Hydrogenotrophic methanogenesis at 7–12 mbar by Methanosarcina barkeri under simulated martian atmospheric conditions

Abstract

Mars, with its ancient history of long-lived habitable environments, continues to captivate researchers exploring the potential for extant life. This study investigates the biosignature potential of Martian methane by assessing the viability of hydrogenotrophic methanogenesis in Methanosarcina barkeri MS under simulated Martian surface conditions. We expose M. barkeri to sustained hypobaria (7–12 mbar), low temperature (0˚C), and a CO₂-dominated gas mixture mimicking the Martian atmosphere. The results demonstrate statistically quantifiable CH₄ production under all tested conditions, including at 7–12 mbar. Transcriptomics reveal that low total pressure and temperature did not significantly impact gene expression, highlighting the resilience of M. barkeri. However, atmospheric gas composition, specifically Mars gas with 2.9% pH₂, led to significant down-regulation of methanogenesis genes, hindering growth over 14 days. Notably, CH₄ production scaled with the partial pressure of H₂, revealing that hydrogen uptake affinity is a stronger predictor of habitability and methanogenic potential than favorable Gibbs free energy of reaction. Our findings suggest that Mars’ subsurface could harbor habitable refugia capable of supporting methanogenesis, sustaining microbial life at low metabolic steady states. These insights challenge assumptions about Martian habitability and have implications for astrobiological exploration, planetary protection, and in situ resource utilization for future human missions.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-86145-1.

Introduction

Redox disequilibria and seasonal variation in planetary atmospheres have been proposed as potential biosignatures in the search for life beyond Earth^1–3. Mars is presently an active testing ground for these hypotheses. In the past two decades, significant attention has been paid to reports of trace methane (CH₄) in the Martian atmosphere^4–9, inspiring debates concerning its occurrence, behavior, and astrobiological implications^10–22. Reports of apparent seasonality in background CH₄ mixing ratios²³ and discoveries of isotopically depleted CH₄ at Gale Crater by Mars Science Laboratory (MSL)¹¹ emphasize that legitimate considerations of Martian CH₄ as a potential biosignature are warranted.

On Earth, microbial methanogenesis plays a significant role in shaping the global CH₄ budget. Nearly 70% of atmospheric CH₄ can be traced to microbial methanogenesis, an ancient metabolism carried out by obligately anaerobic methanogens from the domain Archaea²⁴. The biochemistry, physiology, and kinetic isotope effects of methanogenesis are well characterized^25–27, offering decades of context to thoroughly consider a potential biogenic source of Martian CH₄.

Methanogens’ propensity to survive extreme environmental conditions advocates their use as model organisms to explore the habitability and astrobiological potential of Mars. A growing body of literature assessing microbial methanogenesis under extreme conditions in presumptive Mars Special Regions—i.e., habitable environments for life as we know it²⁸–suggests that modern Mars may not be as hostile to life as previously thought. Methanogen survivability in particular has been assessed with respect to numerous environmental stressors typical of modern Mars, such as desiccation^29–33, starvation³⁴, freeze-thaw cycling^34,35, oxygen and free radical exposure^36,37, elevated radiation dosages³³, high salinity³³, perchlorate exposure^32,38,39, low pressure^30,31, extreme pH⁴⁰, and transient deliquescence of salts⁴¹, among others. Notably, Methanosarcina barkeri (hereafter M. barkeri), a mesophilic methanogen stands out for its versatile metabolic range and robust performance under a range of Mars-relevant stressors^{31,35,38,39,42,43}, making it a favored choice in Mars survival experiments. Its extremotolerance and ability to respond to diverse environmental challenges – often outperforming what might be considered more “ecologically relevant” extremophilic methanogens^{29,30,32,33,35,38,40}—positions M. barkeri as a promising candidate to assess survival and metabolic activities under conditions never encountered during the history of life on Earth. By coupling these survival experiments with RNA sequencing and comparative transcriptomics, we previously revealed that M. barkeri responds to sustained perchlorate exposure by down-regulating the production and recycling pathways of redox-sensitive amino acids and up-regulating genes encoding catalytic molybdoenzymes that may play a role in perchlorate reduction³⁹.

Building upon our previous work³⁹, we present a follow-up study focused on hydrogenotrophic methanogenesis and accompanying gene expression in M. barkeri following 14 days of sustained exposure to simulated Martian near-surface conditions of low atmospheric pressure (7–12 mbar), low temperature (0 °C), and atmospheric composition (hereafter “Mars gas” or “Mars gas mix”) spiked with 2.9% (v/v) H₂ to reflect a shallow subsurface environment with elevated pH₂ conducive to support hydrogenotrophic methanogenesis (92.5% CO₂, 2.9% H_2, 2.6% N₂, 1.9% Ar, 0.16% O₂, 0.03% H₂O)⁴⁴. Experiments were initially conducted with six replicates per condition, though some treatments were later reduced to four replicates (see “Materials and methods”). The choice of M. barkeri for this study is grounded in its robust performance under a range of Mars-relevant stressors^{31,35,38,39,42,43}, making it a promising candidate to assess survival and metabolic activities under environmental conditions that have never been encountered during the history of life on Earth. To constrain the influence of individual factors on the survival and metabolic activities of M. barkeri, we compared CH₄ production rates and changes in gene expression to M. barkeri cultures grown under various combinations of pressure (7–12 mbar vs. 1500 mbar), temperature (0 °C vs. 30 °C) and atmospheric gas mixtures (Mars gas vs. stoichiometrically balanced 80:20 H₂:CO₂ gases) for hydrogenotrophic methanogenesis.

