Ammonia sets limit to life and alters physiology independently of pH in Halomonas meridiana

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Hopton, Peter Nienow, Charles S. Cockell This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6178140/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract S ignificant amounts of ammonia are released anthropogenically, while ammonia is also retained within the water bodies of icy moons that are of astrobiological interest. Ammonia is toxic to many forms of life at high concentrations, and thus it is necessary to understand the habitability impact of ammonia on these environments. The survival limits and physiological response of aerobic bacteria in ammonia, and whether ammonia toxicity is distinct from toxicity by high pH, is poorly understood. Here, we investigate the survival thresholds, growth kinetics, and metabolomic response of Halomonas meridiana in ammonia-water solutions and pH-matched sodium hydroxide solutions. Using closed- and open-air systems to mimic environments with ammonia retention or dispersion, we found complete and partial cell death above 0.05 M ammonia, respectively. In open-air systems, a sub-set of cells survived up to 0.25 M ammonia; metabolomics revealed unique physiological responses to ammonia, including elevation of cyclic compounds and Coenzyme A metabolites, suggesting mechanisms of ammonia toxicity and adaptation. Ammonia and high pH toxicity were found to be distinct. These findings show that ammonia can impose a distinct geobiological limit, potentially constraining habitability of ammonia-rich terrestrial and extraterrestrial environments. Earth and environmental sciences/Planetary science/Astrobiology Biological sciences/Microbiology/Bacteria/Bacterial physiology Biological sciences/Microbiology/Bacteriology Biological sciences/Physiology/Metabolism/Metabolomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ammonia (hereafter considered as total ammonia, ammonia (NH 3 ) + ammonium (NH 4 + )) is a prebiotic component of terrestrial environments. As such, ammonia is the preferred nitrogen source for many bacteria and plays an integral role in the global nitrogen cycle. Yet, despite biochemical significance, the toxicity of NH 3 has been well-documented across the domains of life 1 – 4 . NH 3 is gaseous under temperate conditions. The small size and non-polar properties of NH 3 facilitates passive permeation through membranes 5 – 8 , causing significant intracellular disruption 9 – 11 . The equilibrium between NH 3 and NH 4 + is influenced by factors such as pH (with NH 3 predominating at pH > 9.25), salinity and temperature 12 – 14 . Mono Lake, California, underscores the role of NH 3 in shaping microbial ecosystems; depth-dependent shifts in biodiversity are driven by the spatial distribution of abiotic factors including ammonia, which is predominately in the NH 3 form 15 , 16 . Globally, several dozen tetragrams of ammonia are released annually from agriculture 17 , 18 . Ammonia deposition into soil, water and vegetation occurs as a consequence 19 , 20 . In alkaline soils (pH > 7), application of ammonium fertilizers leads to NH 3 volatilization; NH 4 + is transitioned to gaseous NH 3 and released into the atmosphere. Indeed, applied nitrogen losses of up to 66% have been recorded in alkaline soils as a result of ammonium fertilizer application 21 – 23 . Although toxicological effects are presumed, the downstream impact of NH 3 volatilization on microbial diversity and community structures in alkaline environments, where the relative abundance of NH 3 exceeds NH 4 + , is not well understood. Likewise, the habitability impact of ammonia within extraterrestrial environments is also poorly considered. Ammonia is a primordial constituent of the solar system and has been detected at a volume mixing ratio of 0.61–1.3% on the icy moon Enceladus, orbiting Saturn 29 , 30 . The evidence of a saline, liquid water subsurface ocean 24 , 25 , and other physicochemical conditions suitable for life 26 – 28 , has encouraged strong astrobiological interest in this satellite. With a pH predicted at or above 9 27,28 , the ocean of Enceladus would bear appreciable amounts of toxic NH 3 . Due to the limited research on NH 3 tolerance in bacteria, coupled with the presence of carbon dioxide and hydrogen in the ocean, habitability assessments of Enceladus are currently limited to methanogenic organisms 31 – 33 . To accurately assess the habitability impacts of ammonia, fundamental questions remain. Few studies have systematically investigated the survival limits or physiology of microorganisms under high NH 3 stress. Bacteria such as Bacillus pasteurii can grow in up to 0.5 M NH 3 34 , however this species can utilise ammonia for ATP generation 35 , substrate permeability 36 and oxidation of substrates 37 . Ammonia-oxidising bacteria (AOB) can metabolise NH 3 but are primarily adapted to NH 4 + 38,39 . Without specific adaptations, toxicity at 0.1 M ammonia (pH 9.5) has been observed in B. subtilis and Escherichia coli . However, similar toxicity was noted in sodium chloride solutions of matching pH 40 . The use of neutrophilic organisms has thus far limited our understanding of whether ammonia toxicity is intrinsically pH-based. There is compelling need for research examining the survival limits and physiological response of an alkaline adapted bacteria in ammonia solutions exceeding pH 9.25 (NH 3 > 50%), and comparing with a pH matched counterpart. Here, we use a combination of growth kinetics and cell viability assays to identify habitability limits of Halomonas meridiana Slfth1 in ammonia solutions with high NH 3 content. This organism lacks specific adaptations to ammonia 41 , but possesses alkalitolerant traits relevant for this investigation 42 . We utilise growth systems permitting (“open-air system”) or preventing (“closed-air system”) gaseous escape to mimic environments where ammonia is environmentally dispersed and retained, respectively. By using an alkaline adapted organism, we resolve that ammonia toxicity is independent from pH toxicity and, and, using microscopy and metabolomics analysis, define the specific toxicity effects of ammonia on bacterial physiology independent of pH. Together, these findings advance the understanding of ammonia toxicity and should foster improved habitability assessments of NH 3 -rich environments. Results Physicochemical properties of ammonia and pH-matched solutions Ammonia solutions were prepared from liquid ammonia to molar concentrations of 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 and 1 M, equating to pH values between 8 and 11 (Fig. 1a). Positive control (PC) was unamended yeast media. The percentage abundance of NH 3 to NH 4 + at these concentrations and pH values is given in Fig. 1b. From concentrations of 0.05 M ammonia and higher, NH 3 accounts for over 50% of the total NH 3 /NH 4 + in solution. Solutions of increasing ammonia concentration showed decreased oxygen concentrations; however, this decrease was non-significant from pH-matched counterparts (Fig. 1c). Alterations to water activity with increasing ammonia and pH were also statistically non-significant (Fig. 1d). Ammonia sets a distinct molarity threshold for viability and growth Bacteria were grown in ammonia solutions utilising two system types: a ‘closed-air’ system – to determine growth limits in a perpetually ammonia exposed environments, and an ‘open-air’ system – to determine growth limits in an environment permitting ammonia dispersal. Fig. 2a shows the final colony forming units of H. meridiana after incubation in the closed-air system for 72 h. Colonies were evident following incubation in 0.01 M, 0.025 M and 0.05 M ammonia, but colony number decreased as NH 3 increased relative to NH 4 + (Fig. 1b). A lower number of viable colonies compared to positive control was observed in 0.01 M ammonia ( p < 0.01). No significant difference in colony number was observed when H. meridiana was incubated in 0.025 M compared to positive control ( p = 0.056) or 0.01 M ammonia ( p = 0.323). This concentration of ammonia has a pH within the optimum range for H. meridiana (pH 9) (Fig. 1a). Lower viable cell numbers were observed when incubated in 0.05 M compared to positive control ( p < 0.0001) and 0.025 M ammonia ( p < 0.01). No viable colonies were present following incubation in 0.1 M ammonia or ammonia solutions of higher concentration (Fig. 2a). Fig. 2b. shows the growth dynamics of H. meridiana Slthf1 over 48 h in increasing concentrations of ammonia in an open-air system. Lag phase duration, doubling time ( T d ) and final OD 600 are presented in Fig. 2c-e, respectively. At concentrations exceeding 0.01 M ammonia, lag phase was greater with higher ammonia concentrations; incubation in 0.1 M and 0.25 M ammonia prolongs lag phase time by 6-fold and 25-fold compared to the positive control, respectively (Fig. 2c). The lag phase time in 0.1 M ammonia was higher than that in 0.05 M ammonia ( p < 0.05). Likewise, the lag phase time in 0.25 M ammonia was higher than that in 0.1 M ammonia ( p < 0.0001) (Fig. 2c). T d was also higher with increasing ammonia concentration, with higher T d compared to the positive control observed in 0.05 M ( p < 0.05), 0.1 M ( p ≤ 0.0001) and 0.25 M ( p < 0.0001) solutions (Fig. 2d). The T d was not significantly altered between positive control and 0.01 M ( p = 0.965) or 0.1 M and 0.25 M solutions ( p = 0.9972). Final OD 600 after 48 h growth showed no statistically significant difference from positive control in 0.01 M ( p = 0.932), 0.025 M ( p = 0.330), 0.1 M ( p = 0.248) and 0.25 M solutions ( p = 0.135), but higher OD 600 values were observed in 0.05 M compared to the positive control ( p < 0.01) (Fig. 2e). Cell density was lower in 0.5 and 1 M solutions compared to the positive control (0.5 M, p < 0.01; 1 M, p < 0.001) (Fig. 2e), where no distinguishable growth occurred within 48 hours (Fig. 2b). The prolonged lag phase in cells inoculated into 0.1 M and 0.25 M ammonia indicate bactericidal reduction to the cell population. Alternatively, ammonia may act bacteriostatically, the effects of which cease once ammonia has dispersed. To assess this, temporal cell viability was compared with ammonia concentration over time in 0.1 M (Fig. 2f) and 0.25 M ammonia solutions (Fig. 2g). After 4 h exposure, viable cells in 0.1 M ammonia show a 1000-fold decrease from 0 h (t=28.28, df=4, p < 0.0001) (Fig. 2f). In 0.25 M ammonia, viable cells at 4 h are reduced 10-fold from 0 h (t=6.499, df=4, p < 0.01) (Fig. 2g). Bactericidal reduction to cell populations within 0.1 M ammonia solutions cease between 4 h and 8 h, where a small but non-significant increase in cell viability is observed t=2.421, df=4, p = 0.0727) (Fig. 2f). Likewise, bactericidal effects are absent between 4 h and 8 h in 0.25 M ammonia solutions where cell numbers stabilise (t=0.8934, df=4, p = 0.442) and increase from 8 h to 16 h (t=11.76, df=4, p < 0.001) (Fig. 2g). The increase in cell viability within 0.1 and 0.25 M ammonia solutions at 8 h and 16 h, respectively, aligns with diminished ammonia levels to ≤ 0.05 M at 4 h and 13 hr, respectively (Fig. 2f, 2g). Thus, lag phase extension in the open-air system reflects two events: (1) an immediate bactericidal effect and (2) growth initiation after ammonia levels drop to sub-bactericidal levels (≤ 0.05 M) in surviving populations. Ammonia toxicity is independent from pH toxicity Ammonia is a weak base that raises solution pH with increasing concentration. To delimitate ammonia toxicity from pH toxicity, growth experiments were repeated in NaOH solutions pH-matched to ammonia solutions. Fig. 3a presents the final CFU/mL of H. meridiana in solutions following 72 h closed-air system incubation. No significant differences were observed between cells grown in positive control and pH-matched solutions up to pH 10.78 (equivalent to 1 M ammonia). Fig. 3b presents the growth curve of H. meridiana grown under pH-matched NaOH solutions in an open-air system, with extrapolated parameters of lag phase (Fig. 3c), T d (Fig. 3d) and final OD 600 (Fig. 3e) presented in log 2 fold changes (log 2 FC) from growth in ammonia solutions of the same pH. Significantly lower lag phases were observed in cells grown in pH 8.96, 9.38, 9.73 and 10.18 compared to those grown at 0.025 M (t=4.399, df=3, p < 0.05), 0.05 M (t=11.26, df=3, p < 0.01), 0.1 M (t=7.357, df=3, p < 0.001) and 0.25 M ammonia (t=31.72, df=3, p < 0.0001) (Fig. 3c). The T d compared between cells grown in ammonia and pH-matched counterparts was non-significantly different except for those grown in pH 9.38 (t=3.542, df=3, p < 0.05), which showed a lower T d (Fig. 3d). Final OD 600 at 48 h was lower in pH 8.96 (t=4.882, df=3, p < 0.05), pH 9.38 (t=6.810, df=3, p < 0.01), pH 9.73 (t=4.123, df=3, p < 0.05) and pH 10.18 (t=7.206, df=3, p < 0.01) NaOH-solutions compared to growth in ammonia counterparts, while higher OD 600 was observed in pH 10.49 (t=16.09, df=3, p < 0.001) and pH 10.78 solutions (t=16.74, df=3, p < 0.001) compared to ammonia counterparts (Fig. 3e). These findings confirm that lag phase extension and absent growth in 0.5 and 1 M ammonia cannot be attributed to pH increases. To broaden the pH comparison beyond NaOH, lag phase analysis was used to explore whether the prolonged lag phase in 0.25 M ammonia (Fig. 2c) could be replicated in KOH, Na2SiO3, K2CO3, and Na2CO3 at pH 10.18 (Fig. 3f). No differences in lag phase duration were observed between H. meridiana grown in Na2SiO3 ( p = 0.779) or Na2CO3 ( p = 0.996) compared to NaOH. Longer lag phases were seen in KOH ( p < 0.05) and K2CO3 (p < 0.0001) compared to NaOH, possibly reflecting reduced adaptation to potassium in H. meridiana . All high-pH solutions showed significantly shorter lag phases than 0.25 M ammonia ( p < 0.001), confirming the pH-independent toxicity of ammonia. Ammonia toxicity exerts distinct changes to bacterial morphology Specific bactericidal effects of ammonia on cells can be observed