Microbes used for healthcare, agriculture, or other purposes must be able to withstand extreme conditions, and the manufacturing processes used to make tablets ideally need to be preserved for the long term. MIT researchers have 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 yeasts and bacteria, and showed that these formulas can withstand high temperatures, radiation, and industrial processing that can harm unprotected microbes.
In an even more extreme test, some of the microbes recently returned from a trip to the International Space Station, coordinated by Space Center Houston Science and Research Manager Phyllis Friello, and researchers are now analyzing how well the microbes were able to withstand those conditions.
“The goal of this project is to stabilize organisms for extreme conditions. We’re actually thinking about a broad range of applications, whether it’s space missions, human applications or agricultural uses,” said Giovanni Traverso, an associate professor of mechanical engineering at MIT, a gastroenterologist at Brigham and Women’s Hospital and senior author of the paper.
Miguel Jiménez, a former MIT research scientist who is now an adjunct professor of biomedical engineering at Boston University, is the senior author of the paper, which will appear in Materials from nature.
Surviving extreme conditions
About six years ago, with funding from NASA’s Translational Research Institute for Space Health (TRISH), 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 these products did not contain as many live bacteria as the label claimed.
“What we found was that, while perhaps not surprising, there is a difference that can be significant,” Traverso says. “So the next question was: 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; Ensifera melilotia bacterium that can fix nitrogen in the soil to promote plant growth; Lactobacillus plantaruma bacteria used to ferment food products; and yeast Saccharomyces boulardiiwhich is also used as a probiotic.
When microbes are used for medical or agricultural applications, they are typically dried into a powder using a process called freeze-drying. However, they typically can’t be turned into more useful forms, such as tablets or pills, because this process requires exposure to an organic solvent, which can be toxic to 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 ‘generally regarded as safe’ list of materials and combine them with bacteria and ask if there are ingredients that improve the stability of the bacteria during the freeze-drying process,” Traverso says.
Their system allows them to mix microbes with one of about 100 different ingredients and then grow them to see which ones survive best when stored at room temperature for 30 days. These experiments revealed different ingredients, primarily sugars and peptides, that worked best for each species 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. Researchers found that if they combined caffeine or yeast extract with a sugar called melibiose, they could create a very stable formula of E. coli Nissle 1917. This mixture, which the researchers called formulation D, allowed survival rates of over 10 percent after the microbes were stored for six months at 37 degrees Celsius, while a commercially available formulation of E. coli The Nissle 1917 lost all viability after only 11 days in these conditions.
Formula D could also 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, about 200 micrograys per day).
Researchers don’t know exactly how their formulas protect bacteria, but they hypothesize that the additives may help stabilize bacterial cell membranes during rehydration.
Stress tests
The researchers showed that these microbes can not only survive in harsh conditions, but also maintain their function after these exposures. Ensifera meliloti When exposed to temperatures of up to 50 degrees Celsius, 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 flexora leading cause of diarrhea-related deaths in low- and middle-income countries, when the microbes were grown together on a laboratory plate.
Last year, several strains of these extremophile microbes were sent to the International Space Station, which Jimenez describes as “the ultimate stress test.”
“Even just shipping to Earth for pre-flight validation and storage until flight is part of this test, with no temperature control along the way,” he says.
The samples recently returned to Earth and are being analyzed by Jimenez’s lab. He plans to compare the samples stored inside the ISS with those bolted to the outside of the station, as well as control samples that remained on Earth.
The research was funded by NASA’s Space Health Translational Research Institute, Space Center Houston, MIT’s Department of Mechanical Engineering, and the 711 Human Performance Wing and the Defense Advanced Research Projects Agency.
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.