Results

Methane evolution

High-precision (Fig. S1), controlled incubation experiments (Fig. 1) yielded quantifiable CH₄ from M. barkeri under all assayed conditions, with methane evolution rates ranging from sub-nmol to µmol-levels CH₄ mL⁻¹ d⁻¹ (Fig. 1D). No CH₄ production was observed in either the mechanical blank or media blank controls (see “Materials and methods” for details). The highest CH₄ production (mean ± standard deviation 62.4 ± 14.7µmol CH₄ mL⁻¹) was observed in M. barkeri grown under the baseline reference condition of 30 °C/1500 mbar/80:20 H₂:CO₂, followed by 0 °C/1500 mbar/80:20 H₂:CO₂ (27.7 ± 5.79 µmol CH₄ mL⁻¹) (Fig. 1D, Table S1) and are consistent with previous CH₄ measurements at these temperatures (Fig. S2)³⁹. At 1500 mbar, methane production was significantly lower in 0 °C incubations than at 30 °C, with the 0 °C/80:20 H₂:CO₂ treatment decreasing by ~ 56% and the 0 °C/Mars gas treatment decreasing by ~ 99% (Fig. 1D, Tables S1, S2). Total methanogenesis was higher in M. barkeri at 30 °C/1500 mbar/Mars gas (0.94 ± 0.25 µmol CH₄ mL⁻¹) and 0 °C/1500 mbar/Mars gas (2.96 ± 1.07 nmol CH₄ mL⁻¹) than M. barkeri incubated at 0 °C/7–12 mbar/80:20 H₂:CO₂ (0.51 ± 0.72 nmol CH₄ mL⁻¹) (Fig. 1D, Tables S1, S2, ANOVA, P < 0.01). The lowest CH₄ production (49.2 ± 27.8 pmol CH₄ mL⁻¹) was observed at 0 °C/7–12 mbar/Mars gas but was statistically indistinguishable from the 0 °C/7–12 mbar/80:20 H₂:CO₂ treatment (Fig. 1D, Tables S1, S2, ANOVA, P = 0.21).

Fig. 1.

High precision, controlled incubation conditions, and methane evolution rates. (A) The Planetary Atmosphere Chamber (PAC) system was developed in the Schuerger lab at the University of Florida (74, 75). (B) Twin Methane Assay Chambers (MAC units) were mounted directly to a liquid-nitrogen cold plate. Each MAC unit was fitted with a (1) sample line to a Residual Gas Analyzer (RGA), (2) pressure transducer for real-time monitoring of in situ pressure, (3) a joint gas injection and sampling line, (4) relative humidity (RH) sensor, (5) MAC vent line to the bulk atmosphere within the PAC system, and (6) central void space to support microbial cultures in 6-cm diameter pre-sterilized glass petri dishes. (C) SEM image of M. barkeri cluster collected from a MAC unit following a 14-day incubation at 7–12 mbar. (D) Total CH₄ production (per mL medium) by temperature, pressure, and headspace atmospheric composition. Methane production for individual replicates are indicated by open points, while outliers are represented by black points. The dashed line represents the minimum quantifiable limit (MQL) threshold for the detection CH₄ above the GC-FID background signal. Raw data can be found in Tables S1 and S2 for H₂:CO₂ and Mars gas-mix experiments, respectively.

Nucleic acid recovery, transcriptome quality, and mapping statistics

Quantifiable single-stranded RNA was recovered from cultures grown with 80:20 of H₂:CO₂ (n = 18) and 10 of 12 cultures grown with Mars gas, with the latter treatment type typically recovering RNA at concentrations an order of magnitude lower than the former (Table S3). Due to technical constraints imposed by the design of the PAC/MAC, we prioritized whole sample RNA preservation with RNAlater™ at the termination of the experiments to halt transcriptomic activity. A trade-off of this was that direct cell counts were not possible. TapeStation analysis did not reveal any significant differences between treatments in average fragment size of reverse-transcribed complementary DNA (cDNA) or subsequently prepared sequencing libraries (Table S3).

Shotgun sequencing yielded a total of 2,708,031,962 raw paired-end reads. Of these, 2,461,739,295 paired-end reads passed quality filtering, with an average Phred quality score of 35.45 ± 0.13 and no significant differences in fragment sizes (Student’s paired t-Test, P = 0.71). H₂:CO₂ libraries yielded much deeper sequencing coverage than Mars gas treatments. On average, 52.34% ± 9.01% of H₂:CO₂ and 0.20% ± 0.44% of Mars gas reads mapped back to M. barkeri coding sequence regions (CDS) (Table S3). Regularized for sequencing depth and RNA composition, Mars gas treatments produced ~10 to 100-fold decreases in methanogenesis transcript fragment counts per million mapped read fragments (FPM) relative to H₂:CO₂ treatments (Fig. 2). Factoring for each level of treatment (i.e., pressure, temperature, and atmosphere), a principal component analysis (PCA) identified that 98% of variance in regularized, log-transformed read counts between samples was captured by the first principal component, PC1. The clustering of samples along PC1 strongly correlates with atmospheric composition, indicating that this variable has a dominant influence on gene expression patterns (Fig. 3). Because PCA assumes linear relationships and uses Euclidean distances, it may not capture more complex patterns typical of ecological datasets, such as non-Gaussian distributions. As our dataset contained a large proportion of zero count transcripts, particularly in the Mars gas treatments, an additional non-metric multiscale dimensional analysis (NMDS) based on Bray-Curtis dissimilarity was determined to be a valuable complementary approach, as it preserves rank-based relationships rather than relying on absolute distances. By applying NMDS on variance-stabilized, regularized transcript counts, we confirmed that atmospheric composition was a key factor influencing transcript abundance, with tight clustering of samples incubated with Mars gas (stress value = 0.13, Fig. S3). Using NMDS alongside PCA ensured that we captured both linear and non-linear patterns, providing a more comprehensive understanding of treatment effects across conditions.