in Fig. 4. Under control growth conditions (unaltered yeast media) (Fig. 4a-b), H. meridiana exhibited ribosome-rich cytoplasm, visible nucleoids and clear division of outermembrane, periplasm and inner membrane. Cells exhibited irregular, undulating outermembrane, enlarged periplasmic spaces and PHA-like granules. Upon 2 h treatment of 1 M ammonia (Fig. 4c-d), cells showed intracellular aggregation and loss of ribosomes with some showing cytoplasmic loss and lysis. The periplasmic space volume decreased, and detachment of the inner membrane from the outermembrane was observed. Cells showed expansion of electrolucent cavities, possibly surrounding nucleoids. Condensed material within the electrolucent cavities exhibited splayed morphologies, possibly indicating disruption to DNA supercoiling. Cells exposed to a NaOH solutions pH-matched to 1 M ammonia (10.78 pH) also showed some cell lysis events (Fig. 4e-f). However, there were few morphological differences from cells grown in control media. Differences include uniform outermembrane, and fewer PHA-like granules. Metabolic pathways altered in response to ammonia and high pH exposure The survival of cells in up to 0.25 M ammonia in an open-air system suggests adaptations enabling tolerance to ammonia. Thus, untargeted metabolomics was performed on H. meridiana in unamended yeast media (hereafter denoted ‘control’) and yeast media with 0.25 M ammonia at pH 10.18 (hereafter denoted ‘0.25 M ammonia ’ ) to determine variations in metabolism that may account for the differences observed. To delimitate high pH adaptations from ammonia adaptations, metabolomics was also performed following exposure to yeast media adjusted to pH 10.18 with NaOH (hereafter denoted ‘NaOH pH 10.18’). Growth kinetics and sampling points in these conditions are indicated in Fig. 5a. Principal component analysis (PCA) (Fig. 5b) separated 0.25 M ammonia and NaOH pH 10.18 conditions from the control, with overlap between 0.25 M ammonia and NaOH pH 10.18 conditions indicating metabolic similarity. Univariate volcano analysis identified 23 features significantly altered in 0.25 M ammonia/control (Fig. 5c), 10 features significantly altered in 0.25 M ammonia/NaOH pH 10.18 (Fig. 5d), and 28 features significantly altered in NaOH pH 10.18/control (Fig. 5e). The volcano analysis dataset for each comparison group is shown in Supplementary Table 2-4. The most significantly altered metabolites between these conditions were identified by ANOVA (Supplementary Table 5), depicted as a heatmap (Fig. 5f). Overall, high similarity was found between 0.25 M ammonia and NaOH pH 10.18 exposed samples; both conditions generally exhibited higher levels of unsaturated phospholipid and lower levels of linoleic acid and derivatives. Intermediates that feed glycerophospholipid biosynthesis, CMP-sialic acid (F = 2021.9, p < 0.0001) and glycerol-3-phosphate (F = 2022.2, p < 0.0001), were more abundant compared to the control. Amino acid pathway intermediates indole-3-ethanol (F = 2327.8, p < 0.0001), N, N-dimethylglycine (F = 30.626, p < 0.001) and N-acetylglutamate (F = 25.653, p < 0.01) were also altered compared to control. There were reduced levels of N-acetylserotonin (F = 18876, p < 0.0001) and N-acetyl-L-aspartic acid (F = 24.091, p < 0.01) that could suggest reactions with acetyl donors were less favourable at high pH in both conditions. Ammonia exposure elicits a unique metabolomic response Despite general similarities between cells exposed to 0.25 M ammonia and NaOH pH 10.18, ANOVA indicated five metabolites significantly altered only in 0.25 M ammonia exposed H. meridiana . Box plots of these metabolites are presented in Fig. 6a-e. Samples exposed to 0.25 M ammonia showed significantly higher levels of unidentified metabolites, metabolite i at m/z=215.084 (Fig. 6a) and metabolite ii at m/z=157.0973 (Fig. 6b), compared to NaOH pH 10.18 exposed samples (metabolite i, p < 0.0001; metabolite ii, p < 0.0001) and control samples (metabolite i, F = 2112.3, p < 0.0001; metabolite ii, F = 4865.6, p < 0.0001). Library annotations matched metabolite i to nitrogen-containing heterocyclic compound atrazine (89.6% Q score) and metabolite ii to hydrocarbon 2,7-dimethylnaphthalene (96.5% Q score). Structures of atrazine and 2,7-dimethylnaphthalene are depicted in Fig. 6a and Fig. 6b, respectively. Multivariate analysis also revealed exposure to 0.25 M ammonia increased the relative abundance of pantothenate (F = 28.839, p < 0.001) (Fig. 6c), and amino acids D-allo-isoleucine (F = 28.686, p < 0.001) (Fig. 6d), and alanine (F = 25.391, p < 0.01) (Fig. 6e), compared to those in NaOH pH 10.18 and control conditions. Discussion The influence of ammonia on habitability, particularly to microbes, is underrepresented in scientific literature. Yet, the pollution of ammonia into the environment could shape the underlying microbial community structures and biodiversity of alkaline environments. The presence of ammonia within the subsurface ocean of Enceladus, predicted at an alkaline pH, may also shape the habitability expectations of this satellite. Although the presence of life beyond Earth is speculative, terrestrial organisms can be utilised to explore the boundaries of known life under similar conditions to assess potential for habitability. In this study, we utilise alkalitolerant and halophilic H. meridiana as an analogue organism to investigate the molar concentration thresholds for survival, and physiological changes, that might be present in aerobic bacteria within extreme NH 3 environments. The exposure of H. meridiana to increasing ammonia concentrations revealed that growth at 0.05 M ammonia, where the relative abundance of NH 3 is 62%, sets a consistent habitability limit regardless of whether in a closed-air or open-air system. The tolerance of microbes to ammonia varies and cannot be predicted by genus or species alone; strain-specific responses to stress have been well documented 43 – 48 . However, the molarity limit established in this study aligns with data for other bacteria, including B. subtilis strains T1, T2, T19, T22, T33, T39 34 and Enterobacter cloaecae HNR 9 . The absolute limit for ammonia survival in the literature extends to 0.716 M (NH 4 ) 2 SO 4 at pH 9, survived by strains of B. subtilis and Proteus morgani 34 . However, the relative abundance of NH 3 in these studies is below 40%. Our results characterise a higher habitability limit in NH 3 than previously established for an aerobic bacterium. Independent ammonia and pH toxicity has been characterised in E. coli 40 , yet indistinguishable toxicity has been recorded with B. subtilis , Sporosarcina, Paenibacillus, Staphylococcus, Brevibacillus, Streptomyces, Pseudomonas and Arthrobacter 2 , 40 . Using an alkalitolerant organism, we establish ammonia toxicity is distinct from pH toxicity in H. meridiana , and does not exert toxicity via oxygen displacement or water activity changes. However, the degree of similarly between the metabolomic profiles of cells exposed to 0.25 M ammonia and a pH-matched NaOH solution indicates H. meridiana primarily responds to ammonia using high pH adaptations. Unique physiological responses to ammonia in H. meridiana included the accumulation of two cyclic compounds, one of which bore amine functional groups. The presence of these compounds may be indicative of intracellular NH 3 -driven reactions 49 , 50 . The disruption of such structures to cellular components could be a mechanism of pH-independent toxicity. Indeed, H. meridiana treated with ammonia exhibited aggregation to intracellular components that would suggest significant disruption to cell contents. Elevated levels of alanine, D-allo-isoleucine, and pantothenate were also exclusively observed in ammonia exposed H. meridiana. The presence of an isoleucine derivative and pantothenate suggested modulation to the Coenzyme A pathway that affects the cell membrane 51 , 52 , and amino acid metabolism 52 – 54 . Indeed, D-amino acids have been implicated in cell wall remodelling in Vibrio cholerae and B. subtilis 55 . Alanine dehydrogenase catalyses the production of alanine by a reaction between pyruvate and ammonium 56 – 58 . Thus, alanine could be a by-product of elevated ammonia levels. Notably, D-amino acid aminotransferase catalyses the production of pyruvate and D-glutamate ​from D-alanine and 2-oxoglutarate, for which D-allo-isoleucine can act as an amino donor in Thermotoga maritima 59 . The presence of D-allo-isoleucine may therefore lead to enrichment of pyruvate and, in turn, enrichment of D-alanine or L-alanine via the alanine dehydrogenase pathway, or both. D-alanine may act to impede ammonia permeation; D-alanine can increase rigidity, and decrease permeability, of the peptidoglycan layer 60 – 62 . L-alanine may act to prevent excessive ammonia metabolism; in some bacteria, L-alanine is an allosteric inhibitor of the nitrogen assimilation enzyme glutamine synthetase 63 – 65 . Interpreted from the point of view of planetary habitability, the ammonia toxicity limit established in this study is higher than the predicted 0.01–0.1 M ammonia content of the Enceladus ocean 28 , 30 . The closed-air system is the most accurate representation of icy moon subsurface oceans as ammonia concentrations are presumed to remain constant. With oceanic temperatures estimated at 0 ºC 26 and salinity at 4% 27 , the Enceladus ocean would require a pH of < 9.96 to satisfy the NH 3 < 62% habitability boundary suggested in this study, which is in range of current estimations 27 , 28 . In the terrestrial environment, up to 78 tetragrams of ammonia per annum are expelled globally from agricultural processes 17 , 18 . A water volume of 1 litre would require just 1 gram of ammonia to exceed an ammonia concentration of 0.05 M. In alkaline environments, this may alter bacterial community structures and biodiversity once the relative abundance of NH 3 exceeds thresholds for survival, as has been observed in Mono Lake 15 . However, unless NH 3 deposition is continuous, bactericidal effects may be transient due to the open-air dispersal of NH 3 – repopulation of H. meridiana in an open-air ammonia system was observed at concentrations above 0.05 M ammonia. Without field-based environmental surveys, the effects of NH 3 on terrestrial and extraterrestrial habitability are only estimated. Additionally, without isolation and biological characterisation of metabolites i and ii, we can only speculate the contribution of these molecules in ammonia toxicity. However, we have shown that bacterial growth and viability may become impacted when molar concentrations of ammonia are above 0.05 M, where NH 3 exceeds NH 4 + . Moreover, we deduce that ammonia and pH toxicity are distinct, suggesting a need to modify the current understanding of NH 3 toxicity in bacteria. Together, these findings can be integrated into the current knowledge regarding the impact of ammonia on terrestrial geomicrobiology and may inform future habitability assessments of NH 3 -rich extraterrestrial environments. Materials and Methods Solution preparation Solutions of liquid ammonia were made to the required molar concentrations—0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 M ammonia — in yeast media using 35% NH 3 (Fisher Scientific, CAS Number: 1336-21-6). The pH of the solutions was determined with a Jenway 3510 benchtop pH meter. pH-matched sodium hydroxide solutions (NaOH; Fisher Scientific, CAS Number: 1310-73-2) were prepared by gradual addition of 1 M NaOH into yeast media. To compare growth in different basic solutions at a pH equivalent to that of 0.25 M ammonia (pH 10.18), several bases were selected that have a natural pH at or greater than pH 10.18 and high solubility in water. Thus, yeast media was made to pH 10.18 ± 0.01 using gradual addition of 1 M NaOH, 1 M potassium hydroxide (KOH), 1 M sodium metasilicate (Na 2 SiO 3 ), 1 M potassium carbonate (K 2 CO 3 ) or 1 M sodium carbonate (Na 2 CO 3 ). Growth experiments proceeded as outlined below. pH was matched to within ± 0.01 pH units. All solutions were filter-sterilised through a 0.22-micron pore before use. NH 3 /NH 4 + percentage abundance The percentage concentration of ammonia was determined by indirect calculation based on commonly utilised equations developed by Hampson 12 . Firstly, the ionic strength of the prepared solutions based on known salinity (0.2 M NaCl) was determined by Eq. 1.0 \(\:I=19.9273\left(S\right)/(1000-1.005109\left(S\right))\) Eq. 1.0 Where I = molar ionic strength; S = salinity (parts per thousand, ppt). The stoichiometric acid hydrolysis constant of ammonium ions, pKa s , based on I, was then calculated. pKa s values for given ionic strengths were provided by Hampson 12 . These values were plotted to obtain a linear regression for calculating pKa (Supplementary Fig. 1). pKa s was calculated from Eq. 2.0. \(\:pK{a}^{s}=0.1179\left(I\right)+9.246\) Eq. 2.0 Percentage abundance of NH 3 at given salinity, pH and temperature was then calculated from Eq. 3.0. As salinity, temperature, pressure and pKa s are constant in the experiment, only pH was modified to give percentage abundance of NH 3 at each pH value (Supplementary Table 1). \(\:N{H}_{3}=N{H}_{3}+N{H}_{4}^{+}/(1+{10}^{\left({pKa}^{s}+0.0324\left(298-T\right)+0.0415\left(P\right)/T-1\right)}\) Eq. 3.0 Where P = 1 atm pressure; T = temperature (K) Oxygen measurements Oxygen concentrations (µmol/L) of unamended yeast media, ammonia solutions and pH-matched solutions, prepared as described above, were recorded using an oxygen microsenser with tip diameter of < 2 mm (OX-500, Unisense, Denmark). Solutions were equilibrated for 0.5 h before reading at 28 ºC in 96-well plates sealed with aluminium foil to prevent gas loss. Water activity Unamended yeast media, ammonia solutions and pH-matched solutions were prepared as described above. Solutions were equilibrated for 1.5 h and water activities measured at 28 ºC with a Rotronic HP23-AW water activity meter (Rotoronic AG, Bassersdorf, Switzerland). Bacterial