Ammonia as a parameter shaping habitability on icy moons.

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Learn more: PMC Disclaimer | PMC Copyright Notice FEMS Microbes . 2026 Mar 26;7:xtag015. doi: 10.1093/femsmc/xtag015 Search in PMC Search in PubMed View in NLM Catalog Add to search Ammonia as a parameter shaping habitability on icy moons Cassie M Hopton Cassie M Hopton 1 UK Centre for Astrobiology, School of Physics and Astronomy, University of Edinburgh, James Clerk Maxwell Building, Peter Guthrie Tait Road, Edinburgh EH9 3FD, United Kingdom Find articles by Cassie M Hopton 1, ✉ , Charles S Cockell Charles S Cockell 2 UK Centre for Astrobiology, School of Physics and Astronomy, University of Edinburgh, James Clerk Maxwell Building, Peter Guthrie Tait Road, Edinburgh EH9 3FD, United Kingdom Find articles by Charles S Cockell 2 Editor: Michael Macey Author information Article notes Copyright and License information 1 UK Centre for Astrobiology, School of Physics and Astronomy, University of Edinburgh, James Clerk Maxwell Building, Peter Guthrie Tait Road, Edinburgh EH9 3FD, United Kingdom 2 UK Centre for Astrobiology, School of Physics and Astronomy, University of Edinburgh, James Clerk Maxwell Building, Peter Guthrie Tait Road, Edinburgh EH9 3FD, United Kingdom ✉ Corresponding author. UK Centre for Astrobiology, School of Physics and Astronomy, University of Edinburgh, James Clerk Maxwell Building, Peter Guthrie Tait Road, Edinburgh, EH9 3FD, United Kingdom. E-mail: [email protected] Roles Michael Macey : Editor Received 2025 Dec 30; Revised 2026 Mar 2; Accepted 2026 Mar 18; Collection date 2026. © The Author(s) 2026. Published by Oxford University Press on behalf of FEMS. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. PMC Copyright notice PMCID: PMC13094550  PMID: 42016463 Abstract The search for life now extends beyond the traditional habitable zone to include the icy moons of Jupiter and Saturn. These moons feature ice-covered surfaces overlying substantial oceans formed primarily of liquid water and other potential constituents, such as ammonia. On Earth, ammonia supports biochemistry at low concentrations by providing nitrogen but becomes disruptive at higher concentrations. Ammonia could therefore influence the habitability of extraterrestrial oceans, yet this topic has received limited attention in the literature. This review synthesises current research on ammonia in Saturn’s icy moons, Enceladus and Titan, and its effects on terrestrial life. We summarize the celestial incorporation, speciation, and phase behaviour of ammonia and review data on its occurrence and concentration in icy moon oceans. We examine the role of ammonia in prebiotic chemistry, biochemistry, and toxicity. Focusing on bacteria, we compare known survival limits in ammonia to estimated ammonia concentrations on Enceladus and Titan. We find that bacterial survival limits exceed concentrations estimated on Enceladus, but are below those estimated on Titan, and propose that ammonia measurements are crucial for assessing extraterrestrial habitability. Finally, we highlight outstanding knowledge gaps and challenges that influence our understanding of how ammonia shapes the potential for life beyond Earth. Keywords: astrobiology, icy moons, Enceladus, Titan, ammonia, ocean worlds This review explores how ammonia in the subsurface oceans of Saturn’s icy moons, Enceladus and Titan, could shape conditions for life and influence their potential to host microbes. Introduction A habitable environment can be defined as “an environment capable of supporting the activity of at least one known organism” (Cockell et al. 2016 ). The question of habitability beyond Earth has been the subject of both philosophical and scientific interest for two millennia. As early as the 6 th century BCE, Greek philosopher Anaximander speculated about multiple worlds that may harbour extraterrestrial life, and a water origin of human life on Earth (Rovelli 2023 ). This early curiosity evolved with scientific discovery. In the 17 th century, four moons of Jupiter (Io, Europa, Ganymede, and Callisto) were discovered by Galileo and subsequently dubbed the “Galilean moons” (Soderblom 1980 ), a discovery that expanded the known celestial bodies in the Solar System. By the 20 th century, the role of water in the emergence of life became evident. Stanley Miller and Harold Urey demonstrated that amino acids could form from a mixture of ammonia, methane, hydrogen, and water (Miller 1955 ). This experiment provided evidence for Oparin’s theory of abiogenesis, a “primordial soup” origin of life hypothesis whereby a mixture of inorganic compounds in water gave rise to living organisms (Oparin 1938 ). Almost two decades later, the first physical evidence of extraterrestrial water came from water-bearing minerals detected in lunar samples returned by Apollo 11 (Goles 1971 , Levinson and Taylor 1971 ). Building on this discovery, the Galileo mission subsequently provided data to support a subsurface ocean of liquid water below the thick ice crust of one of the Galilean moons, Europa (Reynolds et al. 1983 , Anderson et al. 1998 , Greeley et al. 1998 ). At the turn of the 21 st century, scientific attention to extraterrestrial water continued, and NASA introduced a new approach as part of the Mars Exploration Program: “Follow the water” (Hubbard et al. 2002 ). The approach was designed initially to discover records of biological processes on Mars. However, in 2005, the Cassini–Huygens mission revealed plumes of water vapour and ice erupting from Saturn’s moon Enceladus (Hunter et al. 2006 ). Now, strong evidence for subsurface oceans of liquid water encased below surface ice is established not only on Europa and Enceladus (Roberts and Nimmo 2008 , Tobie et al. 2008 , Miles et al. 2025 ), but also Ganymede (Showman et al. 2004 , Saur et al. 2015 ), Callisto (Zimmer et al. 2000 , Cochrane et al. 2025 ), Neptune’s moon Triton (Gaeman et al. 2012 ), and the Dwarf planet Ceres, amongst others (McCord and Sotin 2005 ). Saturn’s largest moon, Titan, could also harbour a subsurface liquid ocean (Gabriel et al. 2005 , Baland et al. 2011 , Bills and Nimmo 2011 ) or a high-pressure ice layer containing pockets of partial melt (Petricca et al. 2025 ). With these discoveries, the “follow the water” approach now applies not only to Mars but also to the icy moons of Jupiter, Saturn, and other ocean-bearing planetary bodies across the universe. Beyond the frost line, far from the Sun’s warming influence, the preservation of a liquid layer within icy moons and other celestial bodies could appear paradoxical. Yet, such layers are possible through tidal or radiogenic heating, or the presence of freezing point depressants such as salt or ammonia. Indeed, temperatures are low enough beyond the frost line for volatile compounds like ammonia to condense into solid ice and accrete with planetary bodies. As such, ammonia has been detected in the plumes of Enceladus by Cassini (Waite et al. 2009 ) and on the surface of Ceres (King et al. 1992 ). When considering astrobiology, the presence of such a constituent within the oceans of extraterrestrial bodies would be significant. On Earth, ammonia has been implicated in facilitating prebiotic chemistry as a nitrogen source (Wigley and Brimblecombe 1981 , Martin et al. 2008 , Sojo et al. 2016 , Nishizawa et al. 2021 ), and in modern systems, plays an integral role in the global nitrogen cycle (Fowler et al. 2013 ) and the synthesis of nucleic acids and amino acids (Adeniyi et al. 2023 , Fu et al. 2023 ). However, high concentrations of ammonia are also known to be toxic across all the domains of life (Eno et al. 1955 , Thurston et al. 1981 , Leejeerajumnean et al. 2000 , Hachiya et al. 2021 ). On Earth, what constitutes “high” concentrations of ammonia is not well defined, but could be considered those that exceed 6 ppm (≈ 0.00035 M) in waters and soils (Roney et al. 2004 ). Ammonia could therefore be a prominent factor influencing habitability as we know it if present within icy moon subsurface oceans above this level. Ammonia can exist in two molecular forms: a weak base, ammonia (NH 3 ), and a weak acid, the ammonium ion (NH 4 + ). Henceforth, the term “total ammonia” refers collectively to the concentration of both NH 3 and NH 4 + in an aqueous environment. The position of equilibrium between these two species is dictated primarily by pH; under high pH, NH 3 dominates, and under low pH, NH 4 + dominates. While NH 4 + can disrupt the ionic balance of organisms, the permeation of this species is regulated by membrane transporters (Moser 1987 , Wacker et al. 2014 ), and the tolerance of organisms to NH 4 + is high (i.e., up to and possibly beyond 2 M). For these reasons, NH 4 + is characterized as “non-toxic”. NH 3 is a proton (H + ) acceptor, and has properties (e.g., small size, uncharged) which facilitate passive permeation through biological membranes. As a result, NH 3 readily reacts with H + to raise both extracellular and intracellular pH, disrupt the proton motive force, and form NH 4 + intracellularly (Rose et al. 2005 , Bosoi and Rose 2009 , Angelova et al. 2022 ). High concentrations of NH 4 + delivered through NH 3 permeation can disrupt membrane potential, ionic balance, and metabolism (Wang et al. 2018 , Xiao et al. 2019 , Shi et al. 2020 ). NH 3 is therefore considered toxic and has the potential to significantly disrupt the physicochemical environment as well as the biochemistry within living organisms. To date, 0.5 M is the highest concentration at which a bacterium has been observed to survive in NH 3 (Leejeerajumnean et al. 2000 ). Given these effects, the occurrence of NH 3 may thus limit the habitability potential of extraterrestrial oceans. Focusing on icy moons, it is not yet known if all of these oceans contain significant amounts of NH 3 , if any. Owing to the thermal conditions of the Jovian nebula and current geochemical models of Europa’s ocean, it is unlikely that NH 3 predominates there. Ganymede and Callisto may harbour subsurface waters containing NH 3 , but available compositional constraints remain insufficient to draw firm conclusions. The ocean of Enceladus is estimated to have a pH of ∼10.6 (Glein and Truong 2025 ), and Titan’s ocean may be of a similarly high pH, or greater (Marion et al. 2012 , Brassé et al. 2017 , Leitner and Lunine 2019 ). Both oceans are expected to contain dissolved NH 3 and/or NH 4 + based on current interior and thermal evolution models. For these reasons, this review focuses on Enceladus and Titan, where well-constrained formation and physicochemical conditions are most conducive to elevated NH 3 abundances. Despite the possibility of NH 3 in the oceans, there