Fig. 2.

Fragment counts per million mapped reads (FPM) in the methanogenesis pathway of M. barkeri. For each transcriptomic library, FPM was calculated using the robust median ratio method to account for sequencing differences in library sizes and RNA compositions in samples. (A) FPM from 80:20 H₂:CO₂-incubated experiments. (B) FPM from Mars gas mix-incubated experiments. Genes are ordered spectrally in the legend from red to violet according to relative steps in the hydrogenotrophic methanogenesis pathway (i.e., red, orange = early pathway steps; yellow, green = mid-pathway steps; blue, violet = terminal steps). Note the difference in scale of the y-axes between A and B.

Fig. 3.

Principal component analysis of regularized log (rlog)-transformed read counts between conditions (n = 28 libraries). Each point represents an individual sample and is colored by atmospheric condition (80:20 H₂:CO₂ or Mars gas mix) and shaped by pressure condition (7–12 mbar or 1500 mbar).

Regulation of methanogenesis pathway

Every gene involved in the hydrogenotrophic pathway of methanogenesis was represented in sequencing libraries, implying they were actively transcribed under all 80:20 H₂:CO₂ treatments, including under 0 °C/7–12 mbar (Fig. 4). By contrast, there was a considerable decrease in methanogenic transcriptional activity for M. barkeri under Mars gas mix treatments, with zero counts observed for several genes based on raw counts (Table S3). Accounting for sequencing depth, a regularized log transformation of transcript counts predicted Mars gas mix base mean expression values ranging from 527.4 (mtrG) to 30,103.1 (fmdA) (Table S4), suggesting active transcription of methanogenesis genes was probable in Mars gas mix treatments at 7–12 mbar, albeit at reduced levels when compared to counterpart H₂:CO₂ treatments.

Fig. 4.

Differential gene expression (log₂-fold change) of the methanogenesis pathway in M. barkeri under Mars gas mix headspace relative to the 80:20 H₂:CO₂ headspace control. Text sizes of individual genes are scaled according to average normalized transcript copy numbers. Statistically significant differential expression is indicated by red genes and determined via Wald test (P ≤ 0.05). Related gene subunits are clustered into single enzymes (cytoplasmic = light blue; periplasmic = yellow). Metabolites and pathways directionality are indicated by serifed text and dark grey arrows, respectively. Full names of listed genes and metabolites are found in Tables S7 and S8, respectively. Differential expression graphs with respect to temperature and pressure variables are found in Figures S4 and S5, respectively.

No genes in the hydrogenotrophic pathway of methanogenesis were significantly up-regulated under the Mars atmosphere gas mix in comparison to H₂:CO₂ (Fig. 4, Table S4). However, nearly two-thirds of genes implicated in hydrogenotrophic methanogenesis showed significant down-regulation under the Mars gas mix at 7–12 mbar relative to the 80:20 H₂:CO₂ control (Fig. 4). The largest change in gene expression belonged to enzyme complexes involved in the final reducing step and first catalytic step of methanogenesis, respectively. The beta subunit of methyl-coenzyme M reductase (mcrB), the enzyme complex responsible for reducing methyl-coenzyme M (CH₃-CoM) to CH₄, exhibited the largest change in expression (log₂-fold change ± standard error = – 7.61 ± 2.63; Wald test, P_adj = 1.89E-09). With the exception of mcrG, all other subunits of the mcr complex showed significant down-regulation in expression, ranging from − 5.87 ± 2.40 (mcrC) to – 2.67 ± 0.62 (mcrA). At the outset of the pathway, formylmethanofuran dehydrogenase subunit D (fmdD)—part of a flanking tunnel that shuttles CO₂ inside the enzyme complex—experienced a log2-fold change in expression of – 6.95 ± 2.56 (Wald test, P_adj = 1.45E−07). No significant differences in expression were observed in fmdB or fmdA, which respectively harbor the Mo-containing active site responsible responsible for reducing CO₂ to formate using ferredoxin and forms methanofuran. No subunits of the energy conserving hydrogenase, ech, or [NiFe] hydrogenase, hyp, were observed to be significantly differentially expressed either.

Notably, neither differences in temperature (0 °C vs. 30 °C) nor pressure (7–12 mbar vs. 1500 mbar) imparted any significant change in gene expression within the entire hydrogenotrophic methanogenesis pathway of M. barkeri, regardless of atmospheric gas composition (Wald test, P_adj > 0.05; Figs. S4, S5).