strain selection Halomonas meridiana Slthf1 (DSM 15724; Gram negative) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). The organism was originally isolated at a depth of 2000 m from low temperature hydrothermal fluid in the East Pacific Rise. The strain exhibits halophilic growth up to 22% NaCl, and shows tolerance up to pH 12 42 . The strain is therefore representative of the physiological attributes that could be necessary to grow in saline, alkaline fluids. A complete genome sequence is available for this strain 41 . This organism is not known to have specific adaptations to ammonia; this was a deliberate choice. The purpose of this study was not to study microbial biochemical adaptation to high ammonia, but rather to study whether NH 3 can act as an abiotic factor influencing bacterial growth, survival and physiology. Bacterial culture An aerobic culture of H. meridiana was grown in glass conical Erlenmeyer flasks at 28 ºC in an orbital benchtop shaking incubator set to rotate at 150 RPM. We assume ammonia-rich environments present optimal nutrient conditions (i.e., available organic debris) with a salinity < 2% for the purpose of this study. Thus, H. meridiana Slthf1 was grown in a yeast media consisting of 1g/100 mL Bacto™ yeast extract (Becton, Dickinson and Company), 0.2 M NaCl (1.17% salinity) (Thermo Fisher Scientific, CAS Number: 7647-14-5) and deionised water (dH 2 O). Growth experiments Two growth systems were implemented: ‘closed’ growth experiments were conducted in tightly sealed 15 mL falcon tubes with sufficient headspace (14 mL) to allow aerobic metabolism but prevent ammonia evaporation; ‘open-air’ growth experiments were conducted in 96-well plates to permit ammonia evaporation and to monitor growth dynamics. Growth in closed-air system experiments were assessed by colony forming units (CFU) after 72 h incubation at 28 ºC. Growth in open-air systems were assessed in 96-well plates by optical density (OD) readings in a BMG SPECTROstar Nano Microplate Reader at 600 nm with reading taken every half an hour for 48 h at 28 ºC. Plate wells were prepared with 190 µL of a selected solution and seeded with 10 µL overnight H. meridiana culture to OD 600 = 0.05. Positive control wells were 190 µL unamended yeast media inoculated with 10 µL overnight H. meridiana culture to OD 600 = 0.05. Negative controls had no inoculation. To avoid condensation, plate lids were coated with Triton X-100 (0.05%) in 20% ethanol. Plates were shaken at 200 RPM before each reading. Growth parameters Experiments were conducted to yield three standard microbiological growth parameters: lag phase duration, doubling time ( T d ), and final OD 600 . The lag phase was calculated with the web-based Microbial Lag Phase Duration Calculator 66 , with the following parameters: algorithm = parameter fitting to a model; pre-processing applied: cut data at some time = yes, max time = 24 or 48 hours; smooth data = no; initial biomass = first observation; model to fit = logistic; NLS fitting algorithm = auto; max number of iterations = 1000. T d was calculated by first determining the growth rate in the exponential growth phase ( µ ) as per Eq. 4.0, followed by calculation of T d as per Eq. 5.0. \(\:\mu\:=\left({Log}_{10}\left(N\right)-{Log}_{10}\left({N}_{0}\right)\right)2.303/(t-{t}_{0})\) Eq. 4.0 Where N 0 = OD 600 at the beginning of a selected time interval ( t 0 ) in the exponential growth phase; N = OD 600 at the end of a selected time interval ( t ) in the exponential growth phase. t and t 0 are recorded in minutes. \(\:{T}_{\text{d}}=ln2/\mu\:\) Eq. 5.0 The final OD 600 reached after 48 hours was recorded and used to represent the final cell concentration reached. Cell viability versus OD 600 over 24 h was assessed by PrestoBlue ™ cell viability reagent (Thermo Fisher Scientific, Massachusetts, USA). Calibration curves confirm changes to observed OD 600 corresponds to increasing number of viable cells (Supplementary Fig. 2). Growth or lack of growth was determined by the presence or absence of a defined lag phase, exponential phase, and stationary phase within a 48 h growth period. Ammonia evaporation and cell viability Overnight culture of H. meridiana was inoculated to OD 600 = 0.05 in 0 (positive control), 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 and 1 M ammonia solutions into separate 96-well plates. Plates were incubated at 28 ºC on an orbital shaker for 4, 8, 16 and 24 hours. Ammonia content at these time intervals was recorded by the CHEMetrics High Range VACUette Ammonia test kit (K-1510C) using direct nesslerization. The parts per million (ppm) of ammonia in the sample was determined by colorimetric analysis of the Nessler reaction product at 420 nm 67 . Viability of cells was also assessed at these times by CFU on yeast media agar plates. Transmission electron microscopy An overnight culture of H. meridiana was pelleted, resuspended and exposed for 2 h to the following solutions: unamended yeast media (control), 1 M ammonia and a pH-matched solution (pH 10.78). The matched pH solution was created by gradual addition of 1 M NaOH to yeast media. Following incubation, cells were washed and resuspended in phosphate buffered saline (PBS). Samples were fixed in 3% glutaraldehyde in 0.1 M Sodium Cacodylate buffer, pH 7.3, for 2 hours then washed in three 10-minute changes of 0.1 M Sodium Cacodylate. Specimens were then post-fixed in 1% Osmium Tetroxide in 0.1 M Sodium Cacodylate for 45 minutes, then washed in three 10-minute changes of 0.1 M Sodium Cacodylate buffer. These samples were then dehydrated in 50%, 70%, 90% and 100% ethanol (X3) for 15 minutes each, then in two 10-minute changes in Propylene Oxide. Samples were then embedded in TAAB 812 resin. Sections, 1 µm thick were cut on a Leica Ultracut ultramicrotome, stained with Toluidine Blue, and viewed in a light microscope to select suitable areas for investigation. Ultrathin sections, 60 nm thick were cut from selected areas, stained in Uranyl Acetate and Lead Citrate then viewed in a JEOL JEM-1400 Plus TEM. Representative images were collected on a GATAN OneView camera at 4K resolution. Images were processed using ImageJ 57 Metabolomics sampling and extraction An overnight culture of H. meridiana was inoculated to OD 600 = 0.05 within 0.25 M ammonia, pH-matched yeast media (pH 10.18) or unamended yeast media (control) within a 24-well plate. The pH-matched solution was created by gradual addition of 1 M NaOH to yeast media. Growth occurred within plates at 28 ºC and was assessed by OD 600 reading every half an hour using a BMG SPECTROstar Nano Microplate Reader. Samples were harvested during the log phase of growth at OD 600 = 0.5. All the following procedures occurred at 4°C. Samples were aliquoted into microcentrifuge tubes and quenched by rapid cooling through submersion in a dry ice/70% (v/v) ethanol bath after brief incubation on ice. During cooling, samples were mixed vigorously to prevent freezing. Cells were separated from spent medium by centrifugation at 1,000 g for 10 minutes and supernatant discarded. Metabolite extraction occurred by addition of ice-cold chloroform/methanol/water (1:3:1 ratio). Cell lysis was encouraged by sonication with ice for 5 minutes at 37 kHz within an ultrasonication bath (Elmasonic S 60 H). Extraction mixtures were mixed vigorously at 1,200 rpm on an orbital shaker for 1 h. Following mixing, samples were centrifuged at 13,000 g for 3 minutes. The supernatant, containing metabolites, was collected into sterile microcentrifuge tubes. A pooled quality control sample was generated by combining equal volumes of metabolites from each sample. All samples were stored at − 80°C until analysis. Untargeted metabolomics analysis The untargeted metabolomics analysis was performed using liquid chromatography (LC) coupled to ion mobility (IM) quadrupole time-of-flight (qTOF) mass spectrometry (MS) as described previously (Ref 1). The instrumentation consisted of an Agilent 1290 Infinity II series UHPLC system hyphenated with an Agilent 6560 IM-qTOF with a Dual Agilent Jet Stream Electron Ionization source. LC separation was performed on an InfinityLab Poroshell 120 HILIC-Z, 2.1 mm × 50 mm, 2.7 µm UHPLC column (Agilent Technologies 689775–924) coupled to an InfinityLab Poroshell 120 HILIC-Z, 3.0 mm × 2.7 µm UHPLC guard column (Agilent Technologies 823750–948). A 3.5 min gradient was run using organic buffer (acetonitrile) combined with an aqueous buffer with low pH (10 mM ammonium formate, pH 3) or high pH (10 mM ammonium acetate, pH 9) for positive and negative ionization modes, respectively. Data was acquired using MassHunter Data Acquisition 10.0 software on 1 µL of sample separated on the column with a flow rate of 800 µL/min. The quality control sample was injected five times at the beginning of the experiment to condition the column and after every five test samples to monitor the instrument state throughout data acquisition. Data were acquired in the 50 to 1700 m/z range, with an MS acquisition rate of 0.8 scans/s. The metabolomics analyses were carried out by the EdinOmics research facility (RRID: SCR_021838) at the University of Edinburgh. Metabolomics data processing and statistical analysis The raw data files were processed using the Agilent MassHunter software suite. Briefly, ion multiplexed data files and calibration files were demultiplexed using the PNNL PreProcessor v2020.03.23 (the default settings for demultiplexing, moving average smoothing, saturation repair and spike removal were applied to the data). The data files were recalibrated for accurate mass and drift time using the AgtTofReprocessUi and the IM-MS Browser 10.0, respectively. Molecular features were extracted in Mass Profiler 10.0 with a retention time tolerance of ± 0.3 min, drift time tolerance of ± 1.5% and accurate mass tolerance of ± (5 ppm + 2 mDa). Features were annotated based on accurate mass and collision cross section (CCS) values using McLean CCS Compendium PCDL library 68 . Multivariate and univariate statistical analysis was performed using the MetaboAnalyst 6.0 web-based platform 69 . The input data were log-transformed and Pareto-scaled. The results were visualised using principal component analysis (PCA), box and whisker plots, volcano plots and heatmaps. Further aesthetic adjustments, including font changes and line thickness modifications, were made using Inkscape (version 1.0.1). Chemical structures were drawn using ChemDraw (version 23.1.2) and exported in SVG format for inclusion in the figures. The raw data associated with this dataset is available to view in the Supplementary Dataset. This study focuses on metabolomic changes in ammonia, pH and control samples, but the broader metabolomics profiling also included samples exposed to (NH 4 ) 2 SO 4 . For analysis, only ammonia, pH and control samples were included in this study. The metabolomics of (NH 4 ) 2 SO 4 exposed samples are to be addressed in a separate analysis. Statistics and reproducibility All data were compiled from a minimum of three different cultures inoculated on different days with new media (n = 3–4 biological replicates). The normality of data was assessed with the Shapiro-Wilk test. For comparison of two groups, paired or unpaired two-tailed t-tests were used. The Wilcoxon matched-pairs signed-rank test was used for paired groups where the assumption of normality was violated. An unpaired t test with Welch's correction was applied for groups with unequal variance. For comparison of two or more groups, equal variance was assessed with the Brown-Forsythe test. Samples of equal variance were analysed by analysis of variance (ANOVA) followed by Tukey’s post-hoc test. For samples where variance was not equal, Welch’s ANOVA test with Games-Howell’s post-hoc test was used. Statistical tests are specified in figure legends. Significance was considered when p < 0.05. All statistical analyses were performed using GraphPad Prism version 8.0.2 (GraphPad Software Inc.). Declarations Data Availability All data generated or analysed during this study are included in this published article within the Supplementary Dataset, except when specified as part of the Supplementary Material. Acknowledgements Funding for this research was provided by the Natural Environmental Research Council (NERC) though an E4 Doctoral Training Partnership (DTP) studentship (NE/S007407/1). C.H. and C.S.C thanks the Science and Technology Facilities Council (STFC) for support under grant ST/V000586/1 and ST/Y001788/1. We also acknowledge the support of the Wellcome Trust Multiuser Equipment Grant (WT104915MA) for use of the JEOL JEM-1400 Plus TEM. We acknowledge the EdinOmics research facility at the University of Edinburgh where the metabolomics analyses were carried out. Author information Cassie M. Hopton & Charles S. Cockell - School of Physics and Astronomy, University of Edinburgh, James Clerk Maxwell Building, Peter Guthrie Tait Road, Edinburgh, EH9 3FD, United Kingdom Peter Nienow - School of Geosciences, University of Edinburgh, Drummond St, Edinburgh, EH8 9XP, United Kingdom Author contributions C.H. conducted the experiments, data analysis and paper writing. C.S.C and P.N. jointly supervised this work, provided instruments and materials for experiments, and provided input in the writing of the paper. Corresponding author Cassie M. 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Additional Declarations No competing interests reported. Supplementary Files HoptonSupplementarydataset1.xlsx HoptonSupplementaryMaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 04 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 25 Apr, 2025 Reviews received at journal 24 Apr, 2025 Reviewers agreed at journal 04 Apr, 2025 Reviews received at journal 03 Apr, 2025 Reviewers agreed at journal 22 Mar, 2025 Reviewers invited by journal 20 Mar, 2025 Editor assigned by journal 20 Mar, 2025 Editor invited by journal 20 Mar, 2025 Submission checks completed at journal 19 Mar, 2025 First submitted to journal 19 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6178140","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":432503816,"identity":"b71b07ba-3b17-4ff4-9267-f8e295f8c502","order_by":0,"name":"Cassie M. 