are several properties that underpin Enceladus and Titan as prominent targets in the search for life beyond Earth. First, in addition to liquid water, there is evidence of tidal heating (Carr et al. 1998 , Nimmo et al. 2007 , Roberts and Nimmo 2008 , Chen et al. 2014 , Běhounková et al. 2021 ) or radiogenic heating (Grasset et al. 2000 , Sohl et al. 2014 ) within both icy moons. Hydrothermal chemistries at the ocean floor of Enceladus are conceivable (Hand et al. 2007 , Matson et al. 2007 , Vance et al. 2007 , Zolotov Mikhail Y. 2007 , Hsu et al. 2015 ), akin to the origin-of-life hydrothermal vents on Earth (Hand et al. 2007 , Matson et al. 2007 , Vance et al. 2007 , Zolotov Mikhail 2007 ). Redox chemistry is plausible on Titan (McKay and Smith 2005 , McKay 2016 ). Additionally, all the essential elements for life on Earth, the CHNOPS suite (carbon, hydrogen, nitrogen, oxygen, phosphorus, sulphur), have been detected or are otherwise feasible on Enceladus (Waite et al. 2009 , Waite et al. 2017 , Postberg et al. 2023 , Xu et al. 2025 ). Many of the CHNOPS have also been detected on Titan (Sagan et al. 1992 , Hiscox 2000 , Owen 2000 , Nixon 2024 ). Organics have been directly detected in the plume of Enceladus (Waite et al. 2009 , Postberg et al. 2018 , Khawaja et al. 2025 ), and in the atmosphere of Titan (Lellouch et al. 1989 , Niemann et al. 2005 ). Pressures in the ocean of Enceladus could range from 1 to 30 MPa (Vance et al. 2018 ) and could reach 800 MPa on Titan at the seafloor (Journaux et al. 2020 ). While this may limit habitability prospects to piezotolerant and piezophilic organisms, it is the combination of available heat, energy, nutrients, and organics, in addition to liquid water, that makes these icy moons compelling targets in astrobiology (Fortes 2000 , McKay et al. 2008 ). The potential for habitability on Enceladus and Titan has been widely speculated given the favourable internal conditions presented. However, even with its potential impact on life, the significance of NH 3 is little discussed or represented in astrobiology literature. This interdisciplinary review synthesises current research on NH 3 in the high pH oceans of Saturn’s icy moons, Enceladus and Titan, and examines its effects on terrestrial life. We discuss the geochemical occurrence, chemical behaviour, and biological impacts of total ammonia on Earth, drawing implications for the habitability of these extraterrestrial, ammoniacal oceans. Origin, speciation, and phase behaviour of ammonia NH 3 is a primordial molecule. It is found in celestial bodies across the universe, including stars (Schmidt et al. 2011 , Wong et al. 2018 ), planets (Henderson-Sellers and Schwartz 1980 , Cleland and Rimmer 2022 , Moeckel et al. 2023 , Irwin et al. 2025 ), moons (Nelson et al. 2009 , Waite et al. 2009 , Holler et al. 2017 ), comets (Wyckoff et al. 1989 , Feldman et al. 1993 , Poch et al. 2020 ), asteroids (Pizzarello et al. 2011 , Glavin et al. 2025 ), and distant galaxies (Ao et al. 2011 , Mills and Morris 2013 ), as well as the interstellar medium (Cheung et al. 1968 , Doherty et al. 2022 ). In the near vacuum pressures of space, NH 3 is found primarily as solid ice. Temperatures are below the sublimation point (−73°C) (Glasser 2009 ) and NH 3 ice can be trapped during accretion of cold bodies, including icy moons. Within icy moons, subsequent internal heating can later release NH 3 as a gas as subsurface oceans form (Spohn and Schubert 2003 , Tobie et al. 2012 , Vance et al. 2018 ). NH 3 gas readily dissolves in water (Hales and Drewes 1979 , Dasgupta and Dong 1986 , Simonelli et al. 1998 ). The resulting ammoniacal waters, a mixture of NH 3 and water, can have a freezing point as low as −100°C; dissolution of NH 3 in water interferes with water–water hydrogen bonding, thus acting to lower the freezing point. This antifreeze behaviour has led to the speculation that ammonia–water may maintain subsurface liquid oceans in icy moons, despite cold surface temperatures (Johnson and Nicol 1987 , Croft et al. 1988 , Leliwa-Kopystyński et al. 2002 , Chua et al. 2023 ). Within aqueous environments, NH 3 undergoes a particular reaction. As a H + acceptor, the dissolution of NH 3 into water leads to protonation and the formation of NH 4 + and hydroxide ions (OH − ), according to the following equation: The position of equilibrium between NH 3 and NH 4 + in water is dictated primarily by pH as described by the Henderson–Hasselbalch equation, where low pH promotes the formation of NH 4 + and high pH promotes the formation of NH 3 . However, the speciation of total ammonia is also influenced by salinity, temperature, and pressure. Higher salinity, colder temperatures, and elevated pressures increase the negative logarithm of the acid dissociation constant (pK a ) of the equilibrium, thus favouring the protonation of NH 3 (forming NH 4 + ). Lower salinity, warmer temperatures, and lower pressure exert the opposite effect (Neuhausen and Patrick 1921 , Emerson et al. 1975 , Hales and Drewes 1979 , Dasgupta and Dong 1986 ). Thus, the speciation of total ammonia in icy moon interiors is dictated by these physicochemical parameters (Fig. 1 ). Figure 1. Open in a new tab Abundance of NH 3 and NH 4 + as a function of pH. The pK a of the system was calculated based upon an ionic strength of 0.3 M, a temperature of 0°C, and 0.1 MPa pressure. Pressure may vary, but these ionic strengths and temperatures are parameters expected within the ocean of Enceladus (Glein and Truong 2025 ). A pK a of 10.1 was calculated, indicated by a dotted line from the x -axis. In addition to speciation, pressure can also alter the phase behaviour of NH 3 . On icy moons, gravitational forces acting on thick water layers, combined with the substantial thickness of ice shells in the order of several to hundreds of kilometres, generate hydrostatic pressures of tens to a few hundred MPa at the seafloor, and up to a few GPa at the seafloor of larger moons (Billings and Kattenhorn 2005 , Nimmo and Bills 2010 , Baland et al. 2014 , Čadek et al. 2016 , Lucchetti et al. 2017 , Vance et al. 2018 , Levin et al. 2026 ). In mixtures of ammonia–water at standard pressure, the boiling point can remain above 0°C and may only be depressed by a few degrees from the standard boiling point of water (100°C) when mixed at low concentrations of NH 3 , i.e., 1% NH 3 . However, elevated pressures can increase the boiling point, and also alter the freezing point (Clifford and Hunter 1933 ). For example, at 100 MPa pressure, the freezing point of ammonia–water with a concentration of 1% NH 3 by weight is approximately −9°C, slightly colder than the freezing point at 0.1 MPa (−3°C) (Fig. 2 ). At 300 MPa, the freezing point temperature decreases substantially to −33°C, and so on (Leliwa-Kopystyński et al. 2002 ). Figure 2. Open in a new tab Phase diagram of ammonia–water mixtures. Phase behaviour against the concentration-temperature plane is depicted, showing the influence of pressures at 0.1 MPa, 100 MPa, 200 MPa, and 300 MPa. The ammonia–water solidus at 176.16 K is illustrated with a solid line. A dashed line (red) has been superimposed on the figure to highlight phase behaviour at a temperature of 273.15 K (0°C). Figure adapted from Hogenboom et al. ( 1997 ) with permission from Elsevier. Current models estimate that the temperature at the base of the ice shell on icy moons ranges from −20 °C to near 0°C, although subsurface oceans could be warmer near hydrothermal regions or exhibit inverted temperature gradients (Kargel et al. 2000 , Marion et al. 2003 , Melosh et al. 2004 , Matson et al. 2012 , Sohl et al. 2014 , Glein et al. 2015 ). Given these conditions, NH 3 most likely exists in the liquid phase within icy moon oceans, with warmer regions exhibiting NH 3 gas and some colder regions possibly exhibiting NH 3 ice, depending on the temperature and pressure of the region. If permitted by sub-zero temperatures (e.g., −98°C), ammonia–water may also form solid, crystalline ammonia hydrate structures (Fig. 2 ) (Hogenboom et al. 1997 , Muñoz-Iglesias and Prieto-Ballesteros 2021 ). Given the toxicity of NH 3 , it is critical to understand whether icy moon conditions could permit this species to persist, and if so, the concentrations at which it may occur. For icy moons Enceladus and Titan, there is substantial data from which to extrapolate pressure, temperature, salinity, and pH. By integrating geochemical models, spacecraft observations, and laboratory analogues, we can begin to constrain the abundance and speciation of total ammonia, both of which are essential for assessing habitability. Enceladus: plumes of ammonia In the 1980s, investigations with Voyager 2 of the 6 th largest moon of Saturn, Enceladus, revealed a geologically young surface. Co-occurrence of the densest region of Saturn’s E ring with Enceladus' orbit indicated possible evidence of eruptive activity (Smith et al. 1982 ). Over two decades later, the revelations from Cassini were groundbreaking. Multiple flybys of Enceladus revealed jets of icy particles erupting from 130 km long fractures in the Southern pole, the “tiger stripes”, and confirmed deposition of particles into Saturn’s E ring (Fig. 3 ) (Porco et al. 2006 , Spitale and Porco 2007 ). Analysis of the plumes by two mass spectrometers onboard Cassini, the Ion and Neutral Mass Spectrometer (INMS) and Cosmic Dust Analyzer (CDA), elucidated the presence of water vapour, ice particles, simple organics (e.g., benzene), complex macromolecular organics, and gases such as deuterium, hydrogen, carbon dioxide, carbon monoxide, methane, and NH 3 (Waite et al. 2006 , 2009 , 2017 , Postberg et al. 2018 ). Figure 3. Open in a new tab Aqueous NH 3 within Enceladus. (A) Surface of Enceladus captured by the Cassini Imaging Science Subsystem. Portions of the tiger stripe fractures from which plumes of water emanate are visible, surrounded by a circumpolar belt of mountains. Image credits: NASA/JPL/Space Science Institute. (B) Discrete plumes expelled from Enceladus’ surface as captured by Cassini. These plumes were later confirmed to consist of water and other molecules such as NH 3 . Image credits: NASA/JPL-Caltech/SSI/PSI. The discoveries made by the Cassini mission reshaped our view of Enceladus into a prime candidate for astrobiology and further exploration. In particular, the discovery of vapour and NH 3 on Enceladus posed an enthralling question as to whether there could be liquid water beneath the ice crust. As aforementioned, ammoniacal solutions can remain liquid as low as −100°C, depending on the concentration of NH 3 (Johnson and Nicol 1987 , Croft et al. 1988 , Leliwa-Kopystyński et al. 2002 , Chua et al. 2023 ). The detection of NH 3 by Cassini