing phase on a larger scale.
The new technology is the result of doctoral research by Layse Costa de Souza, carried out at the Center for Molecular Biology and Genetic Engineering ( CBMEG ) at Unicamp, within the framework of a research, development and innovation (R&D&I) agreement between Unicamp, Dartmouth College (USA) and the licensed company. The work is part of the activities of the Advanced Second Generation Biofuel Laboratory (A2G), based at Unicamp and funded by the São Paulo Research Foundation ( FAPESP ) through the São Paulo Excellence Chair ( SPEC ) program. The SPEC international cooperation project aims to attract internationally renowned researchers to create cutting-edge research centers at universities in São Paulo state, such as the A2G laboratory at Unicamp.
The bottleneck and international cooperation
Second-generation (2G) ethanol has the potential to contribute to climate change mitigation and economic development, as well as being a promising intermediate for the production of sustainable aviation fuels and basic chemicals. However, the cost of production is currently higher than that of fossil fuels and first-generation (1G) ethanol.
This economic bottleneck occurs because the current production process, made from residues such as bagasse and sugarcane straw, depends on yeast for fermentation. This method requires prior thermochemical treatment steps at high temperatures and the use of hydrolytic enzymes to break down hemicellulose and transform cellulose into glucose. "Together, these two steps considerably increase the cost of biofuel production," explains Luana Walravens Bergamo, a researcher at CBMEG and a member of the study.
The proposed alternative route stems from what is called Consolidated Bioprocessing (CBP), a strategy that simplifies the production process by replacing yeasts with thermophilic anaerobic bacteria. These microorganisms have the natural ability to break down lignocellulosic biomass and carry out fermentation in a single process, eliminating the need for pretreatment steps.
This alternative has been studied for over three decades by a group coordinated by Professor Lee Lynd of Dartmouth College, focusing on the bacterium Clostridium thermocellum . Although this microorganism degrades cellulose with excellence, its natural ethanol production is very low.
Terragia Biofuel researcher Christopher David Herring explains that current yeasts are very economically useful, but limited in the types of carbohydrates they ferment. The bacteria in the study, however, can ferment a wider variety, but require genetic engineering to achieve the same efficiency.
“These bacteria use a different metabolic pathway to produce ethanol, and although we already knew the enzymes involved in carbon conversion, we knew little about how other enzymes balanced hydrogen and electrons. This gap in knowledge hampered our ability to produce ethanol with the same efficiency as yeasts,” explains the researcher.
Combining the best of two microorganisms
To overcome these obstacles and expand scientific knowledge, the Brazilian researchers and the American researcher worked with a second bacterium, a kind of 'cousin' of Clostridium thermocellum, initially studied. This is Thermoanaerobacterium thermosaccharolyticum, a bacterium that is also anaerobic and thermophilic, but whose great advantage is its ability to produce ethanol. “We therefore had two complementary microorganisms: one that produces a lot of ethanol but does not degrade cellulose, and another that degrades biomass with excellence but does not generate a good yield of biofuel,” comments Bergamo.
Layse's thesis focused on unraveling the metabolism of high-performance bacteria in ethanol production, to identify the key proteins responsible for this efficiency. "Once the pathways capable of supporting this level of production were mapped, we transferred the genes of these proteins to our key CBP process bacteria, the one that degrades cellulose. As a result of this genetic engineering, we were able to obtain a significantly higher ethanol production," says Souza.
How the research was conducted
Much of the experimentation that led to the invention was done while Souza was completing a year of doctoral research at Lynd's lab in the United States, after two years of research in Brazil. There, she mapped metabolic pathways of the beneficial fermenting bacteria, identified key proteins, and, using a plasmid (a circular DNA molecule used as a vector), transferred the gene to the CBP bacteria, which until then had been unable to ferment properly.