Discussion

This study sought to assess the impact of combinations of low temperature, hypobaria, and a CO₂-dominated Martian gas mixture (+ 2.9% pH₂) on the activity and regulation of hydrogenotrophic methanogenesis in M. barkeri. Low pressure methanogenesis for M. barkeri had been previously demonstrated at 50 mbar under optimal growth temperatures in a 10:90 H₂:CO₂ atmosphere³¹. We extend the lower temperature and pressure limits reported for M. barkeri to 0 °C and 7–12 mbar, demonstrating active methanogenesis via corroborating CH₄ production and transcriptional activity for simulated conditions possible for the Martian surface or shallow subsurface. Under low-pressure and low-temperature conditions, we found that methane production rates were statistically indistinguishable between incubations grown under a stoichiometrically balanced 80:20 H₂:CO₂ atmosphere and a simulated Martian atmosphere spiked with H₂ (92.5% CO₂, 2.9% H_2, 2.6% N₂, 1.9% Ar, 0.16% O₂, 0.03% H₂O) (Fig. 1D). Our data demonstrate that hydrogenotrophic methanogenesis is possible at 7–12 mbar in the shallow subsurface on Mars if enough water, mineral nutrients, and H₂ are available.

There was, however, a significant difference in transcriptional activity between M. barkeri incubated under 80:20 H₂:CO₂ versus Mars gas + 2.9% pH₂. Considering methane production rates alone, it appeared hypobaria imparted a significant reduction in methanogenic activity (Fig. 1). However, RNA sequencing and comparative transcriptomics revealed that hypobaria alone did not confer any direct significant regulatory shifts in gene expression (Fig. S5, Table S6). The same phenomenon was observed with respect to temperature (Fig. S4, Table S5), suggesting that transcription was not hindered by exposure to low temperatures. Instead, atmospheric gas composition was the determining factor in regulating hydrogenotrophic methanogenesis at all assayed temperatures and pressures, with significant down-regulation of genes throughout the methanogenesis pathway when M. barkeri was exposed to the Mars gas mix with 2.9% pH₂ (Figs. 3 and 4, Table S4).

The sparseness of the Mars gas transcriptomic libraries does not yield any obvious clues regarding the observed differential responses. We do observe the up-regulation of universal stress proteins and cell surface proteins, suggesting potential responses to oxidative stress. Though aerotolerant amongst methanogens⁴⁵, M. barkeri is an obligate anaerobe and the premise of trace O₂ in the Mars gas mix offers one additional mechanism in which increased pO₂ exacerbates low pH₂ availability thus limiting methanogenesis and associated transcriptional activities. While this makes logical sense, it would be speculative to draw any conclusions in the absence of additional corroborating data. Future investigations and refined experiments with additional time points in the incubation could shed more light on these intricate relationships.

It is important to note that all steps between RNA isolation, cDNA generation, library prep, and sequencing were performed simultaneously and with parallel extraction and sequencing blanks. Given the quality of all libraries, there is no indication that the smaller libraries of the Mars atmospheric gas mixtures were artifacts of degradation from sample handling (e.g., from freeze/thaw cycling of recovered nucleic acids), as the same phenomenon should have been observed for all treatments, not just the Mars gas mix conditions. This is notable in light of (1) the quantifiable CH₄ production rates observed for all Mars gas treatments, and (2) our observation of no statistical differences in methane production rates between H₂:CO₂ and Mars gas mix treatments at 0 °C and 7–12 mbar (Fig. 1D). The lack of transcriptional activity in the Mars gas treatments, therefore, cannot be considered synonymous with metabolic inactivity. Rather, it likely reflects a microbial community conserving energy and limiting transcription in direct response to energy limitation. A consequence of this was reduced nucleic acid yields from RNA recovery, and subsequently, decreased sequencing depth. Despite decreased sequencing coverage (Fig. 2), a conservative but robust statistical analysis of differential expression (see “Materials and methods”) suggested M. barkeri did not prioritize transcription. Collectively, these findings support an inference of significantly slowed growth and are indicative of a microbial community at low metabolic steady state. Analogous terrestrial microbiomes are commonly observed in energy-constrained ecosystems such as the oligotrophic deep subsurface biosphere⁴⁶, and comparable findings here appropriately support arguments of subsurface lithoautotrophic microbial ecosystems as Mars environments of particular astrobiological relevance^47,48.

Observing that CH₄ production generally scaled with the partial pressure of H₂ (pH₂) (Table 1), we infer that H₂ bioavailability—and by association, the minimum H₂ uptake threshold of a hydrogenotrophic methanogen—plays the most critical role in methanogenic activity and, consequently biomass production, of the assessed factors in the present study. Minimum H₂ uptake thresholds, also referred to as hydrogen affinity thresholds, refer to the minimum pH₂ utilizable by H₂-oxidizing microbes for metabolism and are tied to the minimum energy requirements needed to synthesize ATP: Below these threshold concentrations, H₂ consumption stops. Hydrogenotrophic methanogens with energy-conserving cytochromes like M. barkeri have higher H₂ thresholds than methanogens lacking cytochromes (Table 2)^26,49; in other words, they require higher partial pressures of H₂ to sustain metabolism and support growth. It has been demonstrated that by increasing dissolved nickel (Ni), an important trace element for the active site of mcr and the cofactor F₄₃₀, cytochrome-containing methanogens, including M. barkeri, can utilize H₂ at lower partial pressures (Table 2)^50,51, and we note that 0.1 µM Ni was available to M. barkeri in this study. Although Gibbs free energy yields (∆_rG) favorably support hydrogenotrophic methanogenesis under all assayed conditions (Table 3) and are well above the reported minimum free energy requirements (∆G_min) to support microbial metabolism (∆G_min = −12 to −15 kJ mol⁻¹ for static or starving populations and ∆G_min ~ − 20 kJ mol⁻¹ for actively growing populations)^52,53, the range of H₂ partial pressures across treatments described in this study prove to be a more useful metric for assessing metabolic potential. Reported H₂ affinity threshold values for M. barkeri range from 0.13 to 0.19 mbar H₂^50,51, indicating that the ~ 0.2–0.35 mbar pH₂ of the 0 °C/7–12 mbar/Mars gas mix treatment reflects conditions approaching the lower observable H₂ limit for this species. However, the H₂ affinity threshold was not reached, as the observed CH₄ production was insufficient to stoichiometrically decrease pH₂ to the affinity threshold (Table 3). Therefore, hydrogenotrophic methanogenesis remained kinetically feasible throughout the duration of the experiment, though H₂ concentrations likely remained below the minimum substrate concentration (S_min) required to support growth⁵⁴. A future study using a modified MAC design to allow for regular biomass measurements would provide valuable insights in elucidating S_min under these conditions.