therefore provided strong indirect evidence for the existence of subsurface liquid water on Enceladus. Further evidence for a liquid subsurface reservoir with NH 3 was gleaned by the presence of 40 Ar in the plume (Waite et al. 2009 ). Originally in reference to Titan, Engel et al. ( 1994 ) suggested that a water ocean preserved in the liquid state by NH 3 could dissolve sodium and potassium from silicate rock into fluid, leading to eventual decay of potassium-40 into 40 Ar that could later surface into the atmosphere by cryovolcanism. The detection of 40 Ar therefore provided further indications of an ocean containing NH 3 with active chemical exchange with its rocky core. This inference was strengthened by the detection of sodium-rich salts, such as sodium chloride (NaCl) and sodium bicarbonate (NaHCO 3 ), in Saturn’s E ring and freshly ejected plume particles; such salts can only arise from a liquid water origin (Postberg et al. 2009 , 2011 ). Cassini’s INMS measurements of Enceladus’ plumes showed NH 3 mixing ratios between 0.4% and 1.3%. The observed variability is thought to reflect compositional differences among plume eruptions during multiple flybys (Waite et al. 2006 , 2009 , 2017 ). It is notable that the concentrations align well with NH 3 -to-water ratios observed in comets between 0.01% to 1.5% (Wyckoff et al. 1989 , Meier et al. 1994 , Palmer et al. 1996 ), supporting the notion of primordial incorporation of solid NH 3 into Enceladus during its accretion. Mass spectra from the CDA subsequently indicated spectral features consistent with NH 4 + in the ejected ice grains, further supporting the hypothesis of a subsurface ocean with dissolved total NH 3 (Khawaja et al. 2019 ). Incorporating the Cassini NH 3 data, a bulk molecular abundance of total ammonia in the Enceladus ocean between 0.011%–0.169% has been derived, approximating to an oceanic concentration between ≈ 0.01–0.1 M. This corresponds to 0.001%–0.006% NH 3 and 0.01%–0.163% NH 4 + in an ocean between pH 7.95 and 9.05 (Fifer et al. 2022 ). Fifer et al. ( 2022 ) reasoned that the NH 3 plume composition does not directly reflect oceanic NH 3 concentration; volatile gases such as NH 3 likely undergo exsolution from the liquid phase during plume formation, depleting NH 3 concentrations in the plume compared to the ocean. However, the estimations of total ammonia in the ocean of Enceladus are evolving with new data. Phosphate-rich grains have been discovered in the plume ejecta, with species of Na 2 HPO 4 and Na 3 PO 4 reproducing the peak pattern of the CDA spectra most accurately (Postberg et al. 2023 ). Phosphorus in ionic forms of HPO 4 2− and PO 4 3− occur at high pH values between pH 7 and pH 12, and greater than pH 12, respectively. Thus, the presence of these phosphate species constrains the ocean from pH 10.1 to pH 11.6 according to recent geochemical modelling (Glein and Truong 2025 ). Reconstruction of the ocean chemistry place concentrations of NH 3 at 0.0181 molal (≈ 0.0181 M) (Glein and Truong 2025 ). These concentrations are low compared to bulk composition. Thermodynamic modelling of ammonia–water mixtures indicates such concentrations would not be sufficient to sustain liquid water if the ocean temperature were found to be below zero (Croft et al. 1988 , Kargel 1992 , Hogenboom et al. 1997 ). The ocean of Enceladus is thought to be near 0°C at the ice-ocean interface (Matson et al. 2012 , Glein et al. 2015 ). At near 0°C, the pK a for the reaction is ∼10.1 (Bates and Pinching 1949 ). Thus, as per the Henderson–Hasselbalch equation and current pH estimations, the ocean would bear NH 3 predominantly, as per observations from Cassini (Glein and Truong 2025 ). However, at the concentrations estimated, it is unlikely that NH 3 contributes significantly to the maintenance of liquid water. Although NH 3 may facilitate the liquid state in colder regions of the ocean where the temperature may fall slightly below 0°C, internal heat generated by tidal heating is thought to be the primary driver of liquid state preservation (Nimmo et al. 2007 , Roberts and Nimmo 2008 , Chen et al. 2014 ). An ammonia reservoir on titan Until Cassini, the surface of Saturn’s largest moon, Titan, was shrouded in uncertainties as its dense atmosphere concealed the surface (Fig. 4A ). Earth observations indicated a dense atmosphere consisting of methane (Kuiper 1944 ). The atmosphere, denser than that of Earth’s, was unlike that observed on any other known moon, and this motivated inspection by the Voyager 1 spacecraft in 1980 (Coustenis 2014 ). The discoveries made by Voyager 1 revealed a thick nitrogen-methane atmosphere with hydrocarbons and a surface entirely concealed by haze (Smith et al. 1982 ). But in 2005, close flybys of the surface by Cassini unveiled what was long hidden: a hydrologic-like cycle with methane and ethane, resulting in liquid hydrocarbon clouds, rivers, lakes and seas, and methane rainfalls (Stofan et al. 2007 , Turtle et al. 2009 , Poggiali et al. 2024 ), as well as a dynamic surface decorated with drainage networks, fluvial channels (Hörst 2017 ), mountains (Radebaugh et al. 2007 ), and possible cryovolcanoes (Lopes et al. 2007 , Hörst 2017 ). The mission also provided evidence of a subsurface ocean below the ice crust (Lorenz et al. 2008 , Bills and Nimmo 2011 ). Figure 4. Open in a new tab Titan is an abode of NH 3 . (A) Titan captured by the Cassini Imaging Science Subsystem. The thick atmosphere of Titan is visible by a haze across the surface and a lack of surface details. Saturn’s other moon, Tethys, is observed behind Titan. Image credits: NASA/JPL-Caltech/Space Science Institute. (B) False-colour mosaic of Titan obtained by the Visual and Infrared Mapping Spectrometer of Cassini. Colours correspond to atmospheric (red) and surface features (green and blue). The inset image indicates the possible occurrence of a cryovolcano on the surface. Cryovolcanic slurries of water, NH 3 , and methanol have been tentatively observed on the surface. Image credit: NASA/JPL/University of Arizona. A subsurface ocean on Titan had been proposed long before Cassini. The persistence of methane in the atmosphere of Titan, despite continual elimination by photochemical processes, suggested the presence of an internal replenishment mechanism. Lunine and Stevenson ( 1987 ) proposed that Titan harboured a subsurface water ocean maintained in the liquid state by the antifreeze properties of NH 3 . In this model, methane retained during Titan’s accretion was thought to be stored in clathrate hydrates (i.e., crystalline structures including water) and periodically released to the surface by cryovolcanic eruptions. Lunine and Stevenson ( 1987 ) presented an ammonia–water phase diagram that indicated an ocean of 10%–15% NH 3 by weight would be required to maintain the liquid state under Titan conditions. Indeed, cosmochemical models indicate that Titan accreted with solar volatiles and may have incorporated significant levels of NH 3 into its interior (Mousis Olivier et al. 2009 ). The discovery of NH 3 on Enceladus by Cassini circumstantially supports the presence of NH 3 on Titan (Waite et al. 2009 ). As on Enceladus, further indirect evidence of liquid water in contact with the silicate core in Titan’s history was provided by the detection of 40 Ar in the atmosphere (Niemann et al. 2005 ), and an aqueous NH 3 subsurface ocean on Titan could be a plausible source of atmospheric nitrogen (Owen 2000 ). However, the existence of a global liquid reservoir within Titan is currently debated. Recent work suggests Titan’s interior may instead contain high-pressure ice phases and localized, partially melted regions rather than an extensive liquid ocean (Petricca et al. 2025 ). Such environments are likely characterized by low temperatures and high pressures that may be unfavourable for life as we know it. Consequently, the NH 3 -based habitability considerations discussed in this review apply only to scenarios in which liquid or partially liquid ammonia–water phases are present within Titan’s interior. In alignment with internal NH 3 models, cryovolcanic flows observed by the Cassini Titan Radar Mapper exhibited rheological properties consistent with aqueous NH 3 , and possibly methanol, slurries (Fig. 4B ) (Lopes et al. 2007 , Mitri et al. 2008 ). However, more recent models place the abundance of NH 3 at 1.5–5%. A lower NH 3 concentration fits the observed bulk density of Titan (Tobie et al. 2012 , Vance et al. 2018 ), thermal profiles and mechanical behaviour of the ice shell (Sohl et al. 2014 ), and the tidal deformation data provided by Cassini (Leitner and Lunine 2019 ). In addition to NH 3 , radiogenic heating may support the liquid state in Titan’s ocean (Grasset et al. 2000 , Sohl et al. 2014 ). The pH of Titan’s ocean remains undetermined, but is typically modelled at greater than pH 7.3 (Marion et al. 2012 , Brassé et al. 2017 , Leitner and Lunine 2019 ). In an ocean of 5% NH 3 , a pH of 11.83 is expected (Brassé et al. 2017 ). Lower temperatures increase pK a (Zahn 2017a , 2017b , Samuelsen et al. 2019 ), and Titan’s ocean could be −18°C (Sohl et al. 2014 ). As NH 3 dominates above pH 10.1 at 0°C (Bates and Pinching 1949 ), the ocean of Titan would be expected to bear considerable quantities of NH 3 compared to NH 4 + under these conditions. Currently, the presence of a subsurface ammonia–water ocean is largely accepted when modelling Titan’s interior (Grasset and Sotin 1996 , Fortes 2000 , Grasset et al. 2000 , Tobie et al. 2005 ), and found probable by Cassini’s gravitational data (Goossens et al. 2024 ). But it is geochemically plausible that NH 3 dissolved in aqueous solution would react with internal silicates to yield ammonium salts (Kargel 1992 , Marion et al. 2012 ). Indeed, primordial NH 3 inside Titan could react with sulphate produced by water-rock interactions in the rocky core, forming ammonium sulphate [(NH 4 ) 2 SO 4 ] rather than remaining as free NH 3 . An aqueous (NH 4 ) 2 SO 4 ocean would have a density capable of generating Rayleigh–Taylor instabilities within the overlying ice crust, potentially driving the rise of buoyant diapirs as a mechanism for cryovolcanism (Fortes et al. 2007 , Grindrod et al. 2008 ). The presence of (NH 4 ) 2 SO 4 would indicate an oceanic pH less than 10.8, which does not align with current estimations. However, salinity and pressure can increase pK a (Clegg and Whitfield 1995 , Samuelsen et al. 2019 ), and thus the pK a of the equilibrium and the pH required for the transition. These effects could shift the equilibrium toward NH 4 + under high pressure and saline conditions, supporting the persistence of NH 4 + despite the high pH of the ocean. Indeed, a pressure of 800 MPa is