The abundance and distribution of stable isotopes of an element in a substance can provide insights regarding the source, synthesis, and environmental history of that substance. Because isotopic discrimination during chemical reactions can be unique to specific chemical pathways or environmental conditions, isotopic patterns within a substance or between related substances may provide insights into their formation. Biosynthetic pathways can create isotopic patterns that differ from patterns that arise from abiotic processes, but this is not universally true. Isotope patterns are signatures of chemical reactions, so they require additional context to be used as biosignatures. The framework of the Life Detection Knowledge Base discussed herein is used to convey arguments that support or challenge the utility of isotopic patterns for life detection. Examples of carbon and sulfur isotopic patterns in organic materials and minerals are presented to indicate how the life detection criteria “prevalence” and “signal strength” can be applied. In future work, more abiotic processes that might create false-positive life detection claims must be characterized. A broader range of microbial communities, taxa, and biomolecules should be explored for isotopic patterns. Additional elements also warrant investigation as potential isotopic biosignatures and environmental indicators. Studies of sedimentary macromolecular organic matter should be expanded further to provide deeper insights into isotopic abundance patterns.
Get full access to this article
View all access options for this article.
References
1.
AnbarAD. Iron stable isotopes: Beyond biosignatures. Earth Planet Sci Lett, 2004; 217:223–236; doi: 10.1016/S0012-821X(03)00572-7
2.
AponteJC, ElsilaJE, HeinJE, et al.Analysis of amino acids, hydroxy acids, and amines in CR chondrites. Meteorit Planet Sci, 2020; 55(11):2422–2439; doi: 10.1111/maps.13586
BlairN, LeuA, MuñozE, et al.Carbon isotopic fractionation in heterotrophic microbial metabolism. Appl Environ Microbiol, 1985; 50(4):996–1001; doi: 10.1128/aem.50.4.996-1001.1985
6.
BryantRN, HoughtonJI, JonesC, et al.Deconvolving microbial and environmental controls on marine sedimentary pyrite sulfur isotope ratios. Science, 2023; 382(6673):912–915; doi: 10.1126/science.adg6103
7.
CalderJA, ParkerPL. Geochemical implications of induced changes in C13 fractionation by blue-green algae. Geochim Cosmochim Acta, 1978; 37:133–140; doi: 10.1016/0016-7037(73)90251-2
8.
CanfieldDE, RaiswellR. The evolution of the sulfur cycle. Am J Sci, 1999; 299(7–9):697–723; doi: 10.2475/ajs.299.7-9.697
9.
CaponeDG, PopaR, FloodB, et al.Geochemistry: Follow the nitrogen. Science, 2006; 312(5774):708–709; doi: 10.1126/science.1111863
10.
CaiC, LiH, LiK, et al.Thermochemical sulfate reduction in sedimentary basins and beyond: A review. Chem Geol, 2022:607; doi: 10.1016/j.chemgeo.2022.121018
11.
ChanDI, VogelHJ. Current understanding of fatty acid biosynthesis and the acyl carrier protein. Biochem J, 2010; 430(1):1–19; doi: 10.1042/BJ20100462
12.
ChanMA, HinmanNW, Potter-McIntyreSL, et al.Deciphering biosignatures in planetary contexts. Astrobiology, 2019; 19(9):1075–1102; doi: 10.1089/ast.2018.1903
13.
ChimiakL, EilerJ, SessionsA, et al.Isotope effects at the origin of life: Fingerprints of the Strecker synthesis. Geochim Cosmochim Acta, 2022; 321:78–98; doi: 10.1016/j.gca.2022.01.015
14.
ClarkeA. The thermal limits to life on Earth. Intl J Astrobiology, 2014; 13(2):141–154; doi: 10.1017/S1473550413000438
15.
ColmanA, BlakeRE, KarlDM, et al.Marine phosphate oxygen isotopes and organic matter remineralization in the oceans. Proc Natl Acad Sci U S A, 2005; 102(37):13023–13028; doi: 10.1073/pnas.0506455102
16.
CroninJR, PizzarelloS, EpsteinS, et al.Molecular and isotopic analyses of the hydroxy acids, dicarboxylic acids, and hydroxydicarboxylic acids of the Murchison meteorite. Geochim Cosmochim Acta, 1993; 57(19):4745–4752; doi: 10.1016/0016-7037(93)90197-5
17.
DeNiroMJ, EpsteinS. Mechanism of carbon isotope fractionation associated with lipid synthesis. Science, 1977; 197(4300):261–263; doi: 10.1126/science.327543
18.
