Engineers find a way to protect microbes from extreme conditions

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

Their method involves mixing bacteria with food and drug additives from a list of compounds the FDA classifies as “generally regarded as safe.” The researchers identified formulas that help stabilize different types of microbes, including yeast and bacteria, and they 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. The researchers are now analyzing how well the microbes withstood these 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, agricultural applications,” says Giovanni Traverso, an associate professor of mechanical engineering at MIT, a gastroenterologist at Brigham and Women’s Hospital and the study’s lead author.

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

Surviving under extreme conditions

About six years ago, Traverso’s lab began working on new approaches to making beneficial bacteria, such as probiotics and microbial therapies, more resilient. As a starting point, the researchers analyzed 13 commercially available probiotics and found that six of the products did not contain as many live bacteria as the label said.

“What we found was that, perhaps not surprisingly, there is a difference, and it can be significant,” Traverso says. “So the next question was, given that, what can we do to improve 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 support 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 typically dried into a powder through a process called lyophilization. However, they typically can’t be converted into usable forms like a tablet or pill because this process requires exposure to an organic solvent, which can be toxic to the bacteria. The MIT team set out to find additives that could improve the microbes’ ability to survive this type of processing.

“We developed a workflow where we can take materials from the FDA’s list of ‘generally regarded as safe materials’ and combine them with bacteria, and then see if there are any ingredients that improve the stability of the bacteria during the freeze-drying process,” Traverso said.

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

The researchers then chose 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 highly stable formulation of E. coli Nissle 1917.

This mixture, which the researchers dubbed Formula D, produced a survival rate of more than 10 percent after the microbes were stored at 37 degrees Celsius for six months, while a commercially available formula of E. coli Nissle 1917 became fully viable after just 11 days under those conditions.

Formulation D was also shown to be resistant to much higher levels of ionizing radiation, up to 1,000 grays. (The typical radiation dose on Earth is about 15 micrograys per day, and in space it is about 200 micrograys per day.)

The researchers aren’t sure exactly how their formulas protect bacteria, but they suspect the additives may help stabilize bacterial cell membranes during rehydration.

Stress testing

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

They also found that their E. coli Nissle 1917 formulation could inhibit the growth of Shigella flexneri. Shigella flexneri is a leading cause of diarrheal deaths in low- and middle-income countries. The microbes were grown together in a laboratory dish.

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

“Just the transportation on Earth, the pre-flight validation and the storage until the flight are part of this test, without any 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 stored on the ISS with others that were bolted to the outside of the station, as well as with control samples that remained on Earth.

Other authors of the article include Johanna L’Heureux, Emily Kolaya, Gary Liu, Kyle Martin, Husna Ellis, Alfred Dao, Margaret Yang, Zachary Villaverde, Afeefah Khazi-Syed, Qinhao Cao, Niora Fabian, Joshua Jenkins, Nina Fitzgerald, Christina Karavasili, Benjamin Muller, and James Byrne.

This story is republished courtesy of MIT News (web.mit.edu/newsoffice/), a popular site that reports on research, innovation, and education at MIT.