estimated at the seafloor (Journaux et al. 2020 ). Freezing point depression can also be achieved without NH 3 . One of the factors preserving the liquid state of Titan’s ocean could be high salt concentrations (Mitri et al. 2014 ). The possibility of a magnesium sulphate (MgSO 4 ) ocean has been considered, and models of a MgSO 4 ocean align well with Titan’s density (Vance and Brown 2013 , Mitri et al. 2014 , Vance et al. 2018 ). Accretion with MgSO 4 is plausible as C1 and C2 carbonaceous chondrites contain up to 10% MgSO 4 (Fredriksson and Kerridge 1988 ). However, the atmosphere and surface of Titan exhibit reducing conditions. In addition to methane, a suite of reduced organic molecules are present in the atmosphere (e.g., ethane, acetylene, tholins), and there is a lack of oxidants, such as oxygen or carbon dioxide (Kunde et al. 1981 , Maguire et al. 1981 , Sagan et al. 1992 , Niemann et al. 2005 , Nixon 2024 ). Sulphur salts form from oxidation reactions, and thus the redox state of Titan favours NH 3 . Surface analysis by NASA’s upcoming mission to Titan, Dragonfly, may provide clarification into the subsurface ocean composition (Lorenz et al. 2018 , Barnes et al. 2021 ). Ammonia in the solar system Enceladus and Titan represent icy moons of Saturn for which extensive data has been gathered. However, it is not without mention that the Galilean moons of Jupiter’s system, Europa, Ganymede, and Callisto, are also subjects of scientific interest. Europa was first observed from a telescope by Galileo in 1610 (Soderblom 1980 ). Subsequent magnetic field measurements of Europa by the Galileo spacecraft were consistent with a saline liquid layer (Khurana et al. 1998 ), and further surface features revealed the presence of mobile icebergs and ice diapirs (Pappalardo et al. 1998 , Rathbun et al. 1998 , Singer et al. 2021 , Kihoulou et al. 2025 , Lesage et al. 2025 ). As for the other moons, Ganymede is the primary target of ESA’s JUICE (Fletcher et al. 2023 , Poulet et al. 2024 ). Along with Titan, Ganymede and Callisto are the largest known icy satellites, and both moons are expected to feature subsurface oceans or liquid reservoirs (Zimmer et al. 2000 , Kivelson et al. 2002 , Saur et al. 2015 , Cochrane et al. 2025 ). Thermal models indicate that the Galilean satellites likely contain an NH 3 -rich liquid water layer (Lewis 1971 ). However, water or salt brines rather than ammoniacal mixtures likely drive cryovolcanism on Europa (Kargel 1991 ). This is because, unlike the colder Saturn nebula, the thermal properties of the Jovian nebula would have favoured NH 3 gas. Gas is not well incorporated into smaller bodies during accretion, thus preventing significant incorporation of gaseous NH 3 into the Jovian moons, including Europa (Kargel 1991 , Carlson et al. 2009 ). Indeed, nitrogen-bearing species have yet to be conclusively detected on Europa, and evidence of tidal heating indicates an antifreeze component such as NH 3 would not be required to maintain liquid water (Carr et al. 1998 , Chen et al. 2014 , Běhounková et al. 2021 ). On the contrary, Ganymede and Callisto formed in the outer, cooler regions of the Jovian disk, where the incorporation of frozen NH 3 could be plausible (Mousis and Gautier 2004 , Mousis and Alibert 2006 ). Indeed, there is no strong evidence for tidal heating on either of these Jovian moons that could act to preserve liquid water. An antifreeze component within the waters, such as NH 3 , is therefore hypothesized (Khurana et al. 1998 , Mousis et al. 2002 , Spohn and Schubert 2003 , Vance et al. 2018 ). Ammoniacal liquid reservoirs are not confined to the icy moons of Jupiter and Saturn, and are hypothesized across a wide range of Solar System bodies. For example, an ocean is expected on Neptune’s moon, Triton. Thermal-structural models of Triton indicate the strong possibility of a long–lived subsurface ocean, probably enriched in NH 3 . However, the presence of NH 3 in this ocean has not yet been directly confirmed (Gaeman et al. 2012 ). Similarly, Charon, Pluto’s largest moon, and the moons of Uranus (Ariel, Umbriel, Titania, and Oberon) may also host internal liquid water oceans with NH 3 (Brown and Calvin 2000 , Cheng et al. 2014 , Rhoden et al. 2015 , Cochrane et al. 2021 , Castillo‐Rogez et al. 2023 ). As for other planetary bodies, there is evidence of subsurface brines on the Dwarf planet Ceres. Dawn gravity and spectral data indicate localized subsurface brine reservoirs feeding bright surface deposits, and a surface dominated by ammoniated salts and phyllosilicates (Ammannito et al. 2016 , Zolotov 2017 , Raymond et al. 2020 , Singh et al. 2021 , Nathues et al. 2022 ). Pluto may also host a subsurface liquid water ocean, in which liquid water may be maintained by tidal heating or NH 3 (Robuchon and Nimmo 2011 , Nimmo et al. 2016 ). Indeed, NH 3 has been detected on the surface (Dalle et al. 2019 ). More broadly, several Kuiper Belt Objects are predicted to contain subsurface liquid water oceans, in which NH 3 may be a constituent (Hussmann et al. 2006 , Brown 2012 ). However, few of these bodies have confirmed surface detections of NH 3 , and the composition and extent of their internal liquids remain uncertain. While many celestial bodies in the Solar System may host liquid water and NH 3 , compositional data is lacking. These limited physicochemical constraints do not allow for the estimation of the abundance, speciation, or phase behaviour of total ammonia, and thus these bodies were not included as part this review. An essential ingredient for life To understand how NH 3 may influence the habitability of extraterrestrial systems, it is critical to assess how NH 3 shapes the habitability of terrestrial environments. At 25°C, the equilibrium has a pK a of 9.25, and thus a pH above or below this value (salinity and temperature will also adjust marginally this value) dictates whether NH 3 ( > \ pH 9.25) or NH 4 + ( < \ pH 9.25) predominate (Bates and Pinching 1949 , Bower and Bidwell 1978 ). Many environmental systems are temperate and operate at near-neutral pH and standard atmospheric pressure. Thus, in most water, soil and living systems on Earth, NH 3 occurs in gas form. However, the natural levels of NH 3 in water and soil (excluding air) are trace amounts ( < 6 ppm, ≈ 0.00035 M) (Roney et al. 2004 ). As such, there are limited natural environments that mirror a cold and saline aquatic environment with NH 3 present, as is speculated in icy moons. There are, however, a few cases where life has documented to thrive in high ammoniacal environments. In bat caves, decomposition of bat urea drives high concentrations of gaseous NH 3 (McFarlane et al. 1995 ). Yet microbes, along with bats, colonize this habitat (Studier 1966 , Leon et al. 2018 , Newman et al. 2018 ). Similarly, in the alkaline and high pH waters of Mono Lake, United States, concentrations of dissolved NH 3 gas incrementally increase with depth to a final concentration near 0.0005 M at 35 m. Despite high NH 3 concentrations, bacteria have been isolated at these depths (Ward et al. 2000 , Humayoun et al. 2003 ). Both examples indicate that life can persist in concentrated NH 3 environments on Earth. It is even speculated that NH 3 may have facilitated the origin of life on Earth. The origin of life is hypothesized to have arisen from increasingly complex interactions that led to the formation of functional cellular bodies (Monnard and Walde 2015 , Goldman 2023 ). It is known that nitrogen is a key element involved in this process as it is required to produce amino acids in modern systems (Miflin and Lea 1982 , Bender 2012 ). In primordial systems, alkaline hydrothermal vents may have acted as locations for prebiotic chemistry as these sites provide heat, chemical gradients, and mineral catalysts (Martin and Russell 2006 , Russell et al. 2010 , Lane and Martin 2012 , Sojo et al. 2016 ). It is hypothesized that NH 3 produced in early hydrothermal vents could have been the nitrogen source for prebiotic chemistry (Martin et al. 2008 , Sojo et al. 2016 , Nishizawa et al. 2021 ). Indeed, NH 3 has been demonstrated to form amino acids under early Earth conditions (Miller 1955 , Lowe et al. 1963 , Furukawa et al. 2009 ), as well as those present on Titan (Neish et al. 2009 , 2010 ). Very low partial pressures of NH 3 would have been sufficient to sustain prebiotic chemistry in a seawater origin of life scenario (Wigley and Brimblecombe 1981 ). It is of interest to note that hydrothermal-derived NH 3 could have been prevalent as part of the primordial atmosphere within the Hadean era. The concentration of NH 3 in the atmosphere dwindled in the Archean era, when the earliest known life existed, as conditions became more oxidizing, and the rate of NH 3 photodissociation increased as a result of increasing solar luminosity (Brandes et al. 1998 , Nishizawa et al. 2021 , Shang et al. 2023a , 2023b ). It is possible that the heightened levels of NH 3 in the Hadean era may have been one of the factors that precluded the formation of life earlier in Earth’s history. As a nucleophile, NH 3 could destroy prebiotic intermediates such as nucleotides and sugars. Indeed, DNA damage can occur with NH 3 exposure (Zhang et al. 2020 , Tong et al. 2025 ). As a H + acceptor, NH 3 could also act to limit the H + pool for prebiotic chemistry (Aithal et al. 2023 , Pathak et al. 2024 ). Consequently, elevated NH 3 in the Hadean era may have functioned as a chemical barrier to early prebiotic pathways, with its eventual reduction opening the window for life’s emergence in the Archean era. In modern systems, total ammonia sustains habitability by forming a vital part of the nitrogen fixation cycle. NH 3 is formed naturally by diazotrophic bacteria, cyanobacteria, and archaea that perform nitrogen fixation. In this process, atmospheric nitrogen (N 2 ) is reduced to NH 3 (Burris and Roberts 1993 , Ribbe 2011 , Shin et al. 2016 ). NH 3 may also be available as a result of ammonification by decomposition of organic excretion or tissue (including DNA, proteins, and amino acids) by various fungi and prokaryotes (Ladd and Jackson 1982 , Strock 2008 , Singh K 2016 ). However, most of the NH 3 produced rapidly equilibrates to NH 4 + due to the moderate pH of many ecosystems (Fowler et al. 2013 ). At this stage, the total ammonia in an environment may be directly assimilated into microbes and plants and incorporated into the synthesis of amino acids and nucleotides, whereby glutamate, a nitrogen donor, is the first and most central amino acid formed from NH 4 + (Rogers and Aneja 1980 , Smith et al. 1980 , Tesch et al. 1999 , Vo et al. 2013 , Hachiya and Sakakibara 2017 ). Alternatively, the pool of NH 4 + and NH 3 in the