Des MaraisDJ. Isotopic evolution of the biogeochemical carbon cycle during the Proterozoic Eon. Org Geochem, 1997; 27:185–193; doi: 10.1016/S0146-6380(97)00061-2
19.
Des MaraisDJ, NuthJA, AllamandolaLJ, et al.The NASA Astrobiology Roadmap. Astrobiology, 2008; 8(4):715–730; doi: 10.1089/ast.2008.0819
20.
Des MaraisDJ. Isotopic evolution of the biogeochemical carbon cycle during the Precambrian. In: Stable Isotope Geochemistry (ValleyJW, ColeDR., eds.) Rev. Mineral; 2001; pp. 555–578; doi: 10.2138/gsrmg.43.1.555
21.
EglintonTI. Carbon isotopic evidence for the origin of macromolecular aliphatic structures in kerogen. Org Geochem, 1994; 21(6–7):721–735; doi: 10.1016/0146-6380(94)90015-9
22.
EigenbrodeJL, SummonsRE, SteeleA, et al.Organic matter preserved in 3-billion-year-old mudstones at Gale crater, Mars. Science, 2018; 360(6393):1096–1101; doi: 10.1126/science.aas9185
23.
ElsilaJE, DworkinJP, BernsteinMP, et al.Mechanisms of amino acid formation in interstellar ice analogs. Astrophys J, 2007; 660(1):911; doi: 10.1086/513141
24.
ElsilaJE, CharnleySB, BurtonAS, et al.Compound‐specific carbon, nitrogen, and hydrogen isotopic ratios for amino acids in CM and CR chondrites and their use in evaluating potential formation pathways. Meteorit Planet Sci, 2012; 47(9):1517–1536; doi: 10.1111/j.1945-5100.2012.01415.x
FoxAC, EigenbrodeJL, FreemanKH. Radiolysis of macromolecular organic material in Mars‐relevant mineral matrices. J Geophys Res Planets, 2019; 124(12):3257–3266; doi: 10.1029/2019JE006072
27.
FischerWW, FikeDA, JohnsonJE, et al.SQUID–SIMS is a useful approach to uncover primary signals in the Archean sulfur cycle. Proc Natl Acad Sci U S A, 2014; 111(15):5468–5473; doi: 10.1073/pnas.1322577111
28.
FryB, GestH, HayesJM. Isotope effects associated with the anaerobic oxidation of sulfite and thiosulfate by the photosynthetic bacterium, Chromatium vinosum. FEMS Microbiol Lett, 1985; 27(2):227–232; doi: 10.1111/j.1574-6968.1985.tb00672.x
29.
FryB, RufW, GestH, et al.Sulfur isotopes effects associated with oxidation of sulfides by O2 in aqueous solution. Isot Geosci, 1988; 73:205–210; doi: 10.1016/0168-9622(88)90001-2
30.
GarciaAK, CavanaughCM, KacarB. The curious consistency of carbon biosignatures over billions of years of Earth-life coevolution. ISME J, 2021; 15(8):2183–2194; doi: 10.1038/s41396-021-00971-5
31.
GillenC, JeancolasC, McMahonS, et al.The call for a new definition of biosignature. Astrobiology, 2023; 23(11):1228–1237.
32.
GlavinDP, ElsilaJE, McLainHL, et al.Extraterrestrial amino acids and L‐enantiomeric excesses in the CM 2 carbonaceous chondrites Aguas Zarcas and Murchison. Meteorit Planet Sci, 2020; 56(1):148–173; doi: 10.1111/maps.13451
33.
GreenwoodRC, FranchiIA. Alteration and metamorphism of CO3 chondrites: Evidence from oxygen and carbon isotopes. Meteorit Planet Sci, 2004; 39:1823–1838; doi: 10.1111/j.1945-5100.2004.tb00078.x
34.
GrotzingerJP, SumnerDY, KahLC, et al.; MSL Science Team. A habitable fluvio-lacustrine environment at Yellowknife Bay, Gale Crater, Mars. Science, 2014; 343(6169):1242777; doi: 10.1126/science.1242777
35.
HabichtKS, CanfieldDE. Sulfur isotope fractionation during bacterial sulfate reduction in organic-rich sediments. Geochim Cosmochim Acta, 1997; 61(24):5351–5361; doi: 10.1016/S0016-7037(97)00311-6
36.
HaghnegahdarMA, SunJ, HultquistN, et al.Tracing sources of atmospheric methane using clumped isotopes. Proc Natl Acad Sci U S A, 2023; 120(47); doi: 10.1073/pnas.2305574120
37.
HalevyI, JohnstonDT, SchragDP. Explaining the structure of the Archean mass-independent sulfur isotope record. Science, 2010; 329(5988):204–207; doi: 10.1126/science.1190298
38.