Table 1.

H₂ partial pressure (pH₂), and availability (µmoles) under conditions assayed in this study.

Temp. [°C]	Pressure [mbar]	H2 [%]	pH2 [mbar]	H2(g) [µmoles]	H2(aq) [µmoles]
0	1500	80	1200	3.4 × 103	0.04
0	1500	2.9	44	1.2 × 102	1.4 × 10–3
30	1500	80	1200	3.1 × 102	0.03
30	1500	2.9	44	1.1 × 102	1.2 × 10− 3
0	7–12	80	5.6–9.6	37–64	1.8 × 10− 4 to 3.1 10− 4
0	7−12	2.9	0.20–0.35	1.4–2.3	6.6 × 10− 6 to 1.1 × 10− 5
†0	7	1.5 × 10− 3 ± 5.0 × 10− 4	1.8 × 10− 4	–	–

Table 2.

H₂ partial pressure (pH₂), in the 0 °C/7–12 mbar/Mars gas treatment available to M. barkeri compared with reported H₂ affinity thresholds^§ of ^†hydrogenotrophic and ^obligately methylotrophic methanogens.

Species	pH2 [mbar]	Reference no.
†*Methanosarcina barkeri str. MS (+ 0.1 µM Ni)	0.20–0.35	This study
†*Methanosarcina barkeri str. MS (no added Ni)	0.19	51
†*Methanosarcina barkeri str. MS (+ 0.2 µM Ni)	0.13	51
†*Methanosarcina barkeri str. MS	0.16	50
†Methanobacterium formicicum str. JF-1	0.06	49
†Methanobacterium bryantii str. M.O.H.	2.9 × 10− 4 to 1.4 × 10− 3	54
†Methanoculleus bourgensis str. MAB1	0.001	51
^Methanosphaera stadtmanae str. MCB-3	0.01	56
^*Methanimicrococcus blatticola str. PA	< 0.001	56
^Methanomassiliicoccus luminyensis str. B10	< 0.001	56

Cytochrome-containing methanogens are indicated by an asterisk (*).

^§Note that smaller H₂ affinity thresholds indicate the ability to utilize H₂ at lower partial pressures.

Table 3.

Gibbs free energy of reaction (∆_rG), maximum theoretical CH₄ production (assuming complete reaction of limiting substrate), and observed CH₄ production efficiency (as % theoretical max) for hydrogenotrophic methanogenesis under conditions assayed in this study.

Temp. [˚C]	Pressure [mbar]	H2:CO2 [%]	∆rG [kJ/mol C]	Max. theoretical CH4 [µmol]	Avg. ± Std. Err. CH4 production [% Theoretical Max]
0	1500	80:20	− 117.6	859	3.22 ± 0.67
0	1500	2.9:92.5 (Mars mix)	− 113.5	31.1	0.010 ± 0.003
30	1500	80:20	− 118.0	774	8.06 ± 1.90
30	1500	2.9:92.5 (Mars mix)	− 113.5	28.1	3.35 ± 0.91
0	7–12	80:20	− 93.2 to − 95.6	9.37–16.1	< 0.01
0	7–12	2.9:92.5 (Mars mix)	− 89.1 to − 91.6	0.340–0.582	< 0.01

Transcription under these conditions indicates that some energy was available for minimum cellular maintenance activities. An exception would be in the case of replicates in which no quantifiable RNA was recovered (e.g., 0 °C/7–12 mbar/Mars gas-Rep1 and 30 °C/1500 mbar/Mars gas-Rep1). In this case, it would not be unreasonable to infer that these cultures were approaching transcriptional dormancy. Although complete metabolic inactivity or population demise (i.e. cell death) is possible, it cannot be definitively determined with the information available. SEM imaging revealed intact cell clusters from 0 °C/7–12 mbar/Mars gas treatments with no obvious signs of degradation or cell lysis (Fig. 1C). Methanogenesis can still proceed without transcription, as it is the preexisting protein—not the mRNA—that directly mediates methanogenesis. Messenger RNA is nonetheless a valuable proxy for viability, given its short half-life. Though studies on RNA stability in Archaea are limited, research on Methanosarcina acetivorans, a close relative of M. barkeri, reports mRNA half-lives ranging from minutes to hours⁵⁵, making residual mRNA after 14 days negligible. Therefore, we interpret the cessation of transcription of methanogenesis genes as a strong indicator of cells entering a state of dormancy or metabolic stasis at minimum.

While transcriptomics is a powerful tool to assess snapshots of metabolic activity at the time of cell preservation, we acknowledge its limitations. Gene expression should not be considered strictly synonymous with downstream metabolic activity. A future study incorporating proteomics could provide additional insight about the potential of methanogenesis, with the caveat that identifying the presence of proteins would not be synonymous to confirming active metabolism. The argument can be made that the longer half-life of proteins is a worse indicator of recent metabolic activity than mRNA.

