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Life Sciences Research on the Lunar Surface

In Session 3, moderated by planning committee members Krystyn Van Vliet and Jana Stoudemire, four presenters described life science research that has been, is planned to be, or could be carried out on the lunar surface or in lunar orbit. The purpose of the session was to help interested researchers see examples of the kinds of research that could benefit from being performed on the Moon and trigger ideas for future studies. The session also provided some indication of what is required to carry out such research on the Moon through the Commercial Lunar Payload Services (CLPS) program.

The first speaker was Rob Knight, the director of the Center for Microbiome Innovation at the University of California, San Diego, who spoke about microbiome research on the lunar surface. Next was Charles Cockell, a professor of astrobiology at the University of Edinburgh and director of the UK Center for Astrobiology. Cockell described experiments involving microbes that were carried out on the International Space Station (ISS) and that could also be performed on the Moon via CLPS. Clive Svendsen, a professor of regenerative medicine at Cedars Sinai-Medical Center in Los Angeles, then talked about organ chip systems for use in carrying out experiments on the lunar surface and the potential of manufacturing stem cells on the Moon. Last, Anna-Lisa Paul, a professor at the University of Florida, spoke about what China’s Chang’e-4 mission to the Moon in 2019 demonstrated concerning the growth of plants on the Moon.

FOOTPRINTS AND FINGERPRINTS: MICROBIOME AND THE MOON

Knight spoke about three aspects of microorganisms and the Moon: keeping the lunar environment protected from microbes brought from Earth, creating habitable environments on the Moon for the growth of microbes, and using microbes to optimize astronaut nutrition and health in the lunar environment. He noted that the work he does has been made possible by the dramatic cost reduction in sequencing genomes, with the amount of sequencing that would have cost $4 million little more than a decade ago now being possible to do for just 75 cents.

The first aspect of his work that he described was efforts to avoid contaminating places beyond Earth with microbes brought from Earth. “Let’s not find life elsewhere in the universe that we put out there in the first place,” he said, “because it could be totally embarrassing.”

He described studies of the microbiome—the collection of microorganisms—found in various facilities, including the spacecraft assembly facility at the Jet Propulsion Laboratory (JPL) and in a neonatal intensive care unit (NICU). The diversity of the microbiome was higher in the NICU than in the JPL facility assembling the Mars 2020 spacecraft, he said. Knight’s research team also found that the collections of microbes differed significantly between facilities, so that it was possible to distinguish one from the other, based on particular microbes that were unique to each of the environments.

Of note for Mars and lunar exploration, he said, the team identified a number of radiation-resistant microbes in the JPL spacecraft assembly facility. Learning about the sorts of microbes that are found in such facilities—particularly the radiation-resistant ones—will help in developing techniques to eliminate those microbes so that they do not have a chance to hitch a ride to the Moon or Mars.

Turning to the second issue of creating environments that will support desired microbes on the lunar surface, Knight noted that a significant amount of work has been done on the sorts of microbes that are present in a healthy built environment, pointing specifically to a National Academies of Sciences, Engineering, and Medicine report, Microbiomes of the Built Environment: A Research Agenda for Indoor Microbiology, Human Health, and Buildings.¹ Concerning the sorts of microbes that are found in built environments in space, he described a study that examined the microbiome on the ISS on various missions. There was a “core microbiome” found on all missions that consisted of 17 species, both fungi and bacteria. In other words, all human subjects in those studies shared those 17 microbial species, which represented just a small fraction of all of the microbes found on the various individuals on the ISS. This approach should be valuable, Knight said, in determining the sort of microbiome that a healthy space habitat should contain.

In doing this, he continued, one must think not only about keeping unhealthy microbes out of the environment but also about including healthy microbes. He pointed to a study that found that exposure to cows helped protect Amish children against asthma. More generally, the hygiene hypothesis holds that environments that are too clean deprive people of the microbes that are necessary to strengthening the immune system. Thus, in the future it may be the case that certain microbes are included in habitats to improve the health of the inhabitants.

Last, Knight turned to the issue of precision medicine and nutrition for lunar explorers. Studies have found, he said, that individuals living as modern hunter-gatherers have completely different microbiomes from people living a modern lifestyle. The implication is that people today have lost many of the microbes that helped earlier humans maintain their health. The real concern, he said, is that humans living in space or lunar environments could lose even more of the human microbiome, such as microbes that supply essential components for metabolism. Indeed, he said, it has been found that the microbiome plays a role in a number of diseases as well. Thus, making sure that individuals on the lunar surface maintain a healthy microbiome will be crucial.