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(a)\u003cstrong\u003e \u003c/strong\u003eMolar levels of ammonia and corresponding pH values utilised in this study. Column heights and error bars represent mean ± s.d. (n = 3). (b) Calculated percentage abundance of NH\u003csub\u003e3\u003c/sub\u003e/NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in molar levels of ammonia utilised in this study. (c) Oxygen concentration and (d) water activity of ammonia and pH-matched solutions utilised in this study. The upper limit, middle line and lower limit of the boxplots indicate the 25th, 50th (median) and 75th percentiles, respectively. Whiskers represent 1.5× the interquartile ranges. Mean is indicated by a plus sign (+) (n = 4). (c) Decreases to oxygen concentration were found to be non-significant between ammonia and pH-match counterparts: 0.01 M ammonia\u003csub\u003e \u003c/sub\u003evs pH 8.05 (t=1.816, df=3, \u003cem\u003ep\u003c/em\u003e = 0.167); 0.025 M ammonia vs pH 8.96, (t=0.1976, df=3, \u003cem\u003ep \u003c/em\u003e= 0.856); 0.05 M ammonia\u003csub\u003e \u003c/sub\u003evs pH 9.38, (t=0.1722, df=3, \u003cem\u003ep \u003c/em\u003e= 0.874); 0.1 M ammonia\u003csub\u003e \u003c/sub\u003evs. pH 9.73, (t=0.3507, df=3, \u003cem\u003ep \u003c/em\u003e= 0.749); 0.25 M ammonia\u003csub\u003e \u003c/sub\u003evs. pH 10.18, (t=0.5734, df=3, \u003cem\u003ep \u003c/em\u003e= 0.607); 0.5 M ammonia\u003csub\u003e \u003c/sub\u003evs. pH 10.49, (t=2.150, df=3, \u003cem\u003ep \u003c/em\u003e= 0.121); 1 M ammonia\u003csub\u003e \u003c/sub\u003evs pH 10.78, (t=0.2348, df=3, \u003cem\u003ep = \u003c/em\u003e0.830). (d) Alterations to water activity between ammonia and pH-match solutions were also found to be non-significant: 0.01 M ammonia vs pH 8.05, (t=0.7871, df=3, \u003cem\u003ep\u003c/em\u003e = 0.489); 0.025 M ammonia vs pH 8.96, (t=0.1630, df=1, \u003cem\u003ep\u003c/em\u003e = 0.897); 0.05 M ammonia vs pH 9.38, (t=0.4448, df=3, \u003cem\u003ep\u003c/em\u003e = 0.687); 0.1 M ammonia vs. pH 9.73, (t=0.4205, df=3, \u003cem\u003ep \u003c/em\u003e= 0.702); 0.25 M ammonia vs. pH 10.18, (t=1.408, df=3, \u003cem\u003ep\u003c/em\u003e = 0.254); 0.5 M ammonia vs. pH 10.49, (t=0.7924, df=3, \u003cem\u003ep \u003c/em\u003e= 0.486); 1 M ammonia vs pH 10.78, (t=1.185, df=3, \u003cem\u003ep\u003c/em\u003e = 0.321).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6178140/v1/fa98bd363425d0ea662038c5.jpg"},{"id":79080681,"identity":"4522183e-ba65-4bae-990c-4108f5970f0a","added_by":"auto","created_at":"2025-03-24 08:22:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":251543,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival limits of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. meridiana \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein ammonia. \u003c/strong\u003e(a) CFU/mL of \u003cem\u003eH. meridiana \u003c/em\u003egrown in ammonia solutions at 0, 0.01, 0.025, 0.05 and 0.1 M in a closed system for 72 hours. Column heights and error bars represent mean ± s.d. (n = 3). Statistical significance is by one-way ANOVA with Tukey’s multiple comparison test (F (4, 10) = 27.70, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001). (b) Growth curve of \u003cem\u003eH. meridiana \u003c/em\u003eover 48 h in increasing ammonia concentrations. Growth curves represent mean OD\u003csub\u003e600\u003c/sub\u003e values over time ± s.d. (0-0.5 M, n = 4; 1 M, n = 3). Error is indicated by area fill within error bands. Growth parameters of lag phase (c), doubling time (\u003cem\u003eT\u003c/em\u003ed) (d) and final OD\u003csub\u003e600\u003c/sub\u003e at 48 hours (e) extrapolated from (b) are presented. Box plots represent the median as well as the 25% and 75% interquartile ranges. The whiskers represent 1.5× the interquartile ranges. Plus sign (+) indicate the mean and the middle line indicate the median. Welch’s ANOVA with Games-Howell’s multiple comparison test was used in (c) W (3.000, 5.965) = 218.6, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) and (e) W (7.000, 9.736) = 7919, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). Statistics in (d) correspond to one-way ANOVA with Tukey’s multiple comparison test (F (5, 18) = 14.79, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001) (f, g) Time series of ammonia molarity and cell viability over 24 h in 0.1 (f) and 0.25 M ammonia (g). Ammonia molarity is plotted as a line graph with blue diamond markers on the right axis. Molarity at 0.05 M is indicated by a dotted line. Cell viability is plotted as a column bar graph on the left axis. Line and column heights represent mean ± s.d. (n = 3). For cell viability, statistical difference between means is given by unpaired t-test. ns, no significance; *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6178140/v1/371c7ae28fb31c09d35a778f.jpg"},{"id":79080683,"identity":"27debbdb-f93e-4180-92cd-b27d8ffc399e","added_by":"auto","created_at":"2025-03-24 08:22:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":61968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. meridiana \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein NaOH pH-matched solutions. \u003c/strong\u003e(a) CFU/mL of \u003cem\u003eH. meridiana \u003c/em\u003egrown in a closed system for 72 h with solutions of increasing pH that pH-match ammonia solutions of 0 (pH 6.06), 0.01 (pH 8.05), 0.025 (pH 8.96), 0.05 (pH 9.38), 0.1 (pH 9.73), 0.25 (pH 10.18). 0.5 (pH 10.49) and 1 M (pH 10.78). Column heights and error bars represent mean ± s.d. (n = 3-4). Statistical significance is by one-way ANOVA with Tukey’s multiple comparison test (F (7, 16) = 5.292, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). (b) Growth curve of \u003cem\u003eH. meridiana \u003c/em\u003eover 48 h grown in NaOH solutions of increasing pH. Growth curves represent mean OD\u003csub\u003e600\u003c/sub\u003e values over time ± s.d. (n = 4). Error is indicated by area fill within error bands. Growth parameters of lag phase (c), doubling time (\u003cem\u003eT\u003c/em\u003ed) (d) and final OD\u003csub\u003e600\u003c/sub\u003e at 48 hours (e) extrapolated from (b) are presented as log\u003csub\u003e2\u003c/sub\u003e fold change (FC) values comparing pH/ammonia. Statistics correspond to paired two-tailed t-tests comparing the mean value for lag phase, \u003cem\u003eT\u003c/em\u003ed and final OD\u003csub\u003e600\u003c/sub\u003e at each pH condition to the corresponding ammonia molarity as in Fig. 3c-e, respectively, with exception of \u003cem\u003eT\u003c/em\u003ed for pH 9.39 which corresponds to a Wilcoxon matched-pairs signed rank test. Column heights and error bars represent mean ± s.d. (n = 4). (f) Box plots comparing the lag phase of \u003cem\u003eH. meridiana \u003c/em\u003egrown in different pH-matched solutions at pH 10.18. Box plots represent the median as well as the 25% and 75% interquartile ranges. The whiskers represent 1.5× the interquartile ranges. The plus signs (+) indicate the mean and the central line indicate the median (n = 4). Welch’s ANOVA with Games-Howell’s multiple comparison test was used (W (5.000, 8.089) = 170.4). ns, no significance; *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6178140/v1/9929495d0111c7554fd14073.jpg"},{"id":79080687,"identity":"be3a253a-d636-4ae1-afe8-9e5d764aee21","added_by":"auto","created_at":"2025-03-24 08:22:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":836255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTEM of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. meridiana\u003c/strong\u003e\u003c/em\u003e. \u003cem\u003eH. meridiana\u003c/em\u003e after 2 h treatment in control solutions (a, b), 1 M ammonia (c, d), and pH-matched yeast media (pH 10.78, e, f). Yellow arrows: 1, outer membrane; 2, periplasmic space; 3, inner membrane; 4, nucleoid; 5, PHA-like granule; 6, lysed cell; 7, aggregated material; 8, electrolucent cavities.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6178140/v1/f5adf028bd364e30a241a42d.jpg"},{"id":79081483,"identity":"98bac868-dc9b-4a65-9e14-dd7b9bebd8c6","added_by":"auto","created_at":"2025-03-24 08:30:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":256051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolic features significantly altered upon ammonia and high pH exposure in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. meridiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (a) Growth curve of samples cultured for metabolomics processing with each replicate shown. Dotted line at OD\u003csub\u003e600\u003c/sub\u003e=0.5 indicates harvest point. (b) Scores plot of a principal component analysis (PCA). (c-e) Volcano plots depicting metabolites with a fold change greater than 2 and a \u003cem\u003ep\u003c/em\u003e-value lower than 0.05 (adjusted using FDR correction) for 0.25 M ammonia/control (c), 0.25 M ammonia/NaOH pH 10.18 (d) and NaOH pH 10.18/control (e). Relative levels of each metabolite are presented as red dots (high) or blue dots (low). (f) Heatmap of the top 25 metabolites most significantly altered between ammonia, pH and control treated samples. Significance was calculated by one-way ANOVA (alpha=0.05, two-sided) with Tukey’s post-doc testing using Metaboanalyst 6.0. Significance level is indicated next to metabolites. The normalised relative abundance is presented in a gradient from dark blue (high) to yellow (low). All data compiled from three biological replicates (n = 3). PCA, volcano plots and heatmap were generated using MetaboAnalyst 6.0 and amended for visual clarity in Inkscape. **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6178140/v1/c5ced82931b08994109fe6c5.jpg"},{"id":79080689,"identity":"7991c589-2e24-448a-99ad-1a64954b8a0f","added_by":"auto","created_at":"2025-03-24 08:22:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":118114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolic response to ammonia exposure in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. meridiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(a-e) Box plots of five significantly altered metabolites found in 0.25 M ammonia exposed \u003cem\u003eH. meridiana\u003c/em\u003e (ammonia) compared to NaOH pH 10.18 (pH) and control samples (control)\u003cem\u003e. \u003c/em\u003eResults are taken from ANOVA with Tukey post-hoc test (Supplementary Table S5). The upper limit, middle line and lower limit of the boxplots indicate the 25th, 50th (median) and 75th percentiles, respectively. Whiskers represent 1.5× the interquartile ranges. Mean is indicated by a black diamond. Coloured circles represent the values from all samples (n=3 for each group). Box and whiskers were generated using MetaboAnalyst 6.0 and edited for visual clarity in Inkscape. Chemical structures were created using ChemDraw. **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6178140/v1/d9667c68d04514b6bbc06a4e.jpg"},{"id":84242576,"identity":"db6a95b3-51e7-4bb3-a28d-2b4326e44a41","added_by":"auto","created_at":"2025-06-09 16:09:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3010786,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6178140/v1/b17210dd-4810-4b7a-a179-67f6bd43f5bd.pdf"},{"id":79080684,"identity":"e28f76ec-30ee-4225-9a23-506ce9a01c96","added_by":"auto","created_at":"2025-03-24 08:22:20","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":226601,"visible":true,"origin":"","legend":"","description":"","filename":"HoptonSupplementarydataset1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6178140/v1/d527ef039d901bcca225df09.xlsx"},{"id":79081484,"identity":"2434c56f-424d-44ca-b2da-61b0f2af1a26","added_by":"auto","created_at":"2025-03-24 08:30:20","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":204138,"visible":true,"origin":"","legend":"","description":"","filename":"HoptonSupplementaryMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6178140/v1/7157adee7cffc684eb17c679.