The pH₂ levels of all treatments explored in the current study fall within the range of previously reported pH₂ uptake thresholds for M. barkeri and other hydrogenotrophic methanogens^49,51,54 (Tables 1 and 2). Assuming minimum cellular maintenance requirements can be sustained by 0.3 mol ATP produced per mole CH₄, the thermodynamic equilibrium of hydrogenotrophic methanogenesis has been estimated to be reached at pH₂ = 0.0018 mbar²⁶, increasing confidence that CH₄ production observed here at 7–12 mbar may be reflective of de novo methanogenesis; i.e., CH₄ that was produced by M. barkeri during the course of the incubation experiment, rather than being residual dissolved gas transferred from the inoculant culture (Fig. 1D). This is bolstered by the detection of mRNA mapping to methanogenesis genes (Fig. 2), indicating that transcription of the pathway was active at the time of sample collection. Additionally, the combination of the methane assay chamber (MAC) design (Fig. 1B) with the N₂-purging scheme during atmospheric re-equilibration prior to initiating the 14-day assays (see “Materials and methods”) makes it unlikely that observed methane production at 7–12 mbar was an artifact of pre-existing CH₄ exsolving from loaded cell cultures into the MAC headspace. If that were the case, the CH₄ production efficiency—i.e., the percentage of CH₄ produced relative to the theoretical maximum (if the reaction ran to completion)—would have been considerably higher than what we observed (Table 3).

The ideal gas law was used to calculate pH₂ and µmoles of gaseous H₂ in the headspace, H₂(g). Bioavailable dissolved H₂, H₂(aq), was calculated according to Henry’s Law, deriving temperature-dependent Henry’s constants for H₂ via the van’t Hoff equation (SI Materials and Methods and Table S9). ^†Present-day Martian surface atmospheric H₂ at 0 °C for reference.

If life evolved on Mars, the Noachian period would have likely been very habitable for hydrogenotrophic methanogens, with a recent study estimating potential biomass productivity rivaling that of Earth’s Archean ocean⁵⁷. It has been estimated that the modern atmospheric composition of Mars could support, through downward diffusion, a maximum steady-state subsurface biomass of 10²⁷ cells⁵⁸, which is two to three orders of magnitude smaller than recent estimates of prokaryotic biomass in Earth’s continental subsurface^59,60. This may be an underestimate, however. Additional modeling efforts considering advective upward fluxes of subsurface gases (e.g., via barometric pumping through fractured rock)⁶¹ could provide a more accurate estimate of the total supportable methanogenic biomass in the Martian subsurface.

While present-day conditions on the Martian surface are widely accepted to be too hostile to support extant life, at depths shallower than the melting isotherm, ~ 90–200 m depth⁶², microscale features such as fluid inclusions, liquid films on water ice, or brine lenses present potential microhabitats could support methanogenesis. At depth, the subsurface offers the potential of long-lived “habitable refugia” in the form of rock-hosted fracture fluids⁶³. Much like oligotrophic deep biosphere environments on Earth, habitable refugia in the present-day Martian subsurface offer energetic landscapes capable of supporting methanogenic biomass at low, but sustained, metabolic steady state.

On Earth, subsurface lithoautotrophic microbial ecosystems (SLiMEs) have been found inhabiting > 2.0 Ga-aged ultra-deep intra-cratonic hypersaline brines⁶⁴. Microbial communities hosting methanogens have been found to depths > 4.4 km in the continental crust⁶⁵ and > 2.5 km depth below the ocean floor⁶⁶. In these hydrologically isolated communities, there are negligible metabolic communications with surface processes. At many deep sites (e.g., > 3 km below land surface in Tau Tona mine, South Africa), carbon isotopic signatures of microbial lipids and carbon substrates indicate microbial communities are sustained by abiotic methane⁶⁷. However, SLiMEs characterized from boreholes in Beatrix (1.39 km below land surface) and Driefontein (1.05 km below land surface) are notable examples where microbial C1 carbon cycling is not based on a downward flux of surface-derived fixed carbon, or even abiotic CH_4, but rather in situ microbial methanogenesis^46,67. In these communities, methanogenesis is fueled by H₂ generated from water-rock reactions such as radiolysis⁴⁶, serpentinization⁶⁸, the oxidation of ferrous silicates⁶⁹, and seismogenic cataclasis⁷⁰. Serpentine deposits have been found in several locations across the southern Martian highlands^71–73, including in the Nili Fossae region where there have been reports of atmospheric CH₄ plumes⁵. It is presently unknown whether serpentinization is still an active geologic process on Mars. However, CH₄ in serpentinizing systems can either be produced chemoautotrophically by methanogens or abiotically through Fischer-Tropsch-type (FTT) synthesis, specifically, low-temperature (< 150 °C) Sabatier reactions^74–76. In the absence of a solid-phase metallic catalyst, abiotic CH₄ formation via FTT reactions would proceed very slowly under the range of pressure and temperature conditions investigated in this study. Given the 14-day duration of incubations and the absence of a solid-phase metallic catalyst, it is unlikely that any significant abiotic CH₄ formation occurred under the range of pressure and temperature conditions investigated in this study.