HalevyI, FikeDA, PasquierV, et al.Sedimentary parameters control the sulfur isotope composition of marine pyrite. Science, 2023; 382(6673):946–951; doi: 10.1126/science.adh1215
39.
HayesJM, WedekingKW, KaplanIR. Precambrian organic geochemistry, preservation of the record. In: Earth’s Earliest Biosphere (SchopfJW, ed.) Princeton University Press: Princeton, NJ; 1983, pp. 93–134.
40.
HayesJM, PoppBN, TakigikuR, et al.An isotopic study of biogeochemical relationships between carbonates and organic carbon in the Greenhorn Formation. Geochim Cosmochim Acta, 1989; 53(11):2961–2972; doi: 10.1016/0016-7037(89)90172-5
41.
HayesJM. Factors controlling 13C contents of sedimentary organic compounds: Principles and evidence. Marine Geol, 1993; 113(1–2):111–125; doi: 10.1016/0025-3227(93)90153-M
42.
HayesJM. Fractionation of carbon and hydrogen isotopes in biosynthetic processes. In: Stable Isotope Geochemistry (ValleyJW, ColeDR, eds.) Rev Mineral; 2001; 43; pp. 225–277; doi: 10.2138/gsrmg.43.1.225
43.
HurleySJ, WingBA, JasperCE, et al.Carbon isotope evidence for the global physiology of Proterozoic cyanobacteria. Sci Adv, 2021; 7(2):eabc8998; doi: 10.1126/sciadv.abc8998
44.
JohnstonDT, FarquharJ, WingBA, et al.Multiple sulfur isotope fractionations in biological systems: A case study with sulfate reducers and sulfur disproportionators. Am J Sci, 2005; 305:645–660; doi: 10.2475/ajs.305.6-8.645
45.
JohnstonDT, FarquharJ, HabichtKS, et al.Sulphur isotopes and the search for life: Strategies for identifying sulphur metabolisms in the rock record and beyond. Geobiology, 2008; 6:425–435; doi: 10.1111/j.1472-4669.2008.00171.x
46.
KamberBS, WhitehouseMJ. Micro‐scale sulphur isotope evidence for sulphur cycling in the late Archean shallow ocean. Geobiology, 2007; 5(1):5–17; doi: 10.1111/j.1472-4669.2006.00091.x
47.
KaplanIR, RittenbergSC. Microbiological fractionation of sulphur isotopes. J Gen Microbiol, 1964; 34(2):195–212; doi: 10.1099/00221287-34-2-195
48.
KokhMA, AssayagN, MounicS, et al.Multiple sulfur isotope fractionation in hydrothermal systems in the presence of radical ions and molecular sulfur. Geochim Cosmochim Acta, 2020; 285:100–128; doi: 10.1016/j.gca.2020.06.016
49.
Krissansen-TottonJ, BuickR, CatlingDC. A statistical analysis of the carbon isotope record from the Archean to Phanerozoic and implications for the rise of oxygen. Am J Sci, 2015; 315(4):275–316; doi: 10.2475/04.2015.01
50.
LancetMS, AndersE. Carbon isotope fractionation in the Fischer-Tropsch synthesis and in meteorites. Science, 1970; 170(3961):980–982; doi: 10.1126/science.170.3961.980
51.
LeavittWD, HalevyI, BradleyAS, et al.Influence of sulfate reduction rates on the Phanerozoic sulfur isotope record. Proc Natl Acad Sci U S A, 2013; 110(28):11244–11249; doi: 10.1073/pnas.1218874110
52.
LloydMK, McClellandHLO, AntlerG, et al.The isotopic imprint of life on an evolving planet. Space Sci Rev, 2020; 216:1–54; doi: 10.1007/s11214-020-00730-6
53.
LoveGD, StalviesC, GrosjeanE, et al.Analysis of molecular biomarkers covalently bound within Neoproterozoic sedimentary kerogen: In: From Evolution to Geobiology: Research Questions Driving Paleontology at the Start of a New Century (KelleyPH, BambachRK, eds.) Paleontological Society Short Course, Paleontological Society Papers; 2008; pp. 67–83; doi: 10.1017/S1089332600001613
54.
MackoSA, FogelML, HarePE, et al.Isotopic fractionation of nitrogen and carbon in the synthesis of amino acids by microorganisms. Chem Geol (Isotope Geosci Sect), 1987; 65(1):79–92; doi: 10.1016/0168-9622(87)90064-9
55.