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ammonia sets limit to life and alters physiology independently of pH in Halomonas meridiana","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAmmonia (hereafter considered as total ammonia, ammonia (NH\u003csub\u003e3\u003c/sub\u003e)\u0026thinsp;+\u0026thinsp;ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e)) is a prebiotic component of terrestrial environments. As such, ammonia is the preferred nitrogen source for many bacteria and plays an integral role in the global nitrogen cycle. Yet, despite biochemical significance, the toxicity of NH\u003csub\u003e3\u003c/sub\u003e has been well-documented across the domains of life \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. NH\u003csub\u003e3\u003c/sub\u003e is gaseous under temperate conditions. The small size and non-polar properties of NH\u003csub\u003e3\u003c/sub\u003e facilitates passive permeation through membranes \u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, causing significant intracellular disruption \u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The equilibrium between NH\u003csub\u003e3\u003c/sub\u003e and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e is influenced by factors such as pH (with NH\u003csub\u003e3\u003c/sub\u003e predominating at pH\u0026thinsp;\u0026gt;\u0026thinsp;9.25), salinity and temperature \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Mono Lake, California, underscores the role of NH\u003csub\u003e3\u003c/sub\u003e in shaping microbial ecosystems; depth-dependent shifts in biodiversity are driven by the spatial distribution of abiotic factors including ammonia, which is predominately in the NH\u003csub\u003e3\u003c/sub\u003e form \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGlobally, several dozen tetragrams of ammonia are released annually from agriculture \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Ammonia deposition into soil, water and vegetation occurs as a consequence \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In alkaline soils (pH\u0026thinsp;\u0026gt;\u0026thinsp;7), application of ammonium fertilizers leads to NH\u003csub\u003e3\u003c/sub\u003e volatilization; NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e is transitioned to gaseous NH\u003csub\u003e3\u003c/sub\u003e and released into the atmosphere. Indeed, applied nitrogen losses of up to 66% have been recorded in alkaline soils as a result of ammonium fertilizer application \u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Although toxicological effects are presumed, the downstream impact of NH\u003csub\u003e3\u003c/sub\u003e volatilization on microbial diversity and community structures in alkaline environments, where the relative abundance of NH\u003csub\u003e3\u003c/sub\u003e exceeds NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, is not well understood. Likewise, the habitability impact of ammonia within extraterrestrial environments is also poorly considered. Ammonia is a primordial constituent of the solar system and has been detected at a volume mixing ratio of 0.61\u0026ndash;1.3% on the icy moon Enceladus, orbiting Saturn \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The evidence of a saline, liquid water subsurface ocean \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, and other physicochemical conditions suitable for life \u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, has encouraged strong astrobiological interest in this satellite. With a pH predicted at or above 9 \u003csup\u003e27,28\u003c/sup\u003e, the ocean of Enceladus would bear appreciable amounts of toxic NH\u003csub\u003e3\u003c/sub\u003e. Due to the limited research on NH\u003csub\u003e3\u003c/sub\u003e tolerance in bacteria, coupled with the presence of carbon dioxide and hydrogen in the ocean, habitability assessments of Enceladus are currently limited to methanogenic organisms \u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo accurately assess the habitability impacts of ammonia, fundamental questions remain. Few studies have systematically investigated the survival limits or physiology of microorganisms under high NH\u003csub\u003e3\u003c/sub\u003e stress. Bacteria such as \u003cem\u003eBacillus pasteurii\u003c/em\u003e can grow in up to 0.5 M NH\u003csub\u003e3\u003c/sub\u003e \u003csup\u003e34\u003c/sup\u003e, however this species can utilise ammonia for ATP generation \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, substrate permeability \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and oxidation of substrates \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Ammonia-oxidising bacteria (AOB) can metabolise NH\u003csub\u003e3\u003c/sub\u003e but are primarily adapted to NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+ 38,39\u003c/sup\u003e. Without specific adaptations, toxicity at 0.1 M ammonia (pH 9.5) has been observed in \u003cem\u003eB. subtilis and Escherichia coli\u003c/em\u003e. However, similar toxicity was noted in sodium chloride solutions of matching pH \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The use of neutrophilic organisms has thus far limited our understanding of whether ammonia toxicity is intrinsically pH-based. There is compelling need for research examining the survival limits and physiological response of an alkaline adapted bacteria in ammonia solutions exceeding pH 9.25 (NH\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;50%), and comparing with a pH matched counterpart.\u003c/p\u003e \u003cp\u003eHere, we use a combination of growth kinetics and cell viability assays to identify habitability limits of \u003cem\u003eHalomonas meridiana\u003c/em\u003e Slfth1 in ammonia solutions with high NH\u003csub\u003e3\u003c/sub\u003e content. This organism lacks specific adaptations to ammonia \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, but possesses alkalitolerant traits relevant for this investigation \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. We utilise growth systems permitting (\u0026ldquo;open-air system\u0026rdquo;) or preventing (\u0026ldquo;closed-air system\u0026rdquo;) gaseous escape to mimic environments where ammonia is environmentally dispersed and retained, respectively. By using an alkaline adapted organism, we resolve that ammonia toxicity is independent from pH toxicity and, and, using microscopy and metabolomics analysis, define the specific toxicity effects of ammonia on bacterial physiology independent of pH. Together, these findings advance the understanding of ammonia toxicity and should foster improved habitability assessments of NH\u003csub\u003e3\u003c/sub\u003e-rich environments.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhysicochemical properties of ammonia and pH-matched solutions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmmonia solutions were prepared from liquid ammonia to molar concentrations of 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 and 1 M, equating to pH values between 8 and 11 (Fig. 1a). Positive control (PC) was unamended yeast media. The percentage abundance of NH\u003csub\u003e3\u003c/sub\u003e to NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e at these concentrations and pH values is given in Fig. 1b. From concentrations of 0.05 M ammonia and higher, NH\u003csub\u003e3\u003c/sub\u003e accounts for over 50% of the total NH\u003csub\u003e3\u003c/sub\u003e/NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u0026nbsp;\u003c/sup\u003ein solution. Solutions of increasing ammonia concentration showed decreased oxygen concentrations; however, this decrease was non-significant from pH-matched counterparts (Fig. 1c). Alterations to water activity with increasing ammonia and pH were also statistically non-significant (Fig. 1d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAmmonia sets a distinct molarity threshold for viability and growth\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacteria were grown in ammonia solutions utilising two system types: a \u0026lsquo;closed-air\u0026rsquo; system \u0026ndash; to determine growth limits in a perpetually ammonia exposed environments, and an \u0026lsquo;open-air\u0026rsquo; system \u0026ndash; to determine growth limits in an environment permitting ammonia dispersal. Fig. 2a shows the final colony forming units of \u003cem\u003eH. meridiana\u0026nbsp;\u003c/em\u003eafter incubation in the closed-air system for 72 h. Colonies were evident following incubation in 0.01 M, 0.025 M and 0.05 M ammonia, but colony number decreased as NH\u003csub\u003e3\u003c/sub\u003e increased relative to NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (Fig. 1b). A lower number of viable colonies compared to positive control was observed in 0.01 M ammonia\u003csub\u003e\u0026nbsp;\u003c/sub\u003e(\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01). No significant difference in colony number was observed when \u003cem\u003eH. meridiana\u0026nbsp;\u003c/em\u003ewas incubated in 0.025 M compared to positive control (\u003cem\u003ep\u003c/em\u003e = 0.056) or 0.01 M ammonia (\u003cem\u003ep\u003c/em\u003e = 0.323). This concentration of ammonia has a pH within the optimum range for \u003cem\u003eH. meridiana\u0026nbsp;\u003c/em\u003e(pH 9) (Fig. 1a). Lower viable cell numbers were observed when incubated in 0.05 M compared to positive control (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) and 0.025 M ammonia (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). No viable colonies were present following incubation in 0.1 M ammonia or ammonia solutions of higher concentration (Fig. 2a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFig. 2b. shows the growth dynamics of \u003cem\u003eH. meridiana\u0026nbsp;\u003c/em\u003eSlthf1 over 48 h in increasing concentrations of ammonia in an open-air system. Lag phase duration, doubling time (\u003cem\u003eT\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e) and final OD\u003csub\u003e600\u003c/sub\u003e are presented in Fig. 2c-e, respectively. At concentrations exceeding 0.01 M ammonia, lag phase was greater with higher ammonia concentrations; incubation in 0.1 M and 0.25 M ammonia prolongs lag phase time by 6-fold and 25-fold compared to the positive control, respectively (Fig. 2c). The lag phase time in 0.1 M ammonia was higher than that in 0.05 M ammonia (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Likewise, the lag phase time in 0.25 M ammonia was higher than that in 0.1 M ammonia (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001) (Fig. 2c). \u003cem\u003eT\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e was also higher with increasing ammonia concentration, with higher \u003cem\u003eT\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e compared to the positive control observed in 0.05 M (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), 0.1 M (\u003cem\u003ep\u003c/em\u003e \u0026le; 0.0001) and 0.25 M (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001) solutions (Fig. 2d). The \u003cem\u003eT\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e was\u003cem\u003e\u0026nbsp;\u003c/em\u003enot significantly altered between positive control and 0.01 M (\u003cem\u003ep\u003c/em\u003e = 0.965) or 0.1 M and 0.25 M solutions (\u003cem\u003ep\u003c/em\u003e = 0.9972). Final OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003eafter 48 h growth\u0026nbsp;showed no statistically significant difference from positive control in 0.01 M (\u003cem\u003ep\u003c/em\u003e = 0.932), 0.025 M (\u003cem\u003ep\u003c/em\u003e = 0.330), 0.1 M (\u003cem\u003ep\u003c/em\u003e = 0.248) and 0.25 M solutions (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.135), but higher OD\u003csub\u003e600\u003c/sub\u003e values were observed in 0.05 M compared to the positive control (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) (Fig. 2e). Cell density was lower in 0.5 and 1 M solutions compared to the positive control (0.5 M, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01; 1 M, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) (Fig. 2e), where no distinguishable growth occurred within 48 hours (Fig. 2b).\u003c/p\u003e\n\u003cp\u003eThe prolonged lag phase in cells inoculated into 0.1 M and 0.25 M ammonia indicate bactericidal reduction to the cell population. Alternatively, ammonia may act bacteriostatically, the effects of which cease once ammonia has dispersed. To assess this, temporal cell viability was compared with ammonia concentration over time in 0.1 M (Fig. 2f) and 0.25 M ammonia solutions (Fig. 2g). After 4 h exposure, viable cells in 0.1 M ammonia show a 1000-fold decrease from 0 h (t=28.28, df=4, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001) (Fig. 2f). In 0.25 M ammonia, viable cells at 4 h are reduced 10-fold from 0 h (t=6.499, df=4, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) (Fig. 2g). Bactericidal reduction to cell populations within 0.1 M ammonia\u003csub\u003e\u0026nbsp;\u003c/sub\u003esolutions cease between 4 h and 8 h, where a small but non-significant increase in cell viability is observed t=2.421, df=4, \u003cem\u003ep\u003c/em\u003e = 0.0727) (Fig. 2f). Likewise, bactericidal effects are absent between 4 h and 8 h in 0.25 M ammonia\u003csub\u003e\u0026nbsp;\u003c/sub\u003esolutions where cell numbers stabilise (t=0.8934, df=4, \u003cem\u003ep\u003c/em\u003e = 0.442) and increase from 8 h to 16 h (t=11.76, df=4, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) (Fig. 2g). The increase in cell viability within 0.1 and 0.25 M ammonia\u003csub\u003e\u0026nbsp;\u003c/sub\u003esolutions at 8 h and 16 h, respectively, aligns with diminished ammonia levels to \u0026le; 0.05 M at 4 h and 13 hr, respectively (Fig. 2f, 2g). Thus, lag phase extension in the open-air system reflects two events: (1) an immediate bactericidal effect and (2) growth initiation after ammonia levels drop to sub-bactericidal levels (\u0026le; 0.05 M) in surviving populations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAmmonia toxicity is independent from pH toxicity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmmonia is a weak base that raises solution pH with increasing concentration. To delimitate ammonia toxicity from pH toxicity, growth experiments were repeated in NaOH solutions pH-matched to ammonia solutions. Fig. 3a presents the final CFU/mL of \u003cem\u003eH. meridiana\u0026nbsp;\u003c/em\u003ein solutions following 72 h closed-air system incubation. No significant differences were observed between cells grown in positive control and pH-matched solutions up to pH 10.78 (equivalent to 1 M ammonia). Fig. 3b presents the growth curve of \u003cem\u003eH. meridiana\u0026nbsp;\u003c/em\u003egrown under pH-matched NaOH solutions in an open-air system, with extrapolated parameters of lag phase (Fig. 3c), \u003cem\u003eT\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e (Fig. 3d) and final OD\u003csub\u003e600\u003c/sub\u003e (Fig. 3e) presented in log\u003csub\u003e2\u003c/sub\u003e fold changes (log\u003csub\u003e2\u003c/sub\u003eFC) from growth in ammonia solutions of the same pH. Significantly lower lag phases were observed in cells grown in pH 8.96, 9.38, 9.73 and 10.18 compared to those grown at 0.025 M (t=4.399, df=3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), 0.05 M (t=11.26, df=3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), 0.1 M (t=7.357, df=3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) and 0.25 M ammonia (t=31.72, df=3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) (Fig. 3c). The \u003cem\u003eT\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e compared between cells grown in ammonia and pH-matched counterparts was non-significantly different except for those grown in pH 9.38 (t=3.542, df=3, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05), which showed a lower \u003cem\u003eT\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e (Fig. 3d). Final OD\u003csub\u003e600\u003c/sub\u003e at 48 h was lower in pH 8.96 (t=4.882, df=3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), pH 9.38 (t=6.810, df=3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), pH 9.73 (t=4.123, df=3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) and pH 10.18 (t=7.206, df=3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) NaOH-solutions compared to growth in ammonia counterparts, while higher OD\u003csub\u003e600\u003c/sub\u003e was observed in pH 10.49 (t=16.09, df=3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) and pH 10.78 solutions (t=16.74, df=3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) compared to ammonia counterparts (Fig. 3e). These findings confirm that lag phase extension and absent growth in 0.5 and 1 M ammonia cannot be attributed to pH increases.