Turning briefly to personalized, or precision, nutrition, Knight said that the microbiome plays a role there as well. In particular, information on an individual’s microbiome can help predict which foods are healthy or not for that person, particularly with respect to glycemic response. This response plays a role in a number of metabolic diseases.

In conclusion, Knight said that sequencing information on an individual’s microbiome is highly data-intensive—the data from a teaspoon of stool will fill a ton of DVDs—so it will be important to have a high-capacity networking infrastructure to transmit data to Earth if the sequencing is going to be done on the Moon. Alternatively, one may have the computing infrastructure onsite to process all the information generated by the sequencing.

MOBILIZING MICROBES FOR THE MOON

In the next presentation, Cockell continued with the topics of microbes, with a specific focus on doing microbiology experiments on the Moon in the context of the CLPS program. Specifically, he described an experiment his group did on the ISS as an illustration.

Expanding on Knight’s discussion of how microbes could help maintain a healthy environment on the Moon, Cockell listed a number of ways microorganisms can be used productively on the Moon. They can be used to break down waste in life support systems, to produce drugs and biofuels, to assist in soil formation, in the creation of plastics and other industrial products, and in biomining.

Next, Cockell described BioRock, an experiment his group performed on the ISS 2 years ago, as an example of the types of experiments that could be performed on the lunar surface through CLPS. The

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¹ National Academies of Sciences, Engineering, and Medicine, 2017, Microbiomes of the Built Environment: A Research Agenda for Indoor Microbiology, Human Health, and Buildings, The National Academies Press, Washington, D.C.

experiment is done in the context of biomining—the use of microorganisms to extract useful materials from ores. It is a common process on Earth, he said, and about 25 percent of the world’s copper and gold is extracted from rocks using microorganisms. It is energy efficient and avoids the use of toxic compounds such as cyanide, which are often used in the more traditional types of mining.

The BioRock experiment involves a bioreactor in which microbes work on basalt, a common material on the Moon and Mars. The bioreactor is automated so that when it reaches the ISS, a signal is sent to the bioreactor, fluid is injected into its chambers, and the microbes get to work. This miniaturized biological reactor, Cockell said, is the sort of thing that could be put in a lunar lander. It can be used to carry out fundamental microbiology research and to test applications of microbes in the lunar environment.

This sort of biological experiment is wholly compatible with commercial access to space, he added. Indeed, while the BioRock experiment took 10 years from writing the proposal in 2009 to performing it on the space station in 2019, a related experiment, BioAsteroid, which was done completely with commercial space vehicles, took only 1 year from proposal to flying the experiment. “So,” he said, “I would like to just point out that from a scientist’s point of view, these commercial opportunities to access space are really fundamental, not just because they speed up time, but because they allow you to build a research program around spaceflight.” The ability to do studies quickly and reliably—and within a grant cycle—will lead more scientists to get involved with space experiments, Cockell predicted.

The BioRock experiment on the ISS showed that microbes did just as good a job of pulling various rare earth elements from basalt under microgravity conditions or Martian gravity under 1 g. The Martian gravity was mimicked by running the experiment in a centrifuge. Still, Cockell said, it will be important to also do such studies on the Moon itself because “simulated gravity is not quite the same as real gravity, because you’ve got Coriolis effect and different types of sheer stresses inside this miniature centrifuge.”

It will likely never be commercially feasible to get rare earth elements on the Moon and bring them back to Earth, he said. However, mining such elements on the Moon may be important if there is ever a permanent settlement beyond Earth. Microbes have also been shown to be useful in the biomining of vanadium, which has various uses such as in batteries, high-strength steel alloys, and neutron absorption in metals used in reactors.

In answer to a question from Van Vliet during the session’s discussion period, Cockell emphasized the importance of being able to carry out experiments multiple times in order to get sufficient replication to be confident of the results. The tendency on the Moon, he said, will be to want to do new and exciting experiments one after the other. However, from a science point of view it would be better to do a similar—or even the same—experiment over and over again.

In a similar vein, he also spoke in the discussion period about the importance of developing a community of researchers who are familiar with each other’s research and can build on previous work. A problem with research on the ISS, he said, is that each new set of researchers tended to build their own experiment from scratch even if they could have learned from earlier experiments. Ultimately, he said, just having common types of off-the-shelf apparatus that researchers could use for different types of experiments could lead to less waste and greatly increase the ability to carry out lunar science.

