Microbes used for health, agriculture, and other purposes must withstand extreme conditions and the manufacturing processes for long-term storage tablets. MIT researchers have developed a novel method to make these microbes resilient enough to endure such harsh conditions.
Their approach involves mixing bacteria with food and drug additives from a list of compounds classified by the FDA as « generally regarded as safe. » The researchers identified formulations that stabilize various microbes, including yeasts and bacteria, and demonstrated that these formulations could withstand high temperatures, radiation, and industrial treatments that typically harm unprotected microbes.
In an even more extreme test, some microbes recently returned from a trip to the International Space Station, coordinated by Phyllis Friello, Science and Research Manager at Space Center Houston. Researchers are now analyzing how well the microbes survived these conditions.
« The goal of this project is to stabilize organisms under extreme conditions. We are considering a wide range of applications, from space missions to human and agricultural uses, » says Giovanni Traverso, Associate Professor of Mechanical Engineering at MIT, gastroenterologist at Brigham and Women’s Hospital, and senior author of the study.
Miguel Jimenez, a former MIT research scientist and now an Assistant Professor of Biomedical Engineering at Boston University, is the lead author of the study. The paper appears today in Nature Materials.
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 make beneficial bacteria, such as probiotics and microbial therapies, more resilient. They started by analyzing 13 commercially available probiotics and found that six of these products contained fewer live bacteria than the label indicated.
« What we found, unsurprisingly, is that there is a difference, and it can be significant, » says Traverso. « 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, also used as a probiotic.
When used for medical or agricultural purposes, microbes are typically dried into a powder through a process called lyophilization. However, they usually cannot be processed into more useful forms, like tablets or pills, because this requires exposure to an organic solvent, which can be toxic to bacteria. The MIT team sought additives that could improve the microbes’ ability to survive this type of treatment.
« We developed a workflow where we can take materials from the FDA’s ‘generally regarded as safe’ list, mix and match them with bacteria, and ask if there are ingredients that improve bacterial stability during the lyophilization process? » says Traverso.
Their setup allows them to mix microbes with one of 100 different ingredients, then grow them to see which survive best when stored at room temperature for 30 days. These experiments revealed various ingredients, mainly sugars and peptides, that worked best for each microbe species.
The researchers then selected one microbe, E. coli Nissle 1917, for further optimization. This probiotic has been used to treat « traveler’s diarrhea, » a condition caused by consuming water contaminated with harmful bacteria. They found that combining caffeine or yeast extract with a sugar called melibiose created a very stable formulation of E. coli Nissle 1917. This mixture, 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 Nissle 1917 lost all viability after just 11 days under these conditions.
Formulation D also withstood 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 are not exactly sure how their formulations protect the bacteria, but they hypothesize that the additives might help stabilize bacterial cell membranes during rehydration.
Resistance Testing
The researchers then showed that these microbes could not only survive harsh conditions but also retain their function after these exposures. After Ensifer meliloti was exposed to temperatures up to 50 degrees Celsius, the researchers found that it was still able to form symbiotic nodules on plant roots and convert nitrogen to ammonia.
They also found that their formulation of E. coli Nissle 1917 successfully inhibited the growth of Shigella flexneri, a leading cause of diarrhea-related deaths in low- and middle-income countries, when the microbes were co-cultured in a lab dish.
Last year, several strains of these extremophile microbes were sent to the International Space Station, which Jimenez describes as « the ultimate endurance test. »
« Even the shipment on Earth to validation before flight and storage until 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 currently analyzing them. He plans to compare samples stored inside the ISS to those bolted outside the station, as well as control samples kept on Earth.
« This work offers a promising approach to improving the stability of probiotics and/or genetically modified microbes in extreme environments, such as space, which could be used in future space missions to help maintain astronaut health or promote sustainability, for example, by supporting more robust and resilient plants for food production, » says Camilla Urbaniak, a research scientist at NASA’s Jet Propulsion Laboratory, who was not involved in the study.
The research was funded by NASA’s Translational Research Institute for Space Health, Space Center Houston, MIT’s Department of Mechanical Engineering, as well as the 711th Human Performance Wing and the Defense Advanced Research Projects Agency.
Other authors of 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.
