Agnostic Biosignatures: Towards a More Inclusive Life Detection Strategy

Sarah Stewart Johnson1, Heather Graham2, Eric Anslyn3, Pamela Conrad4, Leroy Cronin5,
Andrew Ellington3, Jamie Elsila2, Peter Girguis6, Christopher Kempes7, Eric Libby7, Paul
Mahaffy2, Jay Nadeau8, Mugizi Robert Rwebangira9, and Andrew Steele4
1
Georgetown University (sarah.johnson@gerogetown.edu, 202-687-3893)
2
NASA Goddard Space Flight Center
3
University of Texas at Austin
4
Carnegie Institute of Washington
5
University of Glasgow
6
Harvard University
7
Santa Fe Institute
8
Portland State University
9
Howard University
Abstract
A key scientific question in astrobiology is how to search for signs of life regardless of
underlying biochemistry. Current strategies for biosignature detection rely on identification of
well-established and widely accepted features associated with terrestrial life and signatures of
biologic processes, such as particular classes of molecules and isotopic signatures, enantiomeric
excesses, and patterns within the molecular weights of fatty acids or other lipids. As we begin to
explore icy moons of Jupiter and Saturn and other destinations beyond Earth, a promising
astrobiology research goal is the development of life detection methods that identify
unknowable, unfamiliar features and chemistries that may represent processes of life as-yet
unrecognized. This objective requires us to utilize existing instrumentation in more inclusive
ways, pursue new leads, and synthesize data with probabilistic approaches, as agnostic methods
may trade definitiveness for inclusivity.

Introduction
“Life as we don’t know it” presents a formidable challenge to any astrobiology strategy. How
do we contend with the truly alien? What might the molecular and polymeric building blocks of
life might look like on planetary bodies that are different from our own? Noteworthy advances
have been made in this realm since the publication of the NASA’s last astrobiology strategy, and
significant progress is likely in the next twenty years. Building on foundational work that has been
percolating in the astrobiology community1, this document serves to illustrate some novel ways to
detect chemical life without invoking any particular molecular frameworks.
One promising research area for astrobiology is the detection of “agnostic” biosignatures—in
other words, evidence of biology that doesn’t presuppose a particular biochemistry.

Utilizing Existing Instrumentation in More Inclusive Ways

To cast the widest possible net for life detection, we must broaden not only the range of
measurements we make but also the range of interpretations we allow. Part of this can be achieved
by utilizing high heritage instrumentation or recently proven techniques with the potential to be
developed into space qualified instrumentation in more agnostic ways. For example, flight capable
mass spectrometers have long been flown on spacecraft, designed to search primarily for patterns
among the molecular weights of carbon-bearing organic molecules. However, mass spectrometers
can also be used, for example with tandem mass spectrometry2, to search for chemical complexity
of any type of molecule (organic or inorganic) that would be unlikely or impossible to form
spontaneously.
The term “complexity” is often subjectively used when describing natural and synthetic
chemical structures3. However, chemical complexity can be conceptualized in a more rigorous way

1
e.g. Conrad and Nealson, 2001, Astrobiology; Baross et al, 2007, The Limits of Organic Life in Planetary Systems;
Schulze-Makuch and Irwin, 2008, Life in the universe: expectations and constraints; Scharf et al., 2015,
Astrobiology
2
Goesmann et al., 2017, Astrobiology
3
e.g. Nicolaou et al., 2012, Chem Soc Rev; Ertl and Schuffenhauer, 2009, J Cheminform; Bickerton et al., 2012,
Nature Chemistry

1
if mapped directly to an increase in the number of components in a system and versatility of their
interactions, and if it reflects structural features, including branching, cyclicity, multiple edges,
and heteroatoms.
Recent work at the University of Glasgow has utilized graph theory to understand molecular
structure, therefore serving as a measure of intrinsic complexity, rather than being context
dependent4. By encoding a graph and enumerating graph features like subgraphs or walks,
generational algorithms can be used to count the operations needed to build complexity graphs out
of simpler graphs, thereby computing a Pathway Complexity Index (MCI) for any molecule (see
Figure 1). This work is based on the thesis that the ability of living systems to replicate and evolve
allows for the generation of complex molecules, such as metabolites and co-factors, which would
be highly unlikely to form in any significant quantity in the absence of biology. Work remains to
be done in benchmarking the algorithm, exploring chemical space, and fragmentation mapping
with flight capable ion traps, but repeated measurements above a certain complexity carry the
promise of agnostically detecting whether molecules formed as the result of biology.
Without making assumptions about the chemical structures of molecules, recently published
research suggests there may be a threshold beyond which complex molecules are unlikely to
form without supporting biological machinery.