Looking to the future, Cockell said that his group would like to carry out the BioRock experiment on the Moon, and the CLPS program should provide them with that opportunity. In the long term, he added, the goal is to create a lunar astrobiology lab that makes it possible to carry out a range of biological experiments on the surface of the Moon. To that end, he is currently leading a study that is developing a Lunar BioMission concept for the European Space Agency. As currently envisioned, the mission would carry out a large number of experiments on a variety of organisms, from prokaryotes to single-cell eukaryotes up to multi-cell eukaryote plants and possibly insects. The studies would look at interactions between these organisms and radiation on the lunar surface, lunar gravity, and the regolith, the loose layer of material on the Moon’s surface. The mission would also contain its own analytic facility to carry out biomolecular and genetic analyses measuring such things as the rate of damage to the organisms and their rate of repair.

“Whether it happens or not,” he concluded, “these are the exciting prospects that lie ahead in commercial access to the Moon, for those of us interested in biology.”

ORGAN CHIP SYSTEMS AND FUTURE STEM CELL MANUFACTURING ON THE MOON

Svendsen began his presentation by describing induced pluripotent stem cells (iPSCs). These cells can be created from various types of mature cells, such as skin cells or blood cells. Through a treatment whose development won its creator the Nobel Prize in Physiology or Medicine, these cells are transformed into embryonic-like stem cells that can regenerate themselves endlessly. The cells can, with the correct triggers, transform into any type of cell in the body. They have great promise both in research and in the treatment of disease and injury, with cells harvested from a patient used to provide new cells or even organs that are genetically identical to the patient’s own.

These iPSCs could be used in a variety of space-based or lunar-based experiments. For instance, Svendsen said, working with an astronaut’s cells, it would be possible to create heart cells—even beating heart cells—and take them into space to investigate how radiation affects them.

More generally, the ability to grow various types of human cells makes it possible to simulate different aspects of human biology in order to study how different factors and environments affect function. He described a “lung on a chip” with a layer of lung cells up against a layer of epithelial cells to mimic the interface in the lung between blood vessels and lung cells. The apparatus includes two channels, one that runs alongside the lung tissue and the other alongside the epithelial tissue. Then, to run an experiment modeling lung function, air flows past the lung cells while blood flows past the epithelial cells. In one study, the researchers examined how COVID-19 virus particles in the air attached to the lung tissue and then were attacked by immune cells from the blood flow.

“I think this is going to be an important way of simulating infections in space,” Svendsen said. At present it is not clear how gravity or radiation will affect infections; such studies will help provide an answer.

The researchers are now working to automate these systems for use on space flights, he said. They would love to establish a self-sustaining system on the Moon where they could grow these iPSCs, differentiate them into different types of cells, and carry out experiments on them. Referring to the microbiome studies described by the previous speakers, Svendsen noted that microbes could also be included in these systems, such as a chip with stomach cells. The resulting interactions can be observed under space-based and lunar conditions. These systems also offer the potential for precision health experiments, using cells from a particular astronaut to examine the effects on the biological functions of that individual. For example, if an astronaut was going to have a bad reaction to radiation, it would be possible to predict that before the astronaut went into space.

Last, Svendsen spoke about testing how the growth of iPSCs might be different in low- or microgravity conditions. When these cells are grown in a petri dish, they grow in colonies, but many of them will differentiate into specific types of cells. These differentiated cells must be continually removed, which requires a great deal of manual labor. Another issue is that the iPSCs tend to stick together as the colonies grow. So, Svendsen said, he is interested in whether iPSCs could be grown better in zero g. He suspects that if the cells could free float instead of being pulled down by gravity, it might improve results. There is also the question of how radiation in space might affect the iPSCs as they grow and multiply. He is hoping, he said, for a 2021 launch to put an experiment into space to answer some of these questions.

Ultimately, Svendsen said, he hopes to be able to carry out such experiments on the Moon to see how iPSCs grow in the lunar environment. If that proves to be an advantageous environment, one can envision a lab on the Moon manufacturing stem cells at scale for clinical trials on Earth. However, he acknowledged that, given the transportation issues, there would have to be a significant advantage to working on the Moon.