MarshallSM, MathisC, CarrickE, et al.Identifying molecules as biosignatures with assembly theory and mass spectrometry. Nat Commun, 2021; 12(1):3033; doi: 10.1038/s41467-021-23258-x
56.
MeadowsV, GrahamH, AbrahamssonV, et al.Community report from the biosignatures standards of evidence workshop. arXiv Preprint, 2022; doi: 10.4855/0/arXiv.2210.14293
57.
McCollomTM, SeewaldJS. Carbon isotope composition of organic compounds produced by abiotic synthesis under hydrothermal conditions. Earth Planet Sci Lett, 2006; 243(1–2):74–84; doi: 10.1016/j.epsl.2006.01.027
58.
McDermottJM, OnoS, TiveyMK, et al.Identification of sulfur sources and isotopic equilibria in submarine hot springs using multiple sulfur isotopes. Geochim Cosmochim Acta, 2015; 160:169–187; doi: 10.1016/j.gca.2015.02.016
59.
MillerHM, ChaudhryN, ConradME, et al.Large carbon isotope variability during methanogenesis under alkaline conditions. Geochim Cosmochim Acta, 2018; 237:18–31; doi: 10.1016/j.gca.2018.06.007
60.
MonsonKD, HayesJM. Carbon isotopic fractionation in the biosynthesis of bacterial fatty acids. Ozonolysis of unsaturated fatty acids as a means of determining the intramolecular distribution of carbon isotopes. Geochim Cosmochim Acta, 1982; 46(2):139–149; doi: 10.1016/0016-7037(82)90241-1
61.
Moreras-MartiA, Fox-PowellM, StueekenE, et al.Quadruple sulfur isotope biosignatures from terrestrial Mars analogue systems. Geochim Cosmochim Acta, 2021; 308:157–172; doi: 10.1016/j.gca.2021.06.007
62.
MuyzerG, StamsAJ. The ecology and biotechnology of sulphate-reducing bacteria. Nat Rev Microbiol, 2008; 6(6):441–454; doi: 10.1038/nrmicro1892
63.
NeveuM, HaysLE, VoytekA, et al.The ladder of life detection. Astrobiology, 2018; 18(11):1375–1402; doi: 10.1089/ast.2017.1773
64.
PaisteK, FikeDA, MayikaKB, et al.Sulfur isotopes from the Paleoproterozoic Francevillian Basin record multigenerational pyrite formation, not depositional conditions. Commun Earth Environ, 2024; 5(1):328; doi: 10.1038/s43247-024-01498-1
65.
ParenteauN, HoehlerT, DavilaA, et al.Life detection knowledge base: assessment criteria for potential biosignatures. Astrobiology, 2025; 25(7):474–481; doi: 10.1089/ast.2022.0104
66.
PasquierV, SansjofreP, RabineauM, et al.Pyrite sulfur isotopes reveal glacial-interglacial environmental changes. Proc Natl Acad Sci U S A, 2017; 114(23):5941–5945; doi: 10.1073/pnas.1618245114
67.
PizzarelloS, ShockE. The organic composition of carbonaceous meteorites: the evolutionary story ahead of biochemistry. Cold Spring Harb Perspect Biol, 2010; 2(3):a002105; doi: 10.1101/cshperspect.a002105
68.
PohorilleA, LauG, GliniewiczS, et al.The life detection knowledge base: A community tool for knowledge management and representation. Astrobiology, 2025; 25(7):454–463; doi: 10.1089/ast.2024.0106
69.
PoserA, VogtC, KnöllerK, et al.Stable sulfur and oxygen isotope fractionation of anoxic sulfide oxidation by two different enzymatic pathways. Environ Sci Technol, 2014; 48(16):9094–9102; doi: 10.1021/es404808r
70.
RasmussenC, HoffmanDW. Intramolecular distribution of 13C/12C isotopes in amino acids of diverse origins. Amino Acids, 2020; 52(6–7):955–964; doi: 10.1007/s00726-020-02863-y
71.
RoyerDL, BernerRA, BeerlingDJ. Phanerozoic atmospheric CO2 change: Evaluating geochemical and paleobiological approaches. Earth-Sci Rev, 2001; 54(4):349–392; doi: 10.1016/S0012-8252(00)00042-8
72.
SaltzmanMR, ThomasE, GradsteinFM, et al.Carbon Isotope Stratigraphy. The Geologic Time Scale, 2012; 1:207–232; doi: 10.1016/B978-0-444-59425-9.00011-1
73.
SavidgeWB, BlairNE. Patterns of intramolecular carbon isotopic heterogeneity within amino acids of autotrophs and heterotrophs. Oecologia, 2004; 139(2):178–189; doi: 10.1007/s00442-004-1500-z
74.