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo broaden the pH comparison beyond NaOH, lag phase analysis was used to explore whether the prolonged lag phase in 0.25 M ammonia (Fig. 2c) could be replicated in KOH, Na2SiO3, K2CO3, and Na2CO3 at pH 10.18 (Fig. 3f). No differences in lag phase duration were observed between \u003cem\u003eH. meridiana\u003c/em\u003e grown in Na2SiO3 (\u003cem\u003ep\u003c/em\u003e = 0.779) or Na2CO3 (\u003cem\u003ep\u003c/em\u003e = 0.996) compared to NaOH. Longer lag phases were seen in KOH (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) and K2CO3 (p \u0026lt; 0.0001) compared to NaOH, possibly reflecting reduced adaptation to potassium in \u003cem\u003eH. meridiana\u003c/em\u003e. All high-pH solutions showed significantly shorter lag phases than 0.25 M ammonia (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), confirming the pH-independent toxicity of ammonia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAmmonia toxicity exerts distinct changes to bacterial morphology\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecific bactericidal effects of ammonia on cells can be observed in Fig. 4. Under control growth conditions (unaltered yeast media) (Fig. 4a-b), \u003cem\u003eH. meridiana\u0026nbsp;\u003c/em\u003eexhibited ribosome-rich cytoplasm, visible nucleoids and clear division of outermembrane, periplasm and inner membrane. Cells exhibited irregular, undulating outermembrane, enlarged periplasmic spaces and PHA-like granules. Upon 2 h treatment of 1 M ammonia\u003csub\u003e\u0026nbsp;\u003c/sub\u003e(Fig. 4c-d), cells showed intracellular aggregation and loss of ribosomes with some showing cytoplasmic loss and lysis. The periplasmic space volume decreased, and detachment of the inner membrane from the outermembrane was observed. Cells showed expansion of electrolucent cavities, possibly surrounding nucleoids. Condensed material within the electrolucent cavities exhibited splayed morphologies, possibly indicating disruption to DNA supercoiling. Cells exposed to a NaOH solutions pH-matched to 1 M ammonia\u003csub\u003e\u0026nbsp;\u003c/sub\u003e(10.78 pH) also showed some cell lysis events (Fig. 4e-f). However, there were few morphological differences from cells grown in control media. Differences include uniform outermembrane, and fewer PHA-like granules.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMetabolic pathways altered in response to ammonia and high pH exposure\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe survival of cells in up to 0.25 M ammonia in an open-air system suggests adaptations enabling tolerance to ammonia. Thus, untargeted metabolomics was performed on \u003cem\u003eH. meridiana\u003c/em\u003e in unamended yeast media (hereafter denoted \u0026lsquo;control\u0026rsquo;) and yeast media with 0.25 M ammonia at pH 10.18 (hereafter denoted \u0026lsquo;0.25 M ammonia\u003csub\u003e\u0026rsquo;\u003c/sub\u003e) to determine variations in metabolism that may account for the differences observed. To delimitate high pH adaptations from ammonia adaptations, metabolomics was also performed following exposure to yeast media adjusted to pH 10.18 with NaOH (hereafter denoted \u0026lsquo;NaOH pH 10.18\u0026rsquo;). Growth kinetics and sampling points in these conditions\u003cem\u003e\u0026nbsp;\u003c/em\u003eare indicated in Fig. 5a. Principal component analysis (PCA) (Fig. 5b) separated 0.25 M ammonia and NaOH pH 10.18 conditions from the control, with overlap between 0.25 M ammonia and NaOH pH 10.18 conditions indicating metabolic similarity. Univariate volcano analysis identified 23 features significantly altered in 0.25 M ammonia/control (Fig. 5c), 10 features significantly altered in 0.25 M ammonia/NaOH pH 10.18 (Fig. 5d), and 28 features significantly altered in NaOH pH 10.18/control (Fig. 5e). The volcano analysis dataset for each comparison group is shown in Supplementary Table 2-4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe most significantly altered metabolites between these conditions were identified by ANOVA (Supplementary Table 5), depicted as a heatmap (Fig. 5f). Overall, high similarity was found between 0.25 M ammonia and NaOH pH 10.18 exposed samples; both conditions generally exhibited higher levels of unsaturated phospholipid and lower levels of linoleic acid and derivatives. Intermediates that feed glycerophospholipid biosynthesis, CMP-sialic acid (F = 2021.9, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) and glycerol-3-phosphate (F = 2022.2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001), were more abundant compared to the control. Amino acid pathway intermediates indole-3-ethanol (F = 2327.8, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001), N, N-dimethylglycine (F = 30.626, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) and N-acetylglutamate (F = 25.653, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) were also altered compared to control. There were reduced levels of N-acetylserotonin (F = 18876, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) and N-acetyl-L-aspartic acid (F = 24.091, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) that could suggest reactions with acetyl donors were less favourable at high pH in both conditions. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAmmonia exposure elicits a unique metabolomic response\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDespite general similarities between cells exposed to 0.25 M ammonia and NaOH pH 10.18, ANOVA indicated five metabolites significantly altered only in 0.25 M ammonia exposed \u003cem\u003eH. meridiana\u003c/em\u003e. Box plots of these metabolites are presented in Fig. 6a-e. Samples exposed to 0.25 M ammonia\u003csub\u003e\u0026nbsp;\u003c/sub\u003eshowed significantly higher levels of unidentified metabolites, metabolite i at m/z=215.084 (Fig. 6a) and metabolite ii at m/z=157.0973 (Fig. 6b), compared to NaOH pH 10.18 exposed samples (metabolite i, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; metabolite ii,\u003cem\u003e\u0026nbsp;p\u003c/em\u003e \u0026lt; 0.0001) and control samples (metabolite i, F = 2112.3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; metabolite ii,\u003cem\u003e\u0026nbsp;\u003c/em\u003eF = 4865.6, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). Library annotations matched metabolite i to nitrogen-containing heterocyclic compound atrazine (89.6% Q score) and metabolite ii to hydrocarbon 2,7-dimethylnaphthalene (96.5% Q score). Structures of atrazine and 2,7-dimethylnaphthalene are depicted in Fig. 6a and Fig. 6b, respectively. Multivariate analysis also revealed exposure to 0.25 M ammonia increased the relative abundance of pantothenate (F = 28.839, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) (Fig. 6c), and amino acids D-allo-isoleucine (F = 28.686, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) (Fig. 6d), and alanine (F = 25.391, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) (Fig. 6e), compared to those in NaOH pH 10.18 and control conditions.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe influence of ammonia on habitability, particularly to microbes, is underrepresented in scientific literature. Yet, the pollution of ammonia into the environment could shape the underlying microbial community structures and biodiversity of alkaline environments. The presence of ammonia within the subsurface ocean of Enceladus, predicted at an alkaline pH, may also shape the habitability expectations of this satellite. Although the presence of life beyond Earth is speculative, terrestrial organisms can be utilised to explore the boundaries of known life under similar conditions to assess potential for habitability. In this study, we utilise alkalitolerant and halophilic \u003cem\u003eH. meridiana\u003c/em\u003e as an analogue organism to investigate the molar concentration thresholds for survival, and physiological changes, that might be present in aerobic bacteria within extreme NH\u003csub\u003e3\u003c/sub\u003e environments.\u003c/p\u003e \u003cp\u003eThe exposure of \u003cem\u003eH. meridiana\u003c/em\u003e to increasing ammonia concentrations revealed that growth at 0.05 M ammonia, where the relative abundance of NH\u003csub\u003e3\u003c/sub\u003e is 62%, sets a consistent habitability limit regardless of whether in a closed-air or open-air system. The tolerance of microbes to ammonia varies and cannot be predicted by genus or species alone; strain-specific responses to stress have been well documented \u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45 CR46 CR47\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. However, the molarity limit established in this study aligns with data for other bacteria, including \u003cem\u003eB. subtilis\u003c/em\u003e strains T1, T2, T19, T22, T33, T39 \u003csup\u003e34\u003c/sup\u003e and \u003cem\u003eEnterobacter cloaecae\u003c/em\u003e HNR \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The absolute limit for ammonia survival in the literature extends to 0.716 M (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e at pH 9, survived by strains of \u003cem\u003eB. subtilis and Proteus morgani\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, the relative abundance of NH\u003csub\u003e3\u003c/sub\u003e in these studies is below 40%. Our results characterise a higher habitability limit in NH\u003csub\u003e3\u003c/sub\u003e than previously established for an aerobic bacterium.\u003c/p\u003e \u003cp\u003eIndependent ammonia and pH toxicity has been characterised in \u003cem\u003eE. coli\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, yet indistinguishable toxicity has been recorded with \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eSporosarcina, Paenibacillus, Staphylococcus, Brevibacillus, Streptomyces, Pseudomonas and Arthrobacter\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Using an alkalitolerant organism, we establish ammonia toxicity is distinct from pH toxicity in \u003cem\u003eH. meridiana\u003c/em\u003e, and does not exert toxicity via oxygen displacement or water activity changes. However, the degree of similarly between the metabolomic profiles of cells exposed to 0.25 M ammonia and a pH-matched NaOH solution indicates \u003cem\u003eH. meridiana\u003c/em\u003e primarily responds to ammonia using high pH adaptations. Unique physiological responses to ammonia in \u003cem\u003eH. meridiana\u003c/em\u003e included the accumulation of two cyclic compounds, one of which bore amine functional groups. The presence of these compounds may be indicative of intracellular NH\u003csub\u003e3\u003c/sub\u003e-driven reactions \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. The disruption of such structures to cellular components could be a mechanism of pH-independent toxicity. Indeed, \u003cem\u003eH. meridiana\u003c/em\u003e treated with ammonia exhibited aggregation to intracellular components that would suggest significant disruption to cell contents.\u003c/p\u003e \u003cp\u003eElevated levels of alanine, D-allo-isoleucine, and pantothenate were also exclusively observed in ammonia exposed \u003cem\u003eH. meridiana.\u003c/em\u003e The presence of an isoleucine derivative and pantothenate suggested modulation to the Coenzyme A pathway that affects the cell membrane \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, and amino acid metabolism \u003csup\u003e\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Indeed, D-amino acids have been implicated in cell wall remodelling in \u003cem\u003eVibrio cholerae\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Alanine dehydrogenase catalyses the production of alanine by a reaction between pyruvate and ammonium \u003csup\u003e\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Thus, alanine could be a by-product of elevated ammonia levels. Notably, D-amino acid aminotransferase catalyses the production of pyruvate and D-glutamate ​from D-alanine and 2-oxoglutarate, for which D-allo-isoleucine can act as an amino donor in \u003cem\u003eThermotoga maritima\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The presence of D-allo-isoleucine may therefore lead to enrichment of pyruvate and, in turn, enrichment of D-alanine or L-alanine via the alanine dehydrogenase pathway, or both. D-alanine may act to impede ammonia permeation; D-alanine can increase rigidity, and decrease permeability, of the peptidoglycan layer \u003csup\u003e\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. L-alanine may act to prevent excessive ammonia metabolism; in some bacteria, L-alanine is an allosteric inhibitor of the nitrogen assimilation enzyme glutamine synthetase \u003csup\u003e\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInterpreted from the point of view of planetary habitability, the ammonia toxicity limit established in this study is higher than the predicted 0.01\u0026ndash;0.1 M ammonia content of the Enceladus ocean \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The closed-air system is the most accurate representation of icy moon subsurface oceans as ammonia concentrations are presumed to remain constant. With oceanic temperatures estimated at 0 \u0026ordm;C \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and salinity at 4% \u003csup\u003e27\u003c/sup\u003e, the Enceladus ocean would require a pH of \u0026lt;\u0026thinsp;9.96 to satisfy the NH\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;62% habitability boundary suggested in this study, which is in range of current estimations \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In the terrestrial environment, up to 78 tetragrams of ammonia per annum are expelled globally from agricultural processes \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. A water volume of 1 litre would require just 1 gram of ammonia to exceed an ammonia concentration of 0.05 M. In alkaline environments, this may alter bacterial community structures and biodiversity once the relative abundance of NH\u003csub\u003e3\u003c/sub\u003e exceeds thresholds for survival, as has been observed in Mono Lake \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, unless NH\u003csub\u003e3\u003c/sub\u003e deposition is continuous, bactericidal effects may be transient due to the open-air dispersal of NH\u003csub\u003e3\u003c/sub\u003e \u0026ndash; repopulation of \u003cem\u003eH. meridiana\u003c/em\u003e in an open-air ammonia system was observed at concentrations above 0.05 M ammonia.