environment is oxidized by nitrifying micro-organisms such as ammonia-oxidizing bacteria (AOB) to nitrite, which is subsequently oxidized to nitrate by nitrite-oxidizing bacteria (Wallace and Nicholas 1969 , Schmidt and Belser 1983 , Koops and Pommerening-Röser 2001 , Caranto and Lancaster 2017 ). The nitrogen cycle is completed by denitrifying bacteria such as Paracoccus and Pseudomonas that process nitrate back into N 2 (Alexander 1965 , Ji et al. 2015 , Rajta et al. 2020 ). The presence of NH 3 and NH 4 + in the environment thus plays a crucial role in supporting diverse microbial communities and plants by ensuring the continuous availability of biologically usable nitrogen. Biochemical disruption and disorder While total ammonia forms a vital part of the global nitrogen cycle, high concentrations of NH 3 are widely documented as toxic to life as we know it. The small size and uncharged nature of gaseous NH 3 permits passive, unregulated permeation across lipid membranes (Ritchie and Gibson 1987a , 1987b , Ritchie and Islam 2001 , Brazier 2016 ). Due to the lone pair of electrons on the nitrogen atom, NH 3 may function as a H + acceptor. Under biological pH (i.e., pH 7.4 to 7.8), permeated NH 3 combines with cytoplasmic H + to form NH 4 + . The capture of H + increases cytosolic pH (Bosoi and Rose 2009 ). In eukaryotic cells, such intracellular NH 3 -driven alkalization has been associated with disrupted calcium (Ca 2+ ) signalling (Horie et al. 1995 , Rose et al. 2005 ), disrupted organelle acidification, and with enzyme dysfunction (Moriwaki et al. 2024 ). Additionally, capture of H + can dissipate the proton motive force required for ATP generation (Bai et al. 2001 , Angelova et al. 2022 ), causing oxidative stress by the production of reactive oxygen species (Han et al. 2020 , Angelova et al. 2022 ). In prokaryotes, specific effects of NH 3 -driven reactions remain poorly characterized, but NH 3 transport has been correlated with the dissipation of the transmembrane pH gradient in Methanospirillum hungatei (Sprott et al. 1984 ). Increased NH 4 + and NH 3 have also been correlated with reduced ATP production and electron transport system activity in Enterobacter cloacae HNR (Weng et al. 2022 ). In general, internal alkalization can elicit a stress response in prokaryotes (Schuldiner et al. 1986 ). These cellular toxicity mechanisms are depicted in Fig. 5 . Both higher and lower organisms typically prevent NH 3 toxicity and accumulation by rapid assimilation, enhanced excretion, conversion to less toxic compounds, or scavenging (Givan 1979 , Burkovski 2003 , Ip and Chew 2010 , Chew and Ip 2014 , Haskett et al. 2022 , Okabe et al. 2023 , Kang et al. 2026 ). For example, upon detecting NH 3 , Burkholderia glumae triggers oxalate biosynthesis that can act to neutralize NH 3 (Kang et al. 2026 ). However, in high enough concentrations, NH 3 may overwhelm these defensive mechanisms. Figure 5. Open in a new tab Mechanisms of cellular NH 3 toxicity. Research across the domains of life has revealed several pathways of cellular NH 3 toxicity caused by disrupted internal chemistries. These toxic effects are exerted firstly by the disruption of the intracellular proton pool, as the conversion of occurs following unregulated, passive diffusion of NH 3 . Downstream chemistries such as intracellular pH, the transmembrane proton gradient, and proton motive force can become disrupted as a result. Enzyme and organelle function may also become disrupted by changes in pH. In addition to internal alkalization, the external rise in pH caused by NH 3 has also been implicated as the cause of toxicity in living organisms (Vines and Wedding 1960 , Taglicht et al. 1987 ). Indistinguishable toxicity between NH 3 /NH 4 + solutions and solutions of identical pH has been characterized for Bacillus subtilis, Sporosarcina, Paenibacillus, Staphylococcus, Brevibacillus, Streptomyces, Pseudomonas , and Arthrobacter (Deal et al. 1975 , Kelly et al. 2012 ). In Escherichia coli , results vary. In a study by Deal et al. ( 1975 ), NH 3 /NH 4 + solutions were shown to be more toxic than NaCl solutions at an equivalent pH of 9.5, but indistinct toxicity was observed at pH 10 between the NH 3 /NH 4 + solutions and NH 3 -free solutions at comparable pH. Likewise, for Bacillus subtilis , toxicity between NH 3 /NH 4 + solutions and NaCl solutions was distinct at pH 9 but indistinct at pH 10 (Deal et al. 1975 ). Vines and Wedding ( 1960 ) indicated that, in plants, high pH is not a direct toxicity mechanism of NH 3 per se, but rather a vehicle through which larger amounts of gaseous NH 3 can enter cells and disrupt biological processes. Similarly, in the alkalitolerant extremophile Halomonas meridiana , growth in NH 3 /NH 4 + solutions with a high relative abundance of NH 3 and pH-matched solutions of sodium hydroxide indicated that NH 3 toxicity is distinct from external pH stress (Hopton et al. 2025a ). This study also elucidated that NH 3 induced alterations to metabolites that could suggest cell wall modification, alterations to metabolites within Coenzyme A pathways, and accumulation of compounds that could reflect internal NH 3 -driven reactions (Hopton et al. (Hopton et al. 2025a ). In contrast to NH 3 , the positive charge of NH 4 + prevents passive diffusion through the hydrophobic core of lipid membranes. Instead, NH 4 + is transported through ammonium transport (Amt) membrane proteins (Kim et al. 2012 , Wacker et al. 2014 ), which may also facilitate passive NH 3 diffusion (Soupene et al. 1998 , Soupene et al. 2002 ). Due to similarities in charge, ionic radius, and hydration properties, NH 4 + can compete with potassium ions (K + ) for transport through ion channels (Moser 1987 , Neijssel et al. 1990 , Bosoi and Rose 2009 ). This facilitated diffusion and active transport of NH 4 + regulates cellular uptake, thereby reducing toxicity of NH 4 + relative to NH 3 . However, high accumulations of NH 4 + , such as that caused by cation channel influx (Burckhardt and Frömter 1992 , Ramirez et al. 1999 ) or unregulated permeation of NH 3 , can upset cell homeostasis. NH 4 + can acidify the intracellular medium (Burckhardt and Frömter 1992 , Ramirez et al. 1999 ) and influence membrane potential (Golby et al. 1990 , Wang et al. 2018 ). Elevated NH 4 + can also disrupt K + balance (Sprott and Patel 1986 , Szczerba et al. 2008 , Kong et al. 2014 , Shi et al. 2020 ), which is essential for pH balance, membrane potential, intracellular communication, electrical signalling, as well as osmoprotection [for extended reviews, see Beagle and Lockless ( 2021 ) and Benarroch and Asally ( 2020 ) for bacteria, Johnson et al. ( 2022 ) for plants, and McLean and Wang ( 2021 ) for humans]. The downstream effects of NH 4 + are much more defined in literature across a breadth of species compared to NH 3 . Such effects include disruption of amino acid, lipid, and nucleotide metabolisms in shrimp and bacteria (Xiao et al. 2019 ), production of reactive oxygen species, interruption of Ca 2+ homeostasis, and induction of cell apoptosis in mammalian cells (Wang et al. 2018 ), acidification in plants (Hachiya et al. 2021 ), and decline of motility and energy metabolism in bacteria (Weng et al. 2022 ). Bacteria have also exhibited alterations to amino acid levels (Weng et al. 2022 , Hopton et al. 2025b ) and protein factors involved in translation, translocation, and folding (Sayavedra-Soto et al. 2015 , Zorz et al. 2018 ) under NH 4 + exposure. Accumulating concentrations of NH 4 + , as opposed to increasing NH 3 or pH, have been found to be a contributing factor suppressing the abundance of Anaerobranca, Tepidimicrobium and Proteiniborus in anaerobic digesters (Dai et al. 2016 ). Toxicity occurring by the independent action of NH 4 + is thus possible, although less common due to the regulated transport of this ion. Evidence thus far indicates the overall picture of NH 3 toxicity is one where both NH 3 and NH 4 + act in concert against biological components: NH 3 bypasses membrane regulation and passively diffuses into cells, NH 4 + is formed following the permeation of NH 3 into cells, and damaging cellular effects occur by the actions of both NH 3 and NH 4 + . Biotic models for assessing habitability Icy moons are, relative to conditions on Earth, extreme environments. There are extremes of temperature and pressure exceeding those even possible on Earth. This includes near −200°C icy surfaces (Brown et al. 2006 , Ashkenazy 2019 , Jennings et al. 2019 ) and oceanic pressures of 800 MPa (Journaux et al. 2020 ). Subsurface waters could fall between a salinity of 2 and 20 g/kg on Enceladus, but up to 200 g/kg on Titan. Comparatively, the salinity of seawater on Earth is ∼35 g/kg or 3.5% (Ludwig 2022 ). The waters of Enceladus and Titan are also expected to exceed a pH of 10 (Brassé et al. 2017 , Glein and Truong 2025 ). Organisms that could develop under these extremes of pH, temperature, salinity, and pressure would have to utilize specialized adaptations to survive. On Earth, such organisms are known as extremophiles. Extremophiles on Earth already demonstrate that life can survive in waters at −20°C (Clarke et al. 2013 , Frösler et al. 2017 ), pH 11 (Suzuki et al. 2014 ), and upwards of 200 g/kg salinity (Meinzer et al. 2023 ), for example. Given the ammoniacal waters of icy moons, it is notable that some prokaryotes on Earth display NH 3 -dependent metabolisms. These organisms include ammonia-oxidizing archaea (AOA) and AOB, chemolithotrophs that utilize NH 3 as an energy resource by oxidizing NH 3 to nitrite in the first-rate limiting step of nitrification (Koops et al. 2006 , Zorz et al. 2018 ). However, -phile often implies thriving in highly concentrated or extreme conditions. AOA and AOB are tolerant to high NH 4 + (0.01 M to 0.43 M) but not NH 3 (activity reduction of 15.9% was observed in less than 0.001 M NH 3 ) (Tourna et al. 2011 , Vejmelkova et al. 2012 , Qian et al. 2017 ). When considering the habitability of icy moons, the NH 3 concentrations estimated for their subsurface oceans are low relative to the inferred bulk chemical compositions. This raises questions as to whether life would need NH 3 adaptations to evolve and thrive. Indeed, methanogenic archaea, which are typically without adaptations to high NH 3 , are frequently considered one type of metabolic analogue for life within icy moon oceans. Methanogens have been shown to continue weak metabolic functions in NH 3 levels higher than those expected on Enceladus (≈ 0.0265 M) (Wang et al. 2015 ). Methanogens have also been isolated from an array of extreme environments on Earth, use simple energy sources (e.g., hydrogen and carbon dioxide) to produce methane (methane has been