How will we know if we’ve found Martian microbes?

By Reed Stubbendieck (@bactereedia)

curiosity
Figure 1. Curiosity Mars Rover taking a selfie. [Source]
Drew’s recent blog post spurred a conversation between the two of us about the first extraterrestrial life that humans will encounter. In the end, we both agreed that when/if humans discover aliens, they’ll most likely be microbial.

We are not alone in this assertion. At this very moment, the NASA Mars rover Curiosity (Fig. 1) is currently roaming the surface of the red planet using its suite of instruments to detect and characterize organic molecules that could be indicative of life from ancient aqueous environments. Intriguingly, data already collected by Curiosity has indicated that there are environments on Mars that may have once been habitable for microbial life!

Unfortunately, Curiosity is unable to directly detect living microbes, which begs the question: how will we really know that we’ve found genuine alien microbes?

To address this question, we first need to review the seven fundamental characteristics of life. All living organisms 1) are composed of cells, 2) are ordered, 3) grow, 4) reproduce, 5) pass down genetic information, 6) possess homeostasis, and 7) possess metabolism. In this post, we will consider how growth, reproduction, genetic information, and metabolism are currently used by scientists to detect life both on Earth and in the greater universe.

First and foremost, I am a microbiologist and prefer to follow an old proverb that states, “seeing is believing”. Thus, I would personally be most convinced of life on Mars if I saw an alien microbial colony emerge from a sample cultured on a Petri dish, which would demonstrate the necessary characteristics of growth and reproduction. However, unfortunately this level of evidence is most likely untenable for the foreseeable future.

As an example, on Earth if you directly count the number of bacterial cells from an environmental sample, such as soil or ocean water, using a microscope and then culture that sample on a Petri plate, only ~1% of those bacterial cells will form a visible colony. This phenomenon is known as “The Great Plate Count Anomaly” and has plagued microbiology since its inception. The anomaly is partially caused by how bacteriological medium is prepared, but is more majorly a result of our lack of understanding the nutritional requirements for different individual bacterial cells. Put another way, if we don’t know what Martian microbes like to eat, then we’ll be unable to coax them to reveal themselves.

iChip
Figure 2. The iChip after being removed from the ground. [Source]
On Earth, we’ve developed methods that circumvent and accommodate these picky eaters. For instance, the isolation chip (iChip, Fig. 2) is a relatively recent technology that allows microbes to be cultured in situ (at the place of their origin). This device works by trapping individual microbial cells into tiny wells that are sandwiched between semipermeable membranes. The membranes allow the passage of molecules between the trapped cells and their environment. Thus, the iChip allows microbes to access the nutrients they require without requiring scientists to determine specific requirements and formulate special media. This approach has been used to cultivate up to 50% of the microbes in a soil sample, which led to the discovery of a new antibiotic scaffold from a previously uncultivable bacteria!

Alternatively, as direct culture is a bottleneck for identifying living microbes, culture-independent approaches based on DNA sequencing have exploded in the field of microbial ecology. The primary approach that is used is called amplicon sequencing, which allow us to use specific DNA sequences as barcodes to identify different microbes. An alternative approach is to sequence all of the DNA present in a sample. This approach is called metagenomics and has been used to characterize the genes that are present in different environments on Earth. An advantage of metagenomics over amplicon sequencing is the ability to assemble entire intact genome sequences from environmental samples!

kate_rubins_nanopore
Figure 3. Astronaut Kate Rubins using the Oxford Nanopore MinION Sequencer in Space [Source]
Though once prohibitively expensive and technically challenging, advancements have rapidly decreased the cost of DNA sequencing and shrunken sequencers from the size of a refrigerator to a device that can fit in your hand (which has even been used in space, Fig. 3)! Thus, it may soon be possible to equip our future rovers with their own tiny sequencers. However, there are still important hurdles to overcome before implementing DNA sequence technology. First, direct DNA sequencing can’t distinguish between living and dead microbes. Further, contamination with microbes or microbial DNA from Earth may confound our analyses. Finally, a practical consideration is sampling throughput (the amount of samples that can be processed in a given period of time) and reagent usage.

Whether we attempt to directly culture microbes or sequence their DNA from Martian samples, there’s another practical consideration to discuss: where do we sample? As mentioned above, our rovers will likely carry only a limited amount of reagents for bacterial culture or DNA sequencing. Thus, we need to determine a method to narrow our search space for microbes: we need to identify biosignatures of life.

Fortunately, Curiosity is already equipped with instruments that detect organic molecules. Remember, all living organisms possess metabolism, perhaps we can follow molecules like methane as biosignatures and locate microbes. Unfortunately, because Mars has no atmosphere, the planet surface is bombarded with ultraviolet light, which may destroy volatile biosignatures.

sagan_and_viking
Figure 4. Carl Sagan posing next to a model of a Viking Lander in Death Valley, California. [Source]
As an alternative, we can attempt to identify biosignatures under controlled conditions. One such method was employed in the 1970s during the Viking program. As part of this program, two landers (Fig. 4) were sent to Mars with the mission to search for evidence of life. One experiment performed by the landers was called “Labeled Release”. A Martian soil sample was combined with a mixture of seven radioactive 14C-labeled nutrients and monitored for production of labeled carbon dioxide (14CO2) gas, which would suggest that living organisms had consumed the nutrients and produced the gas as a waste product. The experiment gave mixed results: though both landers initially produced positive results, repeated injections of the labeled nutrients failed to yield additional 14CO2. Though controversial, it is now believed that the 14COobserved in these experiments was produced abiotically. Perhaps future experiments will combine radioactive labeling with culturing or sequencing to identify microbes.

The above list of approaches to identify life is by no means exhaustive. However, I hope this post has highlighted the difficulties that scientists face not only in our search for extraterrestrial life in the universe, but also in characterizing the vast diversity of terrestrial microbes that inhabit on our own planet!