In answer to a question from Van Vliet during the discussion session about technical challenges to growing stem cells on the Moon, Svendsen said that one of the major limitations at this point is that everything must be automated. Once a point is reached where astronauts can play a role, he said, “I think that’s going to exponentially increase the kinds of exciting things we can do,” because there will be more flexibility and options.

THE IMPACT OF THE CHANG’E-4 MISSION ON LUNAR EXPLORATION BIOLOGY PERSPECTIVES

In the next talk, Paul described the Chang’e-4 mission, a Chinese mission to the Moon, and what insights it can provide for conducting lunar biological research. That 2019 mission made the first soft landing on the far side of the Moon, she said. The primary purpose of the mission was to carry out a physical exploration of the surface and subsurface on the far side of the Moon and to carry out low-frequency radio astronomy. There was also a small “biosphere-type payload” developed in collaboration with 28 Chinese universities.

That mini-biosphere payload was a small canister about the size of a 3-pound coffee can, Paul said. It contained soil, air, water, cameras, and a temperature control system to keep the internal environment at a steady temperature. It also held seeds of some common plants—cotton, brassica, potato, and Arabidopsis—as well as some fruit fly eggs and yeast cells. The idea was that the plants would produce oxygen for the system, the fruit flies would breathe the oxygen and produce carbon dioxide, and the yeast cells would metabolize some of the waste of the flies. An identical canister was maintained on Earth so that the growth of the different organisms could be compared between Earth and the Moon.

As it turned out, Paul said, only the plants grew. However, she added that because the work has not yet been published, she is working with unverified information available on the web.

The work was actually quite difficult to do, she said. It required keeping the organisms dormant yet environmentally controlled while on the journey. Developing mechanisms to activate the organisms after landing and to protect them from the Moon’s extreme environment was required. It also required developing a way to monitor the interior of the canister. “The effort that went into this was really pretty extraordinary,” she said.

So why did they choose to try? “The most compelling reason to do this,” Paul said, “is to symbolically take the biosphere that we have here [on Earth] and transplant a tiny bit of that in the container of the biosphere that they put on the surface of the Moon.” Humans have already ventured out into space as “tourists,” she said, but now that the goal is to carry out long-term exploration to the Moon and the Mars. “Before we can really explore past the confines of Earth, we need to understand how our biology behaves in extraterrestrial environments.”

There are a number of challenges to humans establishing a biosphere on the Moon, Paul said. First, the journey itself requires that the organisms survive the microgravity of spaceflight as well as the restricted resources and altered atmosphere. Then, once on the Moon, things really get challenging. Dangerous radiation is hitting the surface. There is no atmosphere, so everything must be done in closed environments. The gravity is one-sixth of Earth’s. The days and nights are 2 weeks long. The temperatures range from around –150°C to +150°C. And almost everything that is needed for that biosphere must be brought from Earth.

A certain amount of testing can be done without going to the Moon with tests on the ISS, lab tests on Earth, or tests in extreme environments such as deserts or Antarctica. However, ultimately testing on the lunar surface will be required to really understand what will be required to create a functioning biosphere on the Moon. The Chang’e-4 mission’s mini-biosphere experiment represented a first step in that direction, she said. Thus, it is valuable to take a closer look at that study.

When Chang’e-4 landed on the Moon on January 3, 2019, the biosphere powered up, water was injected into the system to hydrate the seeds and other organisms, and photography of the interior began. Light for the canister’s interior was provided through a light pipe from the outside rather than from internal light bulbs. It was shut down on January 12 at the onset of the lunar night, and the canister cooled down to –62°C.

By the time of the shutdown, at least one cotton seed had sprouted. It was an extraordinary achievement, Paul said. It showed that it is possible to sprout a plant on the lunar surface. It also demonstrated the differences between a plant sprouting on Earth and one sprouting on the Moon. Although the photographic evidence is somewhat difficult to interpret, the cotton plant that sprouted on

the Moon seems to be showing the hallmarks of a plant under stress when it is growing in suboptimal conditions.

Summing up, Paul said that the Chang’e-4 biosphere experiment offers several lessons for future studies. It is difficult to manage resources, especially in small volumes. Imaging is vitally important, multiple camera angles are essential, and frequent imaging is needed to establish a timeline. It will be important to enable the survival of plants and other organisms over the lunar night. Reduced gravity may affect water behavior more than expected. Keeping experiments simple may help in resource-limited payloads. It will also be crucial to have multiple opportunities to conduct experiments; this cannot be a one-and-done endeavor.