detected on both Enceladus and Titan), and are anaerobic (McKay et al. 2008 , Taubner et al. 2015 ). Due to limited oxygen delivery to the oceans, anaerobic respiration was thought to be a requirement in the oceans of icy moons. These properties have made methanogens suitable study specimens in astrobiology. Models now suggest that oxygen generated on the surface of icy moons by radiation is transported to the subsurface oceans (Teolis et al. 2017 , Ray et al. 2021 , Hesse et al. 2022 , Szalay et al. 2024 , Tinner et al. 2024 ). This process has been proposed to broaden the metabolic possibilities in these oceans to aerobes. Thus, given the low bulk compositions of NH 3 in icy moons, this finding expands biotic models beyond AOB, AOA, or methanogens to also include aerobic extremophilic bacteria that do not have specified NH 3 adaptations. Extremophilic bacteria capable of both aerobic and anaerobic metabolisms are widely documented in the literature [see Pikuta et al. ( 2007 ) or Bowers et al. ( 2009 ) for in-depth reviews]. When choosing suitable biotic models for assessing habitability, it must be considered that, unlike methanogenic archaea, bacteria exhibit a broader range of metabolic pathways that may be compatible with icy moon chemistries (e.g., sulphur oxidation, nitrate reduction, NH 3 oxidation) (Koops et al. 2006 , Yin et al. 2014 , Kilic et al. 2017 ). Bacteria lacking specific NH 3 adaptations have also been isolated at depths of 35 m in the NH 3 -laden (0.0005 M NH 3 ) Mono Lake, as aforementioned (Humayoun et al. 2003 ), as well as the hypersaline and −13°C waters of the ice-sealed Lake Vida (Murray et al. 2012 ). The use of extremophilic, and possibly aerobic, bacteria without specified NH 3 adaptations in limits-of-life research could therefore offer a nuanced perspective on the habitability potential of icy moons. Using bacteria as model organisms, the following sections examine whether the ammoniacal subsurface oceans of icy moons could sustain life by drawing on known bacterial survival thresholds in NH 3 . Bacteria under simulated icy moon extremes As life could be entrained in the ejected ice grains from the plumes of Enceladus, much microbiology research in astrobiology has focused on viability and detectability of microbes following simulated entombment in ice (Kelly et al. 2012 , Bywaters et al. 2020 , Parker et al. 2023 , Klenner et al. 2024 ). Few experiments have provided in vitro assessments of how simulated icy moon conditions with NH 3 could afflict bacterial life. Those that have, have done so from a planetary protection perspective, assessing the risk of surface contamination on icy moons with common terrestrial bacteria carried by spacecraft, not extremophiles. However, this data can still provide valuable constraints on habitability in NH 3 . Molton and Ponnamperuma ( 1972 ) demonstrated survival thresholds of four bacteria, E. coli, Serratia marcescens, Aerobacter aerogenes , and B. subtilis , to a simulated Jovian atmosphere of H 2 (56%), He (43%), CH 4 (0.5%), and NH 3 (0.5%). Pressure-temperature regimes included those that could apply to icy moon interiors (≈ 5 MPa and −13°C, and ≈ 6.8 MPa and 0°C). Survival varied with these conditions. Near-total mortality occurred under the colder treatment (loss of viability as %: E. coli , 97%; S. marcescens , 93%; A. aerogenes , 63%; B. subtilis , 100%), while fewer proportional deaths occurred at 0°C (loss of viability as %: E. coli , 19%; S. marcescens , 50%). Although not designed to assess oceanic habitability, these findings indicate that microbial persistence in NH 3 mixtures may be constrained at sub-freezing temperatures or supported at near 0°C. The multi-extreme conditions employed by Molton and Ponnamperuma ( 1972 ) make it difficult to attribute a single parameter, or a combination of parameters, to bacterial death. Specifically, the influence of NH 3 on bacterial survival limits cannot be established. Subsequent studies, however, have isolated the impact of NH 3 by examining bacterial viability in simple, aqueous NH 3 /NH 4 + solutions under extreme temperatures. Deal et al. ( 1975 ) showed that a 0.1 M NH 3 /NH 4 + solution was toxic to E. coli and B. subtilis at 25°C and pH 9.5–10.5 (NH 3 > 50%). Molar limits of toxicity were not assessed, but cell viability was reduced at a higher rate as pH increased at 25°C. This correlates to an increased relative proportion of NH 3 . Reduction to cell viability still occurred at 0°C, albeit at a slower rate. From this, we can make two assumptions: abundance of NH 3 is proportional to toxicity, and lower temperatures reduce toxicity. The latter phenomenon could be explained by a reduction in kinetic energy associated with lower temperatures that ultimately limits substantial membrane permeation of NH 3 . The parameters utilized in this study (i.e., pH 10.5, temperatures at 0°C and 0.1 M NH 3 /NH 4 + ) align well with the physicochemical properties expected on Enceladus. These findings imply that terrestrial organisms like E. coli and B. subtilis would face significant physiological stress, if not outright mortality, under such conditions. While simulated icy moon environments in vitro have indicated that the presence of NH 3 can contribute to the toxicity of an aqueous solution, these experiments do not define the concentration thresholds for life in aqueous NH 3 . As presented in Table 1 , estimated concentrations of NH 3 /NH 4 + between icy moons vary. In accordance with the toxicological data, aqueous environments with NH 3 , such as those hypothesized for Enceladus and/or Titan, may present a more significant physiological challenge to microbial survival and adaptation if present at sufficient concentrations. However, the definition of “sufficient concentrations” is loose; the concentration thresholds for the growth of bacteria in NH 3 have not yet been properly established. Table 1. NH 3 and NH 4 + concentration and speciation estimated for icy moon subsurface oceans. Icy moon Predominant species 1 Estimated [NH 3 ] Estimated [NH 4 + ] Enceladus NH 3 0.0181 M 2 0.00744 M 2 Titan NH 3 0.96 to 9.6 M 3 Not estimated Open in a new tab 1 Speculated in this review as per physicochemical expectations of oceans. 2 Estimated by Glein and Truong ( 2025 ). 3 Molarity (M) calculated using an oceanic density ( ρ ) of 1,091 g/l (Goossens et al. 2024 ), 1.5% to 15% NH 3 , and the molar mass of NH 3 at 17.031 g/mol (Equation 1 ). (1) Ocean habitability: limits of bacterial life in ammonia There is not a significant body of work that intersects microbiology, icy moon physicochemical conditions, and NH 3 . In lieu of this, survival thresholds in NH 3 /NH 4 + can be gleaned from research in other fields. For example, much existing work regarding survival thresholds of bacteria in NH 3 has been conducted from the perspective of wastewater treatment in anaerobic digesters. Anaerobic digesters are often maintained below pH 9.25, whereby the relative abundance of NH 3 is less than 50% compared to NH 4 + (Dai et al. 2016 , Jiang et al. 2019 , Mutegoa et al. 2020 , Chapleur et al. 2021 ). These conditions are not comparable with those estimated in the oceans of Enceladus and Titan. However, concentration limits of bacterial life in NH 3 can still be drawn from this research. Considering this, the minimal inhibitory concentration (MIC) for a wide variety of bacteria in NH 3 and NH 4 + is presented in Table 2 . Where MIC was not stated outright, an estimate was derived from the pH of the experiment using the pK a of and the Henderson–Hasselbalch equation. In these instances, the MIC was defined as the lowest concentration of NH 3 or NH 4 + that limited visible growth and function. Table 2. Minimal inhibitory concentration (MIC) of NH 3 and NH 4 + across bacterial species. Species Strain MIC (NH 3 , M) MIC (NH 4 + , M) Enterobacter cloacae a HNR ≈ 0.00 049 ≈ 0.0138 Escherichia coli b MG1655 0.0042 0.750 § Corynebacterium glutamicum b ATCC 13 032 0.0112 1.989 § Bacillus subtilis b – 0.0133 0.750 § Escherichia coli c K-12 > \ 0.02, < 0.04 † – Bacillus subtilis c 168 > \ 0.02, < 0.04 † – Enterococcus durans c ST2 > \ 0.02, < 0.04 † – Pseudomonas sp. c – > \ 0.02, < 0.04 † – Enterobacter faecalis d NCTC 00 775 0.025 0.465 Listeria innocua d NCTC 11 288 0.025 0.465 Escherichia coli d NCTC 10 538 0.025 0.465 Halomonas meridiana e Sltfh1 0.03 0.02 Bacillus subtilis d T1, T2, T19, T22, T34, T39, DK-W1, N3, N4 and N5 0.05 0.931 Bacillus cereus d T31 and T38 0.05 0.931 Bacillus megaterium d T3, T4, T21, T34, T37 and T40 0.05 0.931 Pseudomonas aeruginosa d NCTC 10 299 0.05 0.931 Bacillus subtilis f 168 ≈ 0.064 ≈ 1.456 Bacillus subtilis d T5 and DA2 0.15 0.279 Bacillus cereus d NCIMB 9373 0.15 0.279 Sulphate-reducing bacteria g – 0.2 ‡ 0.166 ‡ Bacterial isolate c 4–1 ≈ 0.3 † – Bacterial isolate c 4–2 ≈ 0.3 † – Bacillus subtilis d T20, N1, N2, DA1 and NCIMB 3610 0.3 0.559 Bacillus cereus d T41 0.3 0.559 Bacillus licheniformis d ATCC 39 302 0.3 0.559 Enterococcus faecium d DK-C1 0.3 0.559 Micrococcus luteus d NCDO 0982 0.3 0.559 Staphylococcus aureus d NCDO 0949 0.3 0.559 Salmonella typhimurium d NCIMB 10 248 0.3 0.559 Bacillus subtilis d T36 0.5 0.931 Proteus morgani d NCIMB 00 067 0.5 0.931 Bacillus pumilus d NCIMB 9369 > \ 0.5 0.931 Bacillus pasteurii d NCIMB 8841 > \ 0.5 0.931 Open in a new tab a From Weng et al. ( 2022 ). b From Müller et al. ( 2006 ). c From Tada et al. ( 2021 ). d From Leejeerajumnean et al. ( 2000 ). e From Hopton et al. ( 2025a ). f From Hamill et al. ( 2020 ). g From Dai et al. ( 2017 ). § Growth impairment attributed to osmotic or ionic effects of NH 4 + . † Cultured in NH 3 gas. ‡ MIC derived from NH 3 and NH 4 + concentrations in reactor 3. From Table 2 , it is evident that the NH 3 concentrations estimated on Enceladus fall below the MIC of NH 3 established for many of the bacteria. The intersection between established bacterial MIC of NH 3 and icy moon NH 3 levels is depicted in Fig. 6A . The exceptions are E. cloacae HNR, E. coli MG1655, B. subtilis , and Corynebacterium glutamicum ATCC 13032, which exhibit sensitivity to concentrations of NH 3 below 0.0181 M. It is notable the maximal inhibitory threshold for B. subtilis is 0.5 M. Spore-formation of B. subtilis has been implicated for the survival of this species when frozen in 35% NH 3 (Kelly et al. 2012 ). B. pumilus and B. pasteurii have demonstrated a tolerance up to, but not necessarily limited to, 0.5 M NH 3 , the current upper limit of bacterial survival in NH 3 recorded in literature (Leejeerajumnean et al. 2000 ). The high level of tolerance could be attributed to NH 3 utilization; in B. pasteurii , NH 3 supports substrate oxidation (Wiley and Stokes 1962 ), permeability of substrates (Wiley and