Radiolysis presents a mechanism for active H₂ generation in the Martian subsurface. The Gamma Ray Spectrometer (GRS) onboard the Mars Odyssey orbiter has collected a comprehensive record of radionuclide activities across the surface⁷⁷. From these data, present-day H₂ production rates from radiolysis in groundwater-saturated lithologies have been estimated to be on par with that of Earth’s basaltic oceanic crust^78,79. This includes Jezero Crater, the landing site of Perseverance and primary source of materials destined for the Mars Sample Return program⁸⁰, where bulk radiolytic H₂ production in subsurface fracture fluid has been calculated up to ~ 0.22 nM µm⁻¹ yr⁻¹⁷⁸. Radiolysis, therefore, could be an active source of H₂ as an electron donor in hydrogenotrophic methanogenesis in the Martian subsurface.

The present-day Martian H₂ atmospheric mixing ratio is estimated to be 15 ± 5 ppm⁸¹, roughly one order of magnitude lower than the described hydrogen affinity threshold of any methanogen (Tables 1 and 2). Availability of stable liquid water and nutrients notwithstanding, the Martian near-surface is likely to be limiting for hydrogenotrophic methanogenesis unless there is proximity to H₂ outgassing from the subsurface.

Future research is required to determine if methanogens not dependent on H₂ for metabolism (e.g., acetoclastic and methylotrophic methanogens capable of using alternative reducing equivalents) might survive and produce methane under simulated Martian near-surface conditions. Such research should select methanogens that are tolerant of temperatures close to 0 °C to be active in the thermodynamic window of stable liquid water on Mars⁸², and also possess antioxidative defense systems to combat oxidizing atmospheric pO₂ and perchlorates. M. barkeri possesses all three pathways of methanogenesis (hydrogenotrophic, acetoclastic, and methylotrophic). At low pH₂, a methylotrophic culture of M. barkeri could oxidize methyl groups to CO₂ via the Wood-Ljundahl pathway, creating an energetic advantage over hydrogenotrophic methanogenesis. Methanosarcina species are also considered to be the most aerotolerant of methanogens⁴⁵ and have a proven track record of survival in the presence of perchlorates^38,39,43. Though the jury is still out on the biogenicity of Martian methane, our findings contribute to an emerging picture of a plausibly habitable Mars for life as we know it.

It is remarkable that a methanogen isolated from sewage sludge⁸³ possesses the metabolic plasticity to remain viable and metabolically active under the energetically scant—if not challenging—conditions characteristic of modern Mars. Our findings suggest not only that a methanogenic biosphere in the modern Martian subsurface is possible, but that Methanosarcina barkeri, if given the opportunity to establish itself, could further adapt to these extreme conditions, potentially enhancing its survival and metabolic plasticity. This discovery has significant implications for Planetary Protection measures, particularly in the case of Mars Special Regions (i.e., with respect to spacecraft cleanliness and opportunistic microbial hitchhikers). Looking further ahead to human exploration and colonization of Mars, our findings indicate there may be promising potential to bring along methanogens in future missions for in situ resource generation (i.e., fuel production) and terraforming.

Materials and methods

Microbiological procedures

Cultures of M. barkeri wild-type strain MS (ATCC 51582; obtained from T. Kral, Univ. of Arkansas, Fayetteville, AK USA) were grown in DSMZ 120a media supplemented with 10.0 mg L⁻¹ of ethylenediaminetetraacetic acid (EDTA), 10 ml L⁻¹ of ATCC Trace Mineral Supplement solution (#MD-TMS, ATCC, Manassas, VA USA), and 10 ml L⁻¹ of ATCC Vitamin Supplement solution (#MD-VS; ATCC). All chemicals were obtained from Sigma-Aldrich, Co. (St. Louis, MO USA) unless otherwise noted.

Cultures of M. barkeri cells were grown in crimp-sealed balch anaerobic tubes in 10 mL of DSMZ 120a media and headspaces pressurized to 1500 mbar with an 80:20 mixture of hydrogen (H₂) and carbon dioxide (CO₂). Sodium resazurin (0.5 ml L⁻¹, 0.1% w/v) was used as a low pO₂ indictor reagent. All balch tubes were sparged as required with either 0.2 μm filter-sterilized anoxic N₂ or 80:20 H₂:CO₂ gases. Cultures were grown for 4 weeks at 30 °C before use. Cell densities were estimated by aseptically and anaerobically withdrawing 100 µL of suspended cells in 120a media from each culture, staining the cells with 100 µM aqueous acridine orange (AO), and counting cells in a hemacytometer. Cell densities per culture were approximations because cells of M. barkeri formed medium to large aggregates in the 120a media (Fig. 1C; Table S3). Protocols for preparing M. barkeri cells for SEM imaging are described elsewhere⁸⁴. All anaerobic media and cultures were prepared and handled in a Coy Anaerobic Chamber (Coy Laboratory Products, Grass Lake, MI USA).

For the 1500 mbar treatments, four-week-old cultures of M. barkeri were harvested, centrifuged, and washed thrice with fresh 120a media to purge dissolved CH₄ from the culture media. Fresh cultures were inoculated by aseptically and anaerobically transferring ~ 5.0E+08 cells (as determined by AO stain hemacytometer counts described above) into 35 mL fresh 120a media in 100 mL crimp-sealed borosilicate serum vials and subsequently sparged with either 80:20 H₂:CO₂ gas or the Mars gas mixture according to treatment.

Methane assay chambers (MAC)

Two methane assay chambers (MAC units) were designed in-house and manufactured by EMF, Inc. (Merritt Island, FL USA). Two MAC units were installed into a previously described Planetary Atmospheric Chamber (PAC)^85,86, as shown in Fig. 1A. Each MAC unit was plumbed with gas supply and sampling lines as shown in Fig. 1B.