All tests so far have been conducted on a bench scale, in flasks, without industrial bioreactors. The data has already supported the patent application in the United States and Brazil. In parallel, Souza conducted biochemical assays to prove that the protein is the central piece of the mechanism, in another aspect of the thesis defended at Unicamp.
According to Herring, the new route provides answers about cellular function to questions that previously limited the advancement of the use of these bacteria in production. "In this study, we present a new model of the hydrogen and electron balance mechanism and demonstrate how it can be used to improve the engineering of biomass-fermenting bacteria – an advance that could generate a major economic impact by enabling the efficient conversion of biomass into fuel," comments the scientist.
Hydrogen: from villain to intermediary in production
The central finding of the research contradicts a long-held assumption in microbial biochemistry: hydrogenases, enzymes that produce hydrogen, were seen as competitors to ethanol because they sequester electrons that could go to the final product.
The team identified that a specific hydrogenase, HfsD, has the opposite effect: without it, the cell does not recycle ferredoxin, an essential step in transforming sugar into ethanol, nor does it produce the cofactor NADPH (Nicotinamide Adenine Dinucleotide Phosphate), required for the final stage of the reaction. This cofactor acts as a kind of key that turns on the enzyme's "engine" so it can perform the necessary chemical work. "We identified how to regulate this metabolism so that hydrogen is not a villain, but rather an intermediate in the reaction," describes Souza.
The hypothesis was named redox balance via hydrogen cycling , presented in Souza's doctoral thesis defense at Unicamp in April 2026. Since HfsD generates an excess of cofactor, a second accessory protein needs to balance the process, which explains why the team tested two strains, each with a distinct combination.
“Most metabolic engineering attempts focus on improving the kinetics of a protein or eliminating pathways that compete for carbon. Our hypothesis shows that this is not enough: it is also necessary to balance the cofactors and electron carriers,” Souza summarizes.
Results and upcoming challenges
In bench tests, the modified strains achieved yield increases of up to almost 30% compared to unmodified bacteria. In one of them, named A2G-0053, the yield rose from 59,8% to 88,5% of the theoretical maximum after the insertion of the key hydrogenase, compared to other modified bacteria. “We have already tested other modifications using heterologous proteins. In general, the increase is between 5% and 10%. This was a much more significant result,” assesses Bergamo.
The project focuses on producing ethanol from sugarcane bagasse, but other substrates can also be used, such as agave, corn straw, and eucalyptus leaves. Despite the promising results, further testing is needed to ensure the technology is applicable to industry.
“A common challenge for all strains is knowing the limit the bacteria reach before the ethanol itself has a reverse effect and inhibits production,” says Bergamo. The researchers also cite the need to evaluate the competitiveness of the modified strain against invasive bacteria in an industrial bioreactor. The next step is scaling up, testing whether the bench-scale performance is replicated in larger batches. This stage will be conducted by the American company that licensed the technology.
Inventors Award 2026
In its 19th edition, the Unicamp Inventors Award, organized by Inova Unicamp, recognizes and values inventors who have excelled in the transfer of technologies from the University and in the creation of academic spin-off companies.
Award-Winning Inventors
Luana Walravens Bergamo, Layse Costa De Souza, and Christopher David Herring were awarded in the Licensed Intellectual Property category in 2026.
Check out the complete list of all award winners on the Unicamp Inventors Award website.
2026 Tribute Program
In celebration of the 2026 Inventors Award, Inova Unicamp organized a series of tributes. These included articles highlighting award-winning cases, available to read on the Inova Unicamp and Inventors Award websites.
Continuing the celebrations, on August 4th, Inova will host an in-person event to share challenges, lessons learned, and opportunities in the use of artificial intelligence in innovation processes and the creation of spin-off companies at the University. Participation is open to the public. ( free registration)
The 2026 Inventors Award has the institutional support of Lumina , the Unicamp Endowment Fund, and is sponsored by ClarkeModet and FM2S.
Article originally published on the Inova Unicamp website.
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