Stokes 1963 ), and ATP generation (Jahns 1996 ). These comparisons indicate that the levels of NH 3 speculated in the ocean and plumes may not limit prospects for habitability (Fig. 6B ). However, it should be noted that the survival limit of 0.5 M NH 3 has only been identified in one study. This study did not monitor NH 3 levels, and the result has not been substantiated by any further studies. Figure 6. Open in a new tab Survival of terrestrial life in NH 3 paralleled against known ammoniacal environments on icy moons. (A) Minimal inhibitory concentrations (MIC) of NH 3 in bacteria from Table 2 compared against estimated NH 3 concentrations within the oceans of Enceladus and Titan. NH 3 concentration on the x -axis represents the MIC for bacteria, and the ocean abundance for icy moons. The red dotted line indicates the upper limit of observed bacterial MIC in NH 3 . (B, C) Environments where growth of terrestrial bacteria in similar NH 3 or (NH 4 ) 2 SO 4 levels have been experimentally demonstrated (green, dashed line) or not yet demonstrated (grey, solid line) in vitro are depicted. (B) Schematic overview of the exterior and interior of Enceladus. Subsurface ocean NH 3 concentration determined by Glein and Truong ( 2025 ). Speciation speculated as part of this chapter based upon estimated physicochemical characteristics of the ocean. (C) Cross section of Titan. Left: A putative global subsurface ocean enriched in ammonia–water brines, potentially containing ammonium salts such as (NH 4 ) 2 SO 4 , within which terrestrial bacteria have demonstrated high tolerance. Centre: an alternative interior model in which Titan consists of a slushy high-pressure ice layer with small amounts of partial melting. NH 3 and NH 4 + concentrations are currently unconstrained. Right: a Titan model with a global subsurface ocean that could be comprised of 1.5%–15% NH 3 , calculated at a molarity of 0.96–9.6 M as part of this review. Nonetheless, the survival of B. pumilus and B. pasteurii in up to 0.5 M NH 3 , the maximum concentration utilized in the study by Leejeerajumnean et al. ( 2000 ), suggests survival could possibly exceed 0.5 M NH 3 . This could indicate concentrations of 0.96 M NH 3 (Table 1 ), tentatively calculated within the putative ocean of Titan, could be survived by some species. However, survival in 0.96 M NH 3 , or concentrations exceeding 0.5 M NH 3 , has not been explicitly demonstrated. Thus, based on the data presented in Table 2 , the known NH 3 tolerance in bacteria is lower than the NH 3 concentrations estimated on Titan (Fig. 6A ). This limits habitability prospects in the ocean or likely any adjoining cryovolcanoes (Fig. 6C ). However, if it is true that (NH 4 ) 2 SO 4 predominates in the ocean, the levels of NH 4 + could fall within a range that has been observed to support bacterial survival of Earth. Survival has been documented in up to 1.989 M NH 4 + in C. glutamicum . The concentration of (NH 4 ) 2 SO 4 under the aqueous (NH 4 ) 2 SO 4 ocean model has not been estimated. However, if we assume NH 3 incorporation during accretion was similar under the two models, concentration of (NH 4 ) 2 SO 4 may be comparable. As such, lower threshold levels of 0.96 M to 2 M (NH 4 ) 2 SO 4 may not pose a barrier to habitability. We paint a broad picture of habitability here. However, habitability in ammoniacal environments may be enhanced locally through habitat modification. For example, microenvironments could develop where microbial activity lowers pH, shifting the NH 3 /NH 4 + equilibrium toward NH 4 + , which is less toxic. Additionally, microbes may utilize available NH 3 for biosynthesis, exporting amino acids, or other metabolic processes. These scenarios suggest that native life on such worlds could possess physiological adaptations conferring NH 3 tolerance, highlighting that local chemical modifications and evolutionary adaptations may create habitable niches even in waters that are overall NH 3 -rich. It should be noted that while the physicochemical conditions underpinning the MIC of NH 3 and NH 4 + given in Table 2 may not be entirely comparable to icy moons, some of the bacteria utilized exhibit polyextremophilic attributes ( H. meridiana ), alkaliphilic and halotolerant metabolisms ( B. licheniformis ), or alkalitolerance and halotolerance ( B. subtilis, B. cereus, E. faecium, E. durans, M. luteus ) that are relevant to the high pH and saline properties expected in icy moon oceans. It should be additionally stated that the MIC of NH 3 is not an absolute limit to life. Bacteria could persist in a dormant, non-growing but viable state. However, for the purpose of this review, we consider habitability on the basis of whether an environment can: a) support the emergence of life, and b) support replication and growth. As such, the preceding comparison was not meant to imply the presence of life but rather explore whether the likelihood of habitability could be inferred by comparing the known biological limits in NH 3 with extraterrestrial ammoniacal conditions. Habitability of ammoniacal ice Oceans are not the only environments within icy moons that could hold water, and thus, potential habitats. The ice shells of icy moons could support brine channels, veins, pockets, and fractures (Kargel et al. 2000 , Buffo et al. 2021 , Wolfenbarger et al. 2022a ), Buffo et al. 2023 ). On Earth, such brine networks have shown to sustain life (Cooper et al. 2019 , Buffo et al. 2022 ). Organisms can be entrained into liquid inclusions of ice shells during ice formation. The subsequent labyrinth of brine networks connected to the ocean below can provide liquid water, nutrients, and dissolved gas that preserve viable life (Dieckmann 2002 , Loose et al. 2011 ). Viruses, prokaryotes, and eukaryotes have been isolated from terrestrial sea ice (Maranger et al. 1994 , Brown and Bowman 2001 , Lizotte 2003 , Mueller et al. 2005 ). Chroococcidiopsis CCMEE 029 and CCMEE 171 have been shown capable of surviving in sodium sulphate (Na 2 SO 4 ), Mg 2 SO 4 , and NaCl ice frozen to −40°C (Cosciotti et al. 2019 ). It has therefore been suggested that cryobrine networks in ice shells of icy moons could offer viable habitats, particularly those near the ice-ocean interface where temperatures are more suitable for biological propagation (Kargel et al. 2000 , Wettlaufer 2009 ). While a majority (∼80%) of these brine habitats on Europa may not supply enough nutrients to become inhabited, it is estimated ∼20% could sustain life without active growth under nutrient-limited conditions. A smaller portion of nutrient-rich brine channels, isolated near the ice-ocean interface, may additionally support active growth (Wolfenbarger et al. 2022a ). Such habitable ice environments proximal to the ice-ocean interface have also been suggested on Enceladus (Buffo et al. 2021 ). However, brine distribution within icy shells is also influenced by freeze-concentration processes resulting from sills and diapirs (Neveu et al. 2015 , Wolfenbarger et al. 2022b ). As pure water freezes from ascending ocean-derived melts or intrusions, residual liquids could become increasingly saline and NH 3 -rich, promoting accumulation of dense brines in veins and pockets within the surrounding ice (Neveu et al. 2017 ). Continued freezing could cause expansion and overpressure, contributing to fracture propagation, cryovolcanic transport, and plume or cryolava emplacement at the surface (Neveu et al. 2015 ). Such freeze-concentration may, in many settings, drive brine compositions toward high ionic strength and low water activity conditions that are less favourable for life. Consequently, the habitability of ice-shell brine networks likely varies spatially with thermal and emplacement history, and some veins produced by sill and diapir freezing may be comparatively inhospitable despite containing liquid and nutrients. When considering the permeation of NH 3 into ice shell niches, we can speculate that several scenarios are possible (Fig. 7 ). Brine compositions within ice shells could reflect the composition of the ocean from which the brine is derived. Brine channels, veins, pockets, and fractures within the ice could therefore contain NH 3 . Exclusion from the ice lattice during ocean water freezing may even concentrate NH 3 into the brines (Hammond et al. 2018 ), and pressure from the ice layer may encourage the formation of ammonia hydrates (Hogenboom et al. 1997 , Muñoz-Iglesias and Prieto-Ballesteros 2021 ). Upward shift of brines into the ice shell may promote exsolution of NH 3 (i.e., separation of NH 3 from the liquid phase) and release as a gas. This occurs as per Henry’s Law that states as pressure decreases, as does solubility of gas. However, it should be noted that Henry’s Law is assumed under constant temperature, and temperature would likely change in icy shells. Figure 7. Open in a new tab Penetration of NH 3 into icy moon ice shell habitats. (A) Brine may percolate into channels from the ocean below, delivering chemical constituents, including NH 3 and liquid water, to these habitats. (B) In some instances, NH 3 may concentrate within brine channels or pockets due to exclusion from the ice lattice. (C) Exsolution may occur at regions where liquid water is open to the vacuum or air, such as plume vent regions. (D) These regions may permit volatilization if fractures are open to the surface, leading to either dissolution of NH 3 into intersecting brine channels higher in the ice shelf or (E) release to the surface. (F) NH 3 adsorbed onto the ice shell may migrate to brine channels and pockets. (G) Alternatively, NH 3 could be deposited directly onto and within the ice shell if brine networks are exposed to plumes at the plume vent region. Fractures that are open to the surface may also enable NH 3 volatilization, depending on local pH, pressure, and temperature. Volatilization is a process in which a substance transitions from the liquid to the gas phase, escaping into the surrounding air or vacuum. During this phenomenon, the gaseous substance physically leaves liquid and enters the atmosphere or space. Volatilized NH 3 could migrate along the fracture system and potentially enter other intersecting brine networks within the ice shell. In icy-moon fracture and plume conduits, however, phase partitioning is likely coupled to freezing, boiling, and condensation on cold walls, so NH 3 transport may occur through a combination of gas flow, adsorption to ice, and re-dissolution into intersecting brines rather than simple free volatilization. NH 3 has been shown to adsorb onto ice between −50°C and −25°C (Richter et al. 2025 ). Plume activity at sites like the tiger stripes of Enceladus could feasibly deliver NH 3 gas to ice