\u003c/p\u003e \u003cp\u003eWithout field-based environmental surveys, the effects of NH\u003csub\u003e3\u003c/sub\u003e on terrestrial and extraterrestrial habitability are only estimated. Additionally, without isolation and biological characterisation of metabolites i and ii, we can only speculate the contribution of these molecules in ammonia toxicity. However, we have shown that bacterial growth and viability may become impacted when molar concentrations of ammonia are above 0.05 M, where NH\u003csub\u003e3\u003c/sub\u003e exceeds NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. Moreover, we deduce that ammonia and pH toxicity are distinct, suggesting a need to modify the current understanding of NH\u003csub\u003e3\u003c/sub\u003e toxicity in bacteria. Together, these findings can be integrated into the current knowledge regarding the impact of ammonia on terrestrial geomicrobiology and may inform future habitability assessments of NH\u003csub\u003e3\u003c/sub\u003e-rich extraterrestrial environments.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSolution preparation\u003c/h2\u003e \u003cp\u003eSolutions of liquid ammonia were made to the required molar concentrations\u0026mdash;0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 M ammonia\u003csub\u003e\u0026mdash;\u003c/sub\u003ein yeast media using 35% NH\u003csub\u003e3\u003c/sub\u003e (Fisher Scientific, CAS Number: 1336-21-6). The pH of the solutions was determined with a Jenway 3510 benchtop pH meter. pH-matched sodium hydroxide solutions (NaOH; Fisher Scientific, CAS Number: 1310-73-2) were prepared by gradual addition of 1 M NaOH into yeast media. To compare growth in different basic solutions at a pH equivalent to that of 0.25 M ammonia (pH 10.18), several bases were selected that have a natural pH at or greater than pH 10.18 and high solubility in water. Thus, yeast media was made to pH 10.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 using gradual addition of 1 M NaOH, 1 M potassium hydroxide (KOH), 1 M sodium metasilicate (Na\u003csub\u003e2\u003c/sub\u003eSiO\u003csub\u003e3\u003c/sub\u003e), 1 M potassium carbonate (K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e) or 1 M sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e). Growth experiments proceeded as outlined below. pH was matched to within \u0026plusmn;\u0026thinsp;0.01 pH units. All solutions were filter-sterilised through a 0.22-micron pore before use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eNH\u003csub\u003e3\u003c/sub\u003e/NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e percentage abundance\u003c/h2\u003e \u003cp\u003eThe percentage concentration of ammonia was determined by indirect calculation based on commonly utilised equations developed by Hampson \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Firstly, the ionic strength of the prepared solutions based on known salinity (0.2 M NaCl) was determined by Eq.\u0026nbsp;1.0\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:I=19.9273\\left(S\\right)/(1000-1.005109\\left(S\\right))\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEq.\u0026nbsp;1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhere I\u0026thinsp;=\u0026thinsp;molar ionic strength; S\u0026thinsp;=\u0026thinsp;salinity (parts per thousand, ppt).\u003c/p\u003e \u003cp\u003eThe stoichiometric acid hydrolysis constant of ammonium ions, pKa\u003csup\u003es\u003c/sup\u003e, based on I, was then calculated. pKa\u003csup\u003es\u003c/sup\u003e values for given ionic strengths were provided by Hampson \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These values were plotted to obtain a linear regression for calculating pKa (Supplementary Fig.\u0026nbsp;1). pKa\u003csup\u003es\u003c/sup\u003e was calculated from Eq.\u0026nbsp;2.0.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:pK{a}^{s}=0.1179\\left(I\\right)+9.246\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEq.\u0026nbsp;2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePercentage abundance of NH\u003csub\u003e3\u003c/sub\u003e at given salinity, pH and temperature was then calculated from Eq.\u0026nbsp;3.0. As salinity, temperature, pressure and pKa\u003csup\u003es\u003c/sup\u003e are constant in the experiment, only pH was modified to give percentage abundance of NH\u003csub\u003e3\u003c/sub\u003e at each pH value (Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:N{H}_{3}=N{H}_{3}+N{H}_{4}^{+}/(1+{10}^{\\left({pKa}^{s}+0.0324\\left(298-T\\right)+0.0415\\left(P\\right)/T-1\\right)}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEq.\u0026nbsp;3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhere P\u0026thinsp;=\u0026thinsp;1 atm pressure; T\u0026thinsp;=\u0026thinsp;temperature (K)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOxygen measurements\u003c/h2\u003e \u003cp\u003eOxygen concentrations (\u0026micro;mol/L) of unamended yeast media, ammonia solutions and pH-matched solutions, prepared as described above, were recorded using an oxygen microsenser with tip diameter of \u0026lt;\u0026thinsp;2 mm (OX-500, Unisense, Denmark). Solutions were equilibrated for 0.5 h before reading at 28 \u0026ordm;C in 96-well plates sealed with aluminium foil to prevent gas loss.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWater activity\u003c/h2\u003e \u003cp\u003eUnamended yeast media, ammonia solutions and pH-matched solutions were prepared as described above. Solutions were equilibrated for 1.5 h and water activities measured at 28 \u0026ordm;C with a Rotronic HP23-AW water activity meter (Rotoronic AG, Bassersdorf, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strain selection\u003c/h2\u003e \u003cp\u003e \u003cem\u003eHalomonas meridiana\u003c/em\u003e Slthf1 (DSM 15724; Gram negative) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). The organism was originally isolated at a depth of 2000 m from low temperature hydrothermal fluid in the East Pacific Rise. The strain exhibits halophilic growth up to 22% NaCl, and shows tolerance up to pH 12 \u003csup\u003e42\u003c/sup\u003e. The strain is therefore representative of the physiological attributes that could be necessary to grow in saline, alkaline fluids. A complete genome sequence is available for this strain \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. This organism is not known to have specific adaptations to ammonia; this was a deliberate choice. The purpose of this study was not to study microbial biochemical adaptation to high ammonia, but rather to study whether NH\u003csub\u003e3\u003c/sub\u003e can act as an abiotic factor influencing bacterial growth, survival and physiology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBacterial culture\u003c/h2\u003e \u003cp\u003eAn aerobic culture of \u003cem\u003eH. meridiana\u003c/em\u003e was grown in glass conical Erlenmeyer flasks at 28 \u0026ordm;C in an orbital benchtop shaking incubator set to rotate at 150 RPM. We assume ammonia-rich environments present optimal nutrient conditions (i.e., available organic debris) with a salinity\u0026thinsp;\u0026lt;\u0026thinsp;2% for the purpose of this study. Thus, \u003cem\u003eH. meridiana\u003c/em\u003e Slthf1 was grown in a yeast media consisting of 1g/100 mL Bacto\u0026trade; yeast extract (Becton, Dickinson and Company), 0.2 M NaCl (1.17% salinity) (Thermo Fisher Scientific, CAS Number: 7647-14-5) and deionised water (dH\u003csub\u003e2\u003c/sub\u003eO).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGrowth experiments\u003c/h2\u003e \u003cp\u003eTwo growth systems were implemented: \u0026lsquo;closed\u0026rsquo; growth experiments were conducted in tightly sealed 15 mL falcon tubes with sufficient headspace (14 mL) to allow aerobic metabolism but prevent ammonia evaporation; \u0026lsquo;open-air\u0026rsquo; growth experiments were conducted in 96-well plates to permit ammonia evaporation and to monitor growth dynamics. Growth in closed-air system experiments were assessed by colony forming units (CFU) after 72 h incubation at 28 \u0026ordm;C. Growth in open-air systems were assessed in 96-well plates by optical density (OD) readings in a BMG SPECTROstar Nano Microplate Reader at 600 nm with reading taken every half an hour for 48 h at 28 \u0026ordm;C. Plate wells were prepared with 190 \u0026micro;L of a selected solution and seeded with 10 \u0026micro;L overnight \u003cem\u003eH. meridiana\u003c/em\u003e culture to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.05. Positive control wells were 190 \u0026micro;L unamended yeast media inoculated with 10 \u0026micro;L overnight \u003cem\u003eH. meridiana\u003c/em\u003e culture to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.05. Negative controls had no inoculation. To avoid condensation, plate lids were coated with Triton X-100 (0.05%) in 20% ethanol. Plates were shaken at 200 RPM before each reading.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGrowth parameters\u003c/h2\u003e \u003cp\u003eExperiments were conducted to yield three standard microbiological growth parameters: lag phase duration, doubling time (\u003cem\u003eT\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e), and final OD\u003csub\u003e600\u003c/sub\u003e. The lag phase was calculated with the web-based Microbial Lag Phase Duration Calculator\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, with the following parameters: algorithm\u0026thinsp;=\u0026thinsp;parameter fitting to a model; pre-processing applied: cut data at some time\u0026thinsp;=\u0026thinsp;yes, max time\u0026thinsp;=\u0026thinsp;24 or 48 hours; smooth data\u0026thinsp;=\u0026thinsp;no; initial biomass\u0026thinsp;=\u0026thinsp;first observation; model to fit\u0026thinsp;=\u0026thinsp;logistic; NLS fitting algorithm\u0026thinsp;=\u0026thinsp;auto; max number of iterations\u0026thinsp;=\u0026thinsp;1000. \u003cem\u003eT\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e was calculated by first determining the growth rate in the exponential growth phase (\u003cem\u003e\u0026micro;\u003c/em\u003e) as per Eq.\u0026nbsp;4.0, followed by calculation of \u003cem\u003eT\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e as per Eq.\u0026nbsp;5.0.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabd\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:=\\left({Log}_{10}\\left(N\\right)-{Log}_{10}\\left({N}_{0}\\right)\\right)2.303/(t-{t}_{0})\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEq.\u0026nbsp;4.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhere N\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;OD\u003csub\u003e600\u003c/sub\u003e at the beginning of a selected time interval (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e) in the exponential growth phase; N\u0026thinsp;=\u0026thinsp;OD\u003csub\u003e600\u003c/sub\u003e at the end of a selected time interval (\u003cem\u003et\u003c/em\u003e) in the exponential growth phase. \u003cem\u003et\u003c/em\u003e and \u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e are recorded in minutes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabe\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{T}_{\\text{d}}=ln2/\\mu\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEq.\u0026nbsp;5.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe final OD\u003csub\u003e600\u003c/sub\u003e reached after 48 hours was recorded and used to represent the final cell concentration reached. Cell viability versus OD\u003csub\u003e600\u003c/sub\u003e over 24 h was assessed by PrestoBlue\u003csup\u003e\u0026trade;\u003c/sup\u003e cell viability reagent (Thermo Fisher Scientific, Massachusetts, USA). Calibration curves confirm changes to observed OD\u003csub\u003e600\u003c/sub\u003e corresponds to increasing number of viable cells (Supplementary Fig.