Engineers find way to protect microbes from extreme conditions

Microbes used in healthcare, agriculture, and other applications must be able to withstand extreme conditions, and ideally the manufacturing processes used to make tablets for long-term storage. Now, MIT researchers have developed a new method 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 formulations that help stabilize several different types of microbes, including yeast and bacteria, and showed that these formulations could withstand high temperatures, radiation and industrial treatments that can damage unprotected microbes.

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

“This project was about stabilizing organisms in extreme conditions. We envision a wide range of applications, from space missions to human applications to agricultural uses,” says Giovanni Traverso, associate professor of mechanical engineering at MIT, a gastroenterologist at Brigham and Women’s Hospital and lead author of the study.

Miguel Jimenez, a former MIT research 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.

Surviving extreme conditions

About six years ago, Traverso’s lab began working on new approaches to make helpful bacteria like probiotics and microbial therapies more resilient. To start, the researchers analyzed 13 commercially available probiotics and found that six of the products didn’t contain as much live bacteria as the label claimed.

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

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

When microbes are used for medical or agricultural purposes, they are typically dried and turned into powder through a process called freeze-drying. However, they typically cannot be turned into tablets or pills 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 treatment.

“We developed a workflow that allows us to take materials from the FDA’s list of “generally regarded as safe” materials, mix them and pair them with bacteria, and ask, are there any ingredients that improve the stability of the bacteria during the freeze-drying process?” Traverso explains.

Their device allows them to mix microbes with one of 100 different ingredients, then culture them to see which ones survive best when stored at room temperature for 30 days. These experiments revealed different ingredients, mostly sugars and peptides, that worked best for each species 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,” an illness caused by drinking water contaminated with harmful bacteria. The researchers found that by combining 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, achieved survival rates of more than 10 percent after the microbes were stored for six months at 37 degrees Celsius, while a commercial formulation of E. coli Nissle 1917 lost all viability after just 11 days under these conditions.

Formula D was also able to withstand 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 don’t know exactly how their formulations protect the bacteria, but they hypothesize that the additives may help stabilize bacterial cell membranes during rehydration.

Stress tests

The researchers then showed that these microbes can not only survive harsh conditions, but also retain their function after these exposures. After exposing Ensifer meliloti 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, a leading cause of diarrhea-related deaths in low- and middle-income countries, when the microbes were grown together in a laboratory dish.

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

“Even the simple transportation on Earth until pre-flight validation, as well as storage until flight are part of this test, without any temperature control en route,” he explains.

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

Other authors on the paper 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 article is republished with kind permission from MIT News (web.mit.edu/newsoffice/), a popular site covering the latest research, innovation, and teaching at MIT.