walls. At the ocean-plume interface, brine rises from the ocean into the vent system, where exsolution of NH 3 occurs. Within the plume shaft, NH 3 gas could begin condensing onto the ice walls or become incorporated into the ice shell via plume fallback. A hypothetical scenario might proceed as follows: NH 3 adsorbs onto ice; the adsorbed NH 3 migrates slowly through the ice shell by surface or bulk diffusion; NH 3 may enter liquid inclusions within the shell, where it dissolves into liquid brine. The dispersal and accumulation of NH 3 in brine channels, veins, pockets, or fractures of icy moons is analogous to NH 3 volatilization on Earth. NH 3 losses from ammonium fertilizer application range between 20%–40%, but could be as high as 66%, in pH 7 soils (Powlson and Dawson 2022 ). NH 3 has shown to mobilize from the initial, local site of release to distant environments (Sutton et al. 1998 , Bouet et al. 2005 , Leytem et al. 2024 , Lô et al. 2025 ). Results from the growth of an extremophile, H. meridiana , in proximity to an NH 3 source indicate that NH 3 from icy moon oceans could alter the potential for habitability not just locally within the ocean, but also at a distance in ice shell brine networks where dispersal of NH 3 gas is plausible (Hopton and Cockell 2025 ). Specifically, it was shown that lower concentrations of NH 3 /NH 4 + (i.e., ≤ 0.1 M) improved the growth of H. meridiana when cultivated adjacent to the NH 3 source, possibly by acting as a nitrogen source. Conversely, cultivation of H. meridiana proximal to higher concentrations of NH 3 /NH 4 + (i.e., ≥ 0.5 M) significantly reduced growth and viability. The scale of disruption to growth and viability was both distance-dependent and concentration-dependent; cultures in closer proximity to the NH 3 source showed greater detrimental growth effects, and higher concentrations elicited impacts on growth over a wider spatial range. In the context of icy shells, concentration gradients may arise from a combination of freeze-concentration, fracture transport, and phase partitioning rather than volatilization alone, but nonetheless this factor could influence the propagation of life in a spatial manner. Given the concentration thresholds for life established earlier, these results indicate the habitability of ice shell environments overlying an ocean of ≈ 0.0181 M NH 3 on Enceladus may not be limited by the dispersion of NH 3 into the brine network and could even possibly be enhanced by additional nitrogen input. Conversely, ice shell networks on Titan may be less likely to be habitable when in proximity to the more concentrated ammoniacal ocean. While brine networks higher in the ice shelf could receive little NH 3 dispersed from the ocean, these networks may also receive fewer nutrients to promote growth. Although the exact NH 3 concentrations in icy moon oceans have yet to be determined, the foregoing analysis highlights the need to measure the presence and concentration of NH 3 not just in these oceans, but also within the overlying ice shells, where possibly habitable, and more accessible, niches may exist. Conclusions, challenges, and perspectives The purpose of this review was to present a thorough synopsis of NH 3 within Enceladus and Titan and infer habitability prospects based upon known microbe-ammonia interactions on Earth. Direct analysis of the plumes of Enceladus indicate NH 3 is a constituent of the ocean, and models speculate NH 3 may also be a significant component of the putative ocean of Titan. Both NH 3 and NH 4 + are essential ingredients for life. NH 4 + is the preferred nitrogen source for many living organisms and forms a vital part of amino acid and protein synthesis, as well as the global nitrogen cycle. However, elevated concentrations of NH 3 can perturb internal biochemistry and may act to influence habitability prospects. Bacteria were chosen as biotic models in this review for assessing habitability as a function of NH 3 . Bacteria can survive in extreme conditions but also feature an array of metabolisms that could be suitable for surviving in cold and saline fluids where there is a variety of nutrients and organics. Literature analysis reveals that the highest threshold for growth of a bacteria in NH 3 extends to 0.5 M, with many bacteria showing survival limits around 0.05 to 0.3 M NH 3 . Research has also demonstrated that dispersion of NH 3 gas has little detrimental effect on bacterial growth and survival at ≤0.1 M NH 3 . The ocean of Enceladus is estimated to contain ≈ 0.0181 M NH 3 . The comparison of these two data indicates that the survival thresholds of Earth bacteria lie within the speculated concentrations of total ammonia on Enceladus, and thus NH 3 /NH 4 + in the ocean of Enceladus may not limit the potential for habitability. It is also possible that the habitability of overlying ice shell environments may not be limited by the infiltration or dispersion of oceanic NH 3 into the ice brine networks, if NH 3 concentrations delivered are similar or lower than those in the ocean. Conversely, significant NH 3 abundance (i.e., 0.96 M–9.6 M NH 3 ) may constrain the prospects for habitability on Titan. The known survival limits of terrestrial bacteria are below the speculated concentration of NH 3 in the ocean of Titan. It is possible strains such as B. pumilus and B. pasteurii could grow in concentrations beyond 0.5 M NH 3 , but this has yet to be demonstrated experimentally. NH 3 dispersed from a concentrated source at ≥ 0.5 M NH 3 has also been found to significantly reduce bacterial growth. These comparisons suggest NH 3 may constrain the potential for habitability of both the ocean of Titan and its overlying ice shell. However, the existence of a global subsurface ocean on Titan remains uncertain, and recent models favour cold, high-pressure ice or localized slushy layers that may be inhospitable (Petricca et al. 2025 ). The NH 3 -based habitability considerations discussed here are therefore only applicable if liquid or partially liquid ammonia–water phases occur within Titan’s interior. The comparisons of this review highlight the value of experimentally constraining microbial tolerance to single extraterrestrial parameters. Such data are essential for refining our understanding of biochemical limits to life and for guiding future astrobiological exploration of icy ocean worlds. Yet there are several challenges that prevent more nuanced perspectives. Firstly, we cannot directly compare icy moon oceans to any known environment on Earth. There are terrestrial environments that contain high concentrations of NH 3 effluent. However, these environments do not present the other physicochemical extremes (e.g., pH, temperature, pressure) that would make them suitable as an analogue for icy moon ocean environments. Moreover, we do not yet have precise physicochemical information regarding the oceans. Icy moon oceans are sealed below ice shells typically tens to hundreds of kilometres thick (Billings and Kattenhorn 2005 , Nimmo and Bills 2010 , Baland et al. 2014 , Čadek et al. 2016 , Lucchetti et al. 2017 , Levin et al. 2026 ). While missions could incorporate ice drilling instrumentation, extreme surface radiation and the increasing hardness of ice on icy moons pose significant challenges. Additionally, technological advancements in drilling are limited on Earth as ice sheets reach a maximum thickness of 4.9 kilometres (Fretwell et al. 2013 ). Due to this, the presence and abundances of salts, volatile organics, and the temperature, pH, and pressure of the oceans can only be estimated from surface observations. In turn, conditions within in vitro experiments can only be based upon these estimations or speculative assumptions. This is a particular challenge when considering NH 3 as a habitability factor: What is the concentration of NH 3 within the oceans? What species, NH 3 or NH 4 + , predominates in the oceans? Could the pressure and temperature conditions in the oceans sequester NH 3 into non-toxic solid hydrates? These questions cannot be defined with precision, yet they define toxicity. It is not without mention that, while this review evaluates habitability using terrestrial biology as a reference framework, another question is this: Could NH 3 serve as a biological solvent for life, thus rendering its toxic effects irrelevant? Alternative biochemical systems cannot be excluded. Ammoniacal waters have been proposed as potential solvents for hypothetical non-canonical life, and NH 3 -rich environments could in principle support chemistries distinct from those on Earth (Schulze-Makuch and Irwin 2018 ). Such possibilities fall outside the scope of the present review but underscore that NH 3 may influence habitability in ways not captured by terrestrial tolerance limits alone. Although this review has focused on Enceladus and Titan, NH 3 -bearing aqueous environments may occur across a broader range of solar system bodies, and indeed the universe. Localized brine reservoirs are inferred within Ceres (Raymond et al. 2020 , Nathues et al. 2022 ), and subsurface oceans or brine layers have been proposed for Triton (Gaeman et al. 2012 ), Pluto (Robuchon and Nimmo 2011 , Nimmo et al. 2016 ), Charon (Brown and Calvin 2000 , Cheng et al. 2014 , Rhoden et al. 2015 ), the large moons of Uranus (Cochrane et al. 2021 , Castillo‐Rogez et al. 2023), and other Kuiper Belt Objects (Hussmann et al. 2006 , Brown 2012 ). These environments extend the relevance NH 3 -derived habitability beyond Saturnian moons. They additionally widen the relevance of any planetary protection implications. Elevated NH 3 concentrations may inhibit survival or proliferation of many terrestrial micro-organisms, potentially reducing contamination risk in some environments (Deal et al. 1975 ). Conversely, NH 3 -tolerant or spore-forming microbes carried by spacecraft could persist in local niches comprised of NH 3 (Kelly et al. 2012 ). The consideration of NH 3 is therefore broadly important across icy and volatile-rich worlds for understanding habitability potential and planetary protection. As exploration of icy moons and other extraterrestrial ocean worlds advances, determining the abundance, speciation, and distribution of NH 3 will be central to assessing both the potential for life and the environments in which life might persist. In this sense, NH 3 emerges not just as a chemical constituent of these worlds, but as a parameter shaping their habitability. Contributor Information Cassie M Hopton, UK Centre for Astrobiology, School of Physics and Astronomy, University of Edinburgh, James Clerk Maxwell Building, Peter Guthrie Tait Road, Edinburgh EH9 3FD, United Kingdom. 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