\u0026nbsp;2). Growth or lack of growth was determined by the presence or absence of a defined lag phase, exponential phase, and stationary phase within a 48 h growth period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAmmonia evaporation and cell viability\u003c/h2\u003e \u003cp\u003eOvernight culture of \u003cem\u003eH. meridiana\u003c/em\u003e was inoculated to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.05 in 0 (positive control), 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 and 1 M ammonia solutions into separate 96-well plates. Plates were incubated at 28 \u0026ordm;C on an orbital shaker for 4, 8, 16 and 24 hours. Ammonia content at these time intervals was recorded by the CHEMetrics High Range VACUette Ammonia test kit (K-1510C) using direct nesslerization. The parts per million (ppm) of ammonia in the sample was determined by colorimetric analysis of the Nessler reaction product at 420 nm \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Viability of cells was also assessed at these times by CFU on yeast media agar plates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy\u003c/h2\u003e \u003cp\u003eAn overnight culture of \u003cem\u003eH. meridiana\u003c/em\u003e was pelleted, resuspended and exposed for 2 h to the following solutions: unamended yeast media (control), 1 M ammonia and a pH-matched solution (pH 10.78). The matched pH solution was created by gradual addition of 1 M NaOH to yeast media. Following incubation, cells were washed and resuspended in phosphate buffered saline (PBS). Samples were fixed in 3% glutaraldehyde in 0.1 M Sodium Cacodylate buffer, pH 7.3, for 2 hours then washed in three 10-minute changes of 0.1 M Sodium Cacodylate. Specimens were then post-fixed in 1% Osmium Tetroxide in 0.1 M Sodium Cacodylate for 45 minutes, then washed in three 10-minute changes of 0.1 M Sodium Cacodylate buffer. These samples were then dehydrated in 50%, 70%, 90% and 100% ethanol (X3) for 15 minutes each, then in two 10-minute changes in Propylene Oxide. Samples were then embedded in TAAB 812 resin. Sections, 1 \u0026micro;m thick were cut on a Leica Ultracut ultramicrotome, stained with Toluidine Blue, and viewed in a light microscope to select suitable areas for investigation. Ultrathin sections, 60 nm thick were cut from selected areas, stained in Uranyl Acetate and Lead Citrate then viewed in a JEOL JEM-1400 Plus TEM. Representative images were collected on a GATAN OneView camera at 4K resolution. Images were processed using ImageJ \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMetabolomics sampling and extraction\u003c/h2\u003e \u003cp\u003eAn overnight culture of \u003cem\u003eH. meridiana\u003c/em\u003e was inoculated to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.05 within 0.25 M ammonia, pH-matched yeast media (pH 10.18) or unamended yeast media (control) within a 24-well plate. The pH-matched solution was created by gradual addition of 1 M NaOH to yeast media. Growth occurred within plates at 28 \u0026ordm;C and was assessed by OD\u003csub\u003e600\u003c/sub\u003e reading every half an hour using a BMG SPECTROstar Nano Microplate Reader. Samples were harvested during the log phase of growth at OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5. All the following procedures occurred at 4\u0026deg;C. Samples were aliquoted into microcentrifuge tubes and quenched by rapid cooling through submersion in a dry ice/70% (v/v) ethanol bath after brief incubation on ice. During cooling, samples were mixed vigorously to prevent freezing. Cells were separated from spent medium by centrifugation at 1,000 \u003cem\u003eg\u003c/em\u003e for 10 minutes and supernatant discarded. Metabolite extraction occurred by addition of ice-cold chloroform/methanol/water (1:3:1 ratio). Cell lysis was encouraged by sonication with ice for 5 minutes at 37 kHz within an ultrasonication bath (Elmasonic S 60 H). Extraction mixtures were mixed vigorously at 1,200 rpm on an orbital shaker for 1 h. Following mixing, samples were centrifuged at 13,000 \u003cem\u003eg\u003c/em\u003e for 3 minutes. The supernatant, containing metabolites, was collected into sterile microcentrifuge tubes. A pooled quality control sample was generated by combining equal volumes of metabolites from each sample. All samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eUntargeted metabolomics analysis\u003c/h2\u003e \u003cp\u003eThe untargeted metabolomics analysis was performed using liquid chromatography (LC) coupled to ion mobility (IM) quadrupole time-of-flight (qTOF) mass spectrometry (MS) as described previously (Ref 1). The instrumentation consisted of an Agilent 1290 Infinity II series UHPLC system hyphenated with an Agilent 6560 IM-qTOF with a Dual Agilent Jet Stream Electron Ionization source. LC separation was performed on an InfinityLab Poroshell 120 HILIC-Z, 2.1 mm \u0026times; 50 mm, 2.7 \u0026micro;m UHPLC column (Agilent Technologies 689775\u0026ndash;924) coupled to an InfinityLab Poroshell 120 HILIC-Z, 3.0 mm \u0026times; 2.7 \u0026micro;m UHPLC guard column (Agilent Technologies 823750\u0026ndash;948). A 3.5 min gradient was run using organic buffer (acetonitrile) combined with an aqueous buffer with low pH (10 mM ammonium formate, pH 3) or high pH (10 mM ammonium acetate, pH 9) for positive and negative ionization modes, respectively. Data was acquired using MassHunter Data Acquisition 10.0 software on 1 \u0026micro;L of sample separated on the column with a flow rate of 800 \u0026micro;L/min. The quality control sample was injected five times at the beginning of the experiment to condition the column and after every five test samples to monitor the instrument state throughout data acquisition. Data were acquired in the 50 to 1700 m/z range, with an MS acquisition rate of 0.8 scans/s. The metabolomics analyses were carried out by the EdinOmics research facility (RRID: SCR_021838) at the University of Edinburgh.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMetabolomics data processing and statistical analysis\u003c/h2\u003e \u003cp\u003eThe raw data files were processed using the Agilent MassHunter software suite. Briefly, ion multiplexed data files and calibration files were demultiplexed using the PNNL PreProcessor v2020.03.23 (the default settings for demultiplexing, moving average smoothing, saturation repair and spike removal were applied to the data). The data files were recalibrated for accurate mass and drift time using the AgtTofReprocessUi and the IM-MS Browser 10.0, respectively. Molecular features were extracted in Mass Profiler 10.0 with a retention time tolerance of \u0026plusmn;\u0026thinsp;0.3 min, drift time tolerance of \u0026plusmn;\u0026thinsp;1.5% and accurate mass tolerance of \u0026plusmn; (5 ppm\u0026thinsp;+\u0026thinsp;2 mDa). Features were annotated based on accurate mass and collision cross section (CCS) values using McLean CCS Compendium PCDL library \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Multivariate and univariate statistical analysis was performed using the MetaboAnalyst 6.0 web-based platform \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. The input data were log-transformed and Pareto-scaled. The results were visualised using principal component analysis (PCA), box and whisker plots, volcano plots and heatmaps. Further aesthetic adjustments, including font changes and line thickness modifications, were made using Inkscape (version 1.0.1). Chemical structures were drawn using ChemDraw (version 23.1.2) and exported in SVG format for inclusion in the figures. The raw data associated with this dataset is available to view in the Supplementary Dataset. This study focuses on metabolomic changes in ammonia, pH and control samples, but the broader metabolomics profiling also included samples exposed to (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. For analysis, only ammonia, pH and control samples were included in this study. The metabolomics of (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e exposed samples are to be addressed in a separate analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStatistics and reproducibility\u003c/h2\u003e \u003cp\u003eAll data were compiled from a minimum of three different cultures inoculated on different days with new media (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4 biological replicates). The normality of data was assessed with the Shapiro-Wilk test. For comparison of two groups, paired or unpaired two-tailed t-tests were used. The Wilcoxon matched-pairs signed-rank test was used for paired groups where the assumption of normality was violated. An unpaired t test with Welch's correction was applied for groups with unequal variance. For comparison of two or more groups, equal variance was assessed with the Brown-Forsythe test. Samples of equal variance were analysed by analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post-hoc test. For samples where variance was not equal, Welch\u0026rsquo;s ANOVA test with Games-Howell\u0026rsquo;s post-hoc test was used. Statistical tests are specified in figure legends. Significance was considered when \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All statistical analyses were performed using GraphPad Prism version 8.0.2 (GraphPad Software Inc.).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article within the Supplementary Dataset, except when specified as part of the Supplementary Material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for this research was provided by the Natural Environmental Research Council (NERC) though an E4 Doctoral Training Partnership (DTP) studentship (NE/S007407/1). C.H. and C.S.C thanks the Science and Technology Facilities Council (STFC) for support under grant ST/V000586/1 and ST/Y001788/1. We also acknowledge the support of the Wellcome Trust Multiuser Equipment Grant (WT104915MA) for use of the JEOL JEM-1400 Plus TEM. We acknowledge the EdinOmics research facility at the University of Edinburgh where the metabolomics analyses were carried out.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCassie M. Hopton \u0026amp; Charles S. Cockell - \u003cem\u003eSchool of Physics and Astronomy, University of Edinburgh, James Clerk Maxwell Building, Peter Guthrie Tait Road, Edinburgh, EH9 3FD, United Kingdom\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePeter Nienow - \u003cem\u003eSchool of Geosciences, University of Edinburgh, Drummond St, Edinburgh, EH8 9XP, United Kingdom\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.H. conducted the experiments, data analysis and paper writing. C.S.C and P.N. jointly supervised this work, provided instruments and materials for experiments, and provided input in the writing of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCassie M. Hopton, [email protected], ORCID ID: 0000-0003-2830-7787\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors confirm that this research was carried out without any commercial or financial affiliations that could be perceived as a potential competing interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVines, H. M. \u0026amp; Wedding, R. T. Some Effects of Ammonia on Plant Metabolism and a Possible Mechanism for Ammonia Toxicity 1234. \u003cem\u003ePlant. 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MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e52\u003c/b\u003e, W398\u0026ndash;W406 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6178140/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6178140/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eS\u003c/b\u003eignificant amounts of ammonia are released anthropogenically, while ammonia is also retained within the water bodies of icy moons that are of astrobiological interest. Ammonia is toxic to many forms of life at high concentrations, and thus it is necessary to understand the habitability impact of ammonia on these environments. The survival limits and physiological response of aerobic bacteria in ammonia, and whether ammonia toxicity is distinct from toxicity by high pH, is poorly understood. Here, we investigate the survival thresholds, growth kinetics, and metabolomic response of \u003cem\u003eHalomonas meridiana\u003c/em\u003e in ammonia-water solutions and pH-matched sodium hydroxide solutions. Using closed- and open-air systems to mimic environments with ammonia retention or dispersion, we found complete and partial cell death above 0.05 M ammonia, respectively. In open-air systems, a sub-set of cells survived up to 0.25 M ammonia; metabolomics revealed unique physiological responses to ammonia, including elevation of cyclic compounds and Coenzyme A metabolites, suggesting mechanisms of ammonia toxicity and adaptation. Ammonia and high pH toxicity were found to be distinct. These findings show that ammonia can impose a distinct geobiological limit, potentially constraining habitability of ammonia-rich terrestrial and extraterrestrial environments.\u003c/p\u003e","manuscriptTitle":"Ammonia sets limit to life and alters physiology independently of pH in Halomonas meridiana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 08:22:15","doi":"10.21203/rs.3.rs-6178140/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-25T10:41:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-24T21:09:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41178865194401598184943843670904332384","date":"2025-04-04T13:27:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-03T13:19:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114651835933216462775927257044405702102","date":"2025-03-22T15:17:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-20T13:02:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-20T12:44:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-20T11:08:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-19T10:00:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-19T09:59:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b47b6de7-613a-4dd1-90ca-0ebfded63bb5","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46065113,"name":"Earth and environmental sciences/Planetary science/Astrobiology"},{"id":46065114,"name":"Biological sciences/Microbiology/Bacteria/Bacterial physiology"},{"id":46065115,"name":"Biological sciences/Microbiology/Bacteriology"},{"id":46065116,"name":"Biological sciences/Physiology/Metabolism/Metabolomics"}],"tags":[],"updatedAt":"2025-06-09T16:02:19+00:00","versionOfRecord":{"articleIdentity":"rs-6178140","link":"https://doi.org/10.1038/s41598-025-03858-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-06-04 15:57:30","publishedOnDateReadable":"June 4th, 2025"},"versionCreatedAt":"2025-03-24 08:22:15","video":"","vorDoi":"10.1038/s41598-025-03858-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-03858-z","workflowStages":[]},"version":"v1","identity":"rs-6178140","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6178140","identity":"rs-6178140","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}