Figure 1: What is the simplest way to construct a molecule from its parts, accounting for the
simplifying feature of duplication? As an example, an algorithm can be used to break biphenyl
into a six-step construction process, therefore assigning an MCI of 6. Among natural products,
synthetic drugs, amino acids, metabolites, and other chemical compounds, there appears to be a
MCI threshold of 15, above which no molecules tested thus far have an abiotic origin.
Along similar lines, sequencing technologies have been developed by NASA’s ASTID,
MATISSE, and COLD-Tech instrument development programs as a way to search for nucleic
acids based on a shared ancestry hypothesis and monitor terrestrial contamination5. Rapid
advances in miniaturization have led to stand-alone sequencers, like the Oxford Nanopore
MinION, which was recently demonstrated on the International Space Station6.
While this approach is specific to a particular class of molecules (nucleic acids, including those
with nonstandard bases), work at Georgetown University, the University of Texas at Austin, and
NASA Goddard Space Flight Center has begun to lay the foundation to harness the power of
sequencing to explore sample complexity, regardless of whether life is based on nucleic acids.
This concept, as detailed in a forthcoming paper in Astrobiology7, builds on the fact that
oligonucleotides naturally form secondary and tertiary structures that can have affinity and
specificity for a variety of molecules, from peptides and proteins8, to a wide variety of small
organic molecules9, to inorganics such as mineral surfaces10 and individual metals11. Binding

4
Marshall et al., 2018, Philosophical Transactions of the Royal Society A
5
Carr et al., 2016, IEEE Aerospace Conference; Mojarro et al., 2016, LPSC; Bywaters et al., 2017, AbSciCon
6
Castro-Wallace et al., 2017, Scientific Reports
7
Johnson et al., in press, Astrobiology
8
Sun and Zu, 2015, Molecules

2
patterns of nucleic acids, independent of their biological function, can thereby be used to probe
and report on any chemical environment, opening up a new way to detect agnostic biosignatures.
DNA sequences as short as 15 nucleotides in length (but more commonly 30-80 nucleotides in
length) can form complex structures that, like antibodies, will bind to analytes, from simple
inorganics or minerals to highly complicated cell surfaces12 (See Figure 2).

Figure 2: A new concept for life detection harnesses the power of DNA sequencing, but not to look
for nucleic acid based life. 1) DNA strands are mixed with samples. 2) Many diverse folded
oligonucleotides will bind to a complex surface, such as a cell membrane whereas far fewer will
bind to a simple, repeating, inorganic crystalline structure. 3) Bound sequences can be amplified
and sequenced, revealing the diversity of binding sites within a sample. No prior knowledge of the
surface attributes or about the 3D structures of the binding nucleic acids is required, thereby
enabling an extension beyond terrestrial conceptions of what life may look like.

By accumulating large numbers of binding sequences that reflect different compounds in a

mixture, statistical data analyses of oligonucleotide sequences and sequence counts enable patterns
associated with increasing levels of complexity to be analyzed. This pattern recognition, known as
“chemometrics,” represents a set of protocols that can be applied to find patterns in chemical data
sets13, which in turn can be used to fingerprint nonterran biosignatures.
A newly developed approach could distinguish samples with chemistries suggestive of
biology—to “read” patterns of molecules, for example arising from the vast amount of
information stored on the surface of a primitive microbial cell, and to do it with great
sensitivity.
Additional work is required to hone the chemical assays and refine the chemometrics, but
without presupposing any particular molecular framework, this life detection approach could be
used from Mars to the far reaches of the solar system, all within the framework of a miniaturized
chip drawing little heat and power. While the amount of biomass produced on Ocean Worlds may
be limited14, utilizing the power of PCR, this technique could be capable of amplifying the signal
associated with an exceedingly small input. Further refinements in NextGen chemometrics may

12
e.g. Jayasena, 1999, Clinical Chemistry
13
e.g. Goodwin et al., 2015, Angewandte Chemie; Hughes et al., 2008, Chemistry–A European Journal; Pai and
Ellington, 2009, Biosensors and Biodetection; Stewart et al., 2011, Chembiochem; Umali and Anslyn, 2010, Current
opinion in chemical biology; Wright et al., 2005, Angewandte Chemie; Zamora-Olivares et al., 2014, Angewandte
Chemie
14
McCollum, 1999, JGR

3
be able to not only generate the binding “fingerprint” of a surface but also reveal associated
physical structures.
Several other agnostic biosignatures have been surmised that could take advantage of high
heritage instrumentation, including non-chemical methods. For instance, holographic microscopy
with computational modeling of non-random motion to identify isolated structures, including those
capable of meaningful movement and/or responding to taxis, could also serve as a non-Earth-
centric approach (for more detail, see the white paper submitted by Jay Nadeau and colleagues).

