Using chirality as a biomarker for extraterrestrial life

All known forms of life show a strong preference for using and producing L-amino acids and D-carbohydrates, although chemical reactions do not distinguish between stereoisomers of a compound. Thus, many astrobiologists¹ now advocate recognizing asymmetric chirality as a biomarker. However, newly proposed methods suffer from common problems.

First, abiotic syntheses of chiral amino acids with strong excesses of one isomer have recently been demonstrated.^2,3 Furthermore, the conversion of one stereoisomer to the other over time may cause ancient remnants of life to be missed, and life that is not based on preferential chiral biochemistry would also be missed. Methods that rely on chirality cannot determine whether the organism detected is alive or a fossil. Finally, chiral preference, like all other biomarkers, will fail the test of Occam's razor. Such evidence will be deemed more likely the result of abiotic happenstance than of the development of life.

Chirality was first applied⁴ to the detection of life in the development of the Viking Mission Labeled Release (LR) experiment flown to Mars in 1976. The LR injected radioactive nutrients into soils and then monitored them for expired radioactive gas as evidence of living microorganisms. If a positive response was observed, a duplicate sample of the soil was heated to 160°C to kill microorganisms but not destroy any chemical likely to have caused the response. A negative response from this control would confirm the first response as biological.

Carefully selected organic nutrients (listed in Table 1) were uniformly labeled with ¹⁴C. They were rigorously tested on a wide range of pure and mixed cultures and soils. There was never a false positive, a false negative, or an ambiguous result from the thousands of tests run throughout the 20-year LR development period.

Two of the LR's nutrient compounds, alanine and lactate, occur as stereoisomers. However, alien life might express chiralities opposite to ours.⁴ We planned to include such life in our search by applying the individual isomers to individual soil samples. However, this plan could not be executed because of the added instrumentation it required. Therefore, both isomers of each compound were incorporated into the nutrient solution to avoid missing such life forms.

The LR landed on Mars in 1976. Positive responses, similar in amplitude and kinetics to those of some terrestrial soils, were followed by negative controls, thereby satisfying the pre-mission criteria for life. Additional ad-hoc experiments developed a unique thermal profile for the active agent, as follows. The amplitudes and kinetics of all positive responses fell within the ranges obtained in LR tests of Earth soils. On the second nutrient injection, ~20% of the already evolved gas left the chamber (probably absorbed by wetted soil) and was gradually re-evolved. Soil heated to 51°C for 3h produced several small sporadic peaks (~5%–10% of positive); soil heated to 46°C for 3h produced kinetics that were positive but reduced in amplitude by ~70%. Soils maintained for 3 and 5 months in the dark at ~7–10°C under ambient Martian atmosphere, pressure, and humidity produced nil responses, and soil protected from UV by overlying rock produced typical active responses. Each of these findings can be explained by a range of known micro-organisms. However, no abiotic experiment has ever matched this profile.

All of these results support, or are consistent with, a biological agent. However, largely because the Viking Molecular Analysis gas chromatograph-mass spectrometer (GCMS) experiment failed to find organic matter, the consensus was that the LR had reacted to some Martian chemical, not life. Over time, events have indicated otherwise. For example, the sensitivity and capabilities of the GCMS have been questioned.⁵ Also, liquid water has been found on Mars.⁶ Furthermore, terrestrial extremophiles have been found living under Mars-like conditions. All chemical or physical laboratory attempts have failed to duplicate the Mars LR test and control data, and no sustainable theory has offered a non-biological explanation of the LR data. While some astrobiologists, including the author,^7,8 now support the biological interpretation or at least think it possible,⁹ the consensus remains negative.

To resolve this issue, years ago a proposal was made¹⁰ to adapt the LR to perform as originally desired: to apply only one enantiomer of selected compounds to each soil sample. Thus, like the Viking LR experiment, the enhanced version, the Twin Wireless Experiment for Extraterrestrial Life (TWEEL, named for a mythical vertical ascent and landing Martian bird¹¹), relied on continuing, sols-long reactions instead of obtaining a snapshot of the chirality of a molecule, as the biomarker methods do. If a continuing reaction in TWEEL were found in only one of the two isomers of a compound applied to a soil sample, this would be very strong evidence for metabolism and life. Finding a chiral preference opposite to ours would be the evidence of truly alien life that we originally sought to provide.

However, we have since realized that the TWEEL would miss an alien life form if it had an achiral metabolism. Accordingly, a further enhancement is now proposed to overcome this problem. The TWEEL 2 instrument, like the original TWEEL, consists of a set of small, individual, self-sustained dart-like probes. Packaged in a canister, they would be heat-sterilized before launch, which would occur from a landed spacecraft. They would be launched into the wind to preclude contamination from terrestrial bugs that may have been brought by the spacecraft.

The aerodynamics will land the probes nose first. Penetration stops will keep them from sinking. The impact of landing will drive soil into the sample chambers of each probe. In the original TWEEL, the incoming soil would be wetted with the opposite enantiomers of the test nutrient, which would be contained in two ampoules. However, the TWEEL 2 provides the option of applying the nutrient to the sample after the control substance is added to the soil or after imposition of an environmental condition as a control. Applying Viking-type heat sterilization would thus be possible.

These new controls are implemented by a remote radio-controlled activator that breaks an externally connected ampoule on command, wetting the sample with the nutrient. Any labeled gas rising from the sample passes through a permeable barrier that prevents the beta detectors from seeing the liquid and counting its radioactivity. It also prevents dust or aerosols from carrying radioactive material to the detector, which cumulatively monitors the evolution of labeled gas for radio relay to the spacecraft. A single TWEEL 2 probe, as currently conceived, is depicted in Figure 1.

Figure 1. The Twin Wireless Experiment for Extraterrestrial Life 2 (TWEEL 2), a chiral-labeled release dart with a heated (or other time-delayed) control. On deployment, they would be ejected through the cover of the canister containing them, maintaining their sterility. They would be launched upwind to preclude possible microbial contamination from the landed spacecraft.

Multiple controls are proposed (in addition to each chiral compound's own enantiomer control), including stepwise heating to determine the thermal endpoint of the active agent. Other environmental control conditions could include moisture, humidity, and atmospheric composition. Additional controls could consist of adding anti-metabolites to the samples. Toxic metals, cyanide, antibiotics, and enzyme inhibitors are candidates, as is the de-coupling agent 2,4-dinitrophenol. While these are more Earth-centric, a variety of them could offer the evidence sought, especially because a negative reaction from only one of them, following a positive test reaction from its test chamber or another TWEEL 2, would supply strong additional evidence for life. Results could begin a study of comparative biology with terrestrial forms.

Arizona State University has made an Astrobiology Science and Technology Instrument Development proposal to NASA for proof of concept and initial development of the instrument. The project would run for three years, with the possibility of continuing to flight development for deployment aboard a small Scout-type mission.