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Microbes that are used for healthcare, agriculture or other applications must be able to withstand extreme conditions and ideally the manufacturing processes used to make tablets for long-term storage. Researchers at MIT have now developed a new way to make microbes tough enough to withstand these extreme conditions.

Their method involves mixing bacteria with food and drug additives from a list of compounds that the FDA classifies as “generally regarded as safe.” The researchers identified formulas that help stabilize several different types of microbes, including yeast and bacteria, and showed that these formulas can withstand high temperatures, radiation and industrial processing that can damage unprotected microbes.

In an even more extreme test, some of the microbes recently returned from a trip to the International Space Station coordinated by Phyllis Friello, manager of science and research at Space Center Houston, and now the researchers are analyzing how well the microbes were able to withstand those conditions.

“This project was about stabilizing organisms for extreme conditions. We’re really thinking about a wide range of applications, whether it’s space missions, human applications, or agricultural purposes,” said Giovanni Traverso, an associate professor of mechanical engineering at MIT, a gastroenterologist at Brigham and Women’s Hospital, and senior author of the study.

Miguel Jimenez, a former MIT scientist who is now an assistant professor of biomedical engineering at Boston University, is the lead author of the paper, which appears in Natural materials.

Survival in extreme conditions

About six years ago, Traverso’s lab began working on new approaches to make beneficial bacteria such as probiotics and microbial therapeutics more resistant. As a starting point, the researchers analyzed 13 commercially available probiotics and found that six of these products did not contain as much live bacteria as stated on the label.

“What we found is that—perhaps not surprisingly—there is a difference, and it can be significant,” says Traverso. “So the next question was, given that, what can we do to help the situation?”

For their experiments, the researchers chose four different microbes to focus on: three bacteria and one yeast. These microbes are Escherichia coli Nissle 1917, a probiotic; Ensifer meliloti, a bacterium that can fix nitrogen in the soil to aid plant growth; Lactobacillus plantarum, a bacterium used to ferment food products; and the yeast Saccharomyces boulardii, which is also used as a probiotic.

When microbes are used for medical or agricultural applications, they are usually dried into a powder through a process called lyophilization. However, they usually cannot be made into more useful forms such as a tablet or pill because this process requires exposure to an organic solvent that can be toxic to bacteria. The MIT team set out to find additives that could improve the microbes’ ability to survive this kind of processing.

“We developed a workflow where we can take materials from the FDA’s list of ‘generally considered safe’ materials and mix and match them with bacteria and ask if there are ingredients that increase the stability of the bacteria during the lyophilization process ?” Traverso says.

Their setup allows them to mix microbes with one of about 100 different ingredients and then grow them to see which survive best when stored at room temperature for 30 days. These experiments reveal different ingredients, mostly sugars and peptides, that work best for each type of microbe.

The researchers then selected one of the microbes, E. coli Nissle 1917, for further optimization. This probiotic has been used to treat “traveler’s diarrhea,” a condition caused by drinking water contaminated with harmful bacteria. The researchers found that if they combined caffeine or yeast extract with a sugar called melibiose, they could create a very stable formulation of E. coli Nissle 1917.

This mixture, which the researchers called formulation D, allowed survival rates greater than 10% after the microbes were stored for six months at 37 degrees Celsius, while the commercially available formulation of E. coli Nissle 1917 lost any viability after only 11 days under these conditions conditions.

Formulation D is also able to withstand much higher levels of ionizing radiation, up to 1000 g. (A typical dose of radiation on Earth is about 15 micrograys per day, and in space it is about 200 micrograys per day.)

The researchers don’t know exactly how their formulations protect the bacteria, but they hypothesize that the supplements may help stabilize bacterial cell membranes during rehydration.

Stress tests

The researchers then showed that these microbes could not only survive the harsh conditions, but also maintain their function after these exposures. After Ensifer meliloti were exposed to temperatures of up to 50 degrees Celsius, the researchers found that they were still able to form symbiotic nodules on plant roots and convert nitrogen into ammonia.

They also found that their formulation of E. coli Nissle 1917 was able to inhibit the growth of Shigella flexneri, one of the leading causes of diarrhea-related deaths in low- and middle-income countries, when the microbes were grown together in the laboratory plate.

Last year, several strains of these extremophile microbes were sent to the International Space Station, which Jimenez described as “the ultimate stress test.”

“Even just the delivery to Earth to the pre-flight validation and the storage to the flight are part of this test, with no temperature control along the way,” he says.

The samples recently returned to Earth and Jimenez’s lab is now analyzing them. He plans to compare samples that were kept inside the ISS with others that were attached to the outside of the station, as well as control samples that remained on Earth.

Other authors of the paper include Johanna L’Heurre, Emily Colaia, Gary Liu, Kyle Martin, Husna Ellis, Alfred Dao, Margaret Yang, Zachary Villaverde, Afeefa Hazi-Sayed, Chinhao Cao, Niora Fabian, Joshua Jenkins, Nina Fitzgerald, Christina Caravassili , Benjamin Muller and James Byrne.

This story is republished courtesy of MIT News (web.mit.edu/newsoffice/), a popular site that covers news about MIT research, innovation and teaching.