SchidlowskiM. Carbon isotopes as biogeochemical recorders of life over 3.8 Ga of Earth history: Evolution of a concept. Precamb Res, 2001; 106(1–2):117–134; doi: 10.1016/S0301-9268(00)00128-5
75.
ScottJH, O’BrienDM, EmersonD, et al.An examination of the carbon isotope effects associated with amino acid biosynthesis. Astrobiology, 2006; 6(6):867–880; doi: 10.1089/ast.2006.6.867
SephtonMA, WatsonJS, MeredithW, et al.Multiple cosmic sources for meteorite macromolecules? Astrobiology, 2015; 15(10):779–786; doi: 10.1089/ast.2015.1331
78.
ShuaiY, XieH, ZhangS, et al.Recognizing the pathways of microbial methanogenesis through methane isotopologues in the subsurface biosphere. Earth Planet Sci Lett, 2021; 566:116960; doi: 10.1016/j.epsl.2021.116960
79.
SimMS, BosakT, OnoS. Large sulfur isotope fractionation does not require disproportionation. Science, 2011; 333(6038):74–77; doi: 10.1126/science.1205103
80.
SimMS, OnoS, BosakT. Effects of iron and nitrogen limitation on sulfur isotope fractionation during microbial sulfate reduction. Appl Environ Microbiol, 2012; 78(23):8368–8376; doi: 10.1128/AEM.01842-12
81.
SimMS, OgataH, LubitzW, et al.Role of APS reductase in biogeochemical sulfur isotope fractionation. Nat Commun, 2019; 10(1):44; doi: 10.1038/s41467-018-07878-4
SimonetBR, BrennerS, PetersKE, et al.Thermal alteration of cretaceous black shale by diabase intrusions in the Eastern Atlantic—II. Effects on bitumen and kerogen. Geochim Cosmochim Acta, 1981; 45(9):1581–1602; doi: 10.1016/0016-7037(81)90287-8
84.
ShenJ, ZerkleAL, ClaireMW. Nitrogen cycling and biosignatures in a hyperarid Mars analog environment. Astrobiology, 2022; 22(2):127–142; doi: 10.1089/ast.2021.0012
85.
StamMC, MasonPR, PalludC, et al.Sulfate reducing activity and sulfur isotope fractionation by natural microbial communities in sediments of a hypersaline soda lake (Mono Lake, California). Chem Geol, 2010; 278(1–2):23–30; doi: 10.1016/j.chemgeo.2010.08.006
86.
TeeceMA, FogelML, DollhopfME, et al.Isotopic fractionation associated with biosynthesis of fatty acids by a marine bacterium under oxic and anoxic conditions. Org Geochem, 1999; 30(12):1571–1579; doi: 10.1016/S0146-6380(99)00108-4
87.
VickersP, CowieC, DickSJ, et al.Confidence of life detection: The problem of unconceived alternatives. Astrobiology, 2023; 23(11):1202–1212; doi: 10.1089/ast.2022.0084
88.
VeizerJ, AlaD, AzmyK, et al.87Sr/86Sr, δ13C and δ18O evolution of Phanerozoic seawater. Chem Geol, 1999; 161(1–3):59–88; doi: 10.1016/S0009-2541(99)00081-9
89.
WingBA, HalevyI. Intracellular metabolite levels shape sulfur isotope fractionation during microbial sulfate respiration. Proc Natl Acad Sci U S A, 2014; 111(51):18116–18125; doi: 10.1073/pnas.1407502111
YuenG, BlairN, Des MaraisDJ, et al.Carbon isotope composition of low molecular weight hydrocarbons and monocarboxylic acids from Murchison meteorite. Nature, 1984; 307(5948):252–254; doi: 10.1038/307252a0
92.
ZerkleAL, FarquharJ, JohnstonT, et al.Fractionation of multiple sulfur isotopes during phototrophic oxidation of sulfide and elemental sulfur by a green sulfur bacterium. Geochim Cosmochim Acta, 2009; 73(2):291–306; doi: 10.1016/j.gca.2008.10.027
93.
ZhangZ, SachsJ, MarchettiA. Hydrogen isotope fractionation in freshwater and marine algae: II. Temperature and nitrogen limited growth rate effects. Org Geochem, 2009; 40(3):428–439; doi: 10.1016/j.orggeochem.2008.11.002
94.
ZyakunAM, LuninaON, PrusakovaTS, et al.Fractionation of stable carbon isotopes by photoautotrophically growing anoxygenic purple and green sulfur bacteria. Microbiology, 2009; 78:757–768; doi: 10.1134/S0026261709060137