Pursuing New Leads
Other concepts remain at a nascent stage. While biological phenomena, from biomolecular
production to growth and biosynthesis, have indelible “biosignatures,” it is also true that these
compounds and processes are, in essence, well-coordinated chemical reactions. Metabolically
active organisms, by necessity, maintain themselves at chemical disequilibrium from the
environment. This disequilibrium can be detected and the biogenicity of this signal assessed.
Redox reactions are typical mechanisms for terran organisms to create energy and terran life can
use organic carbon as a reductant and a diversity of soluble oxidants including oxygen, nitrate,
sulfate and carbon dioxide. An agnostic approach to life detection would not limit
bioelectrochemical observations to just these compound pairs though. Rather, disequilibrium
redox chemistries that are inconsistent with abiotic redox reactions could be used as an indicator
of active metabolism.
Many microbes can utilize an active anode as an
electron acceptor in the same way as they utilize
insoluble Fe(III) oxides as an electron acceptor
during respiration. Because the response
measured from those electrons being transferred
from organic substrates is characteristically
different and more sustained than the response
measured from electrons generated by abiotic
oxidation, this signal could be used as an
agnostic biosignature.
Figure 3. This experiment (from Nie et al.,
An illustration of these observations are the 2015) illustrates how abiotic and biological
results reported by Nie et al.15 (Figure 3). These signals are apparent even in simple cases.
simple experiments “fed” microbial communities
iron sulfide mine tailings and found a marked and sustained increase in voltage (and thus net
coulombs recovered) in the reactor with a microbial community compared to a sterile control.
These reactions can also be divorced from observations of cellular activity. Microbial extracellular
electron transfer (EET) has been observed in terran life, where organic redox-active molecules
shuttle electrons to insoluble mineral oxides16. Assuming these reactions are the basis of energy
production for life in any environment, the subsequent development of bioelectrochemical
detection instruments offer a new way to study physiology in nature. These methods and
technologies can be used in remote habitats (both well-established extraterrestrial analogs and

15
Nie et al., 2015, RSC Advances
16
Lies et al., 2005, AEM; Watanabe et
al., 2009, Current Opinion in Biotechnology

4
recently discovered habitats that are of ocean worlds relevance) to produce a robust set of
electrochemical criteria that can be used to agnostically differentiate between biological and
abiotic electrochemical reactions.

Probabilistic Approaches to Data Analysis

While it is necessary to broaden our scope and design inclusive life detection strategies, these
approaches may be less definitive than, say, uncovering a hopane or DNA sequence. A data
interpretation scheme that considers expectations and likelihoods and establishes critical
thresholds for life detection based upon probabilistic models is thereby key.
Life detection may best be viewed along a spectrum of certainty, as opposed to a binary “life”
versus “no life” model.
Modern space missions typically include packages of instruments, results of which should be
considered in tandem. Multiple inputs from multiple types of measurements can be combined to
assess certainty. It may be that no single signature will serve as unequivocal evidence for
extraterrestrial life, but rather that data from a variety of approaches will be required. For instance,
a Bayesian network for which the output is the probability there is a biosignature given the
measurement data (i.e., P(biosignature | Data) can be utilized to assess the probability of life, and
thus convert measurements into likelihoods and thresholds. The results of this data treatment
would enable the community to make recommendations for particular suites of techniques best
suited for particular types of samples. A Bayesian net could help identify complementary analyses
without redundancy. Simultaneous analysis of multiple sources of data could lead to useful higher
order likelihoods.
Expectations for abiotic signals can be set by developing challenging null models. For
instance, models of nonterran physical and physiological environments can generate a large space
of synthetic data representing a wide variety of possibilities for life. These models, which do not
pre-suppose terran chemistry, heritage, or physiology, can help the community build “life-
relevant” expectations for our collected data. Theoretical models can also inform the limits of
biology in foreign environments, anticipate necessary trade-offs indicative of alternate life
strategies, and help us to understand minimum sample sizes necessary to provide robust statistical
analysis for the results. A theoretical approach that focuses on combining inclusive principles with
physical and chemical laws to define feasibility regimes. Required sample sizes could be estimated
by “sampling” observations from an artificial “universe” where probabilities are known, and an
inference using a Bayes Net could be generated to see how close they come to the probabilities in
the model. Studies that carefully consider the abiotic mimics of biosignatures and what tools and
metrics can distinguish them from life are also of critical importance.

Conclusion
Deeper in Solar System, the likelihood that life shares a common heritage with Earth
diminishes. Thereby, a fundamental scientific question for the astrobiology community is how to
develop life detection approaches that inform our search for life without presupposing any
particular molecular framework. Agnostic biosignature detection concepts need to be advanced
individually but also joined in a unified data interpretation program informed by probabilistic
models. International partnerships among teams of biologists and chemists, computer scientists
and mathematicians, as well as planetary scientists and veteran instrument scientists will help to
ensure the astrobiology community most effectively realizes this promising research goal.