Initial assays were performed to determine potential abiotic CH₄ evolution in the MAC units. In the first preliminary experiment, a mechanical blank (i.e., empty MAC units with no media or cells) was stabilized under all combinations of temperature and pressure conditions investigated in this study and left for 14 days before sampling and analyzing headspace gases. The second preliminary experiment repeated this process with the inclusion of filter-sterilized DSMZ 120a media blank (i.e., no cells).

For the 7–12 mbar treatments, four-week-old cultures of M. barkeri were harvested, centrifuged, washed thrice with fresh 120a media (i.e., to purge dissolved CH₄ from the culture media), combined, and aseptically and anaerobically processed such that 10 mL of 120a media inoculated with ~ 5.0E+08 cells (derived from AO stain hemacytometer counts described above) were available for each MAC unit. The MAC units were precooled to ~ 7–8 °C with a liquid nitrogen cryogenic system. With filter-sterilized anoxic N₂ gas flowing into the MAC units, the tops were briefly opened and 10 mL of M. barkeri cells placed within sterile 6-cm glass petri dishes. The MAC units were fitted with new copper gaskets, sealed, and chilled to 4 °C. Each MAC unit was loaded separately with anoxic and viable cultures of M. barkeri to maintain cultures in an oxygen-free state. All cultures showing pink coloration after the setup protocol were discarded and the process restarted (i.e., resazurin dye indicated absorption of trace O₂ into the media).

After both MAC units were anaerobically loaded with viable cultures of M. barkeri, the temperature of the MAC and PAC systems was lowered to 0 °C (± 0.5 °C) and 7.0 mbar (± 0.2 mbar) of N₂. While the internal MAC units were stabilizing at Martian temperature and pressure, the N₂ gases within the headspaces of the units were purged three times with fresh N₂ (over approx. 5 min), and then purged thrice more with either the 80:20 H₂:CO₂ or Mars gas + 2.9% pH₂ mixtures (over approx. 5 min). An Opto-22 data logging system (Opto-22, Inc., Temecula, CA USA) was used to record the internal temperatures, pressures, and relative humidities (RH) in headspaces above M. barkeri cultures within both MAC units. Cultures of M. barkeri cells in the MAC units were then maintained for 14 days at 0 °C and 7–12 mbar and then sampled at day 14 for the accumulation of CH₄ in the headspace gases.

Over the course of the 14-day experiments (described below), liquid water evaporated from the DSMZ 120a medium and increased the headspace pressures within the MAC units to 12 mbar. The system could not maintain the MAC headspace pressures at lower than 12 mbar without evacuating accumulated CH₄. Therefore, we report that these experiments were conducted between 7 and 12 mbar.

Although a Residual Gas Analyzer (RGA) was present in the PAC (Fig. 1B), it could only provide a qualitative indication of CH₄ due to a peak interference between O and CH₄ at 16 atomic mass units (amu). Therefore, we used a gas chromatograph (Trace 1310, Thermo-Fisher Scientific, Waltham, MA USA) fitted with a flame ionization detector (FID) for quantitative measurements, as it allowed for more accurate calibration and detection of CH₄. Methane concentrations within the MAC unit headspaces were collected by pumping the headspace gases from the MAC units directly into 10 cc Tedlar bags using a low-pressure 2-stage pump (model MPU3920-N813, KNF Manufacturing, Trenton, NJ USA). The gases within the Tedlar bags were immediately sampled and injected into the GC-FID. The same gas sampling procedure was followed for the 1500 mbar treatments. Preliminary trials with gas mixtures doped with known concentrations of CH₄ indicated that removing the headspace gases from the MAC units at 7 mbar, collecting them outside the Mars chamber at 1015 mbar (i.e., average atmospheric pressure on the Florida coast where the PAC is located), injecting them into the GC-FID system had a minimum detection level of 10 ppm CH₄ in the MAC atmospheres. Thus, all readings below 10 ppm were considered below the minimum quantifiable limit (MQL) and treated as zeroes in the data spreadsheets. Standard curves for measuring CH₄ in the MAC units are given in Fig. S1.

Two separate experimental treatments were conducted in the PAC at 7–12 mbar to simulate Mars surface pressure. The first treatment involved 80:20 H₂:CO₂ headspace gas mixtures within the MAC units; i.e., at Mars surface pressure but under a stoichiometrically-balanced atmosphere to support hydrogenotrophic (H₂:CO₂-based) methanogenesis. The second treatment also tested for hydrogenotrophic methanogenesis at 7–12 mbar, but under a Mars gas mixture⁴⁴ supplemented with H₂. The Mars gas mixture was composed of 92.5% CO₂, 2.9% H₂, 2.59% N₂, 1.94% Ar, 0.16% O₂, 0.03% H₂O (Boggs Gases, Titusville, FL), more closely representing present-day Martian shallow subsurface. The choice of 2.9% H₂ balanced the goal of creating a Mars gas mixture with H₂ concentrations near reported H₂ affinity threshold values for M. barkeri (Table 2) and safety limits imposed by the vendor to ensure the gas mixture remained stable and non-flammable. Each treatment included six replicates; however, since the PAC could only accommodate two MAC units (i.e., two replicates) per run (Fig. 1B), the experiment was repeated three times per treatment to achieve the full set of replicates.

Statistical analysis of CH₄ evolution data

Methane evolution data for both experiments were analyzed with the statistical analysis software package SAS, v9.4 (SAS Institute, Inc., Cary, NC USA). Two out of the six replicates in the 0 °C/7–12 mbar/Mars gas treatment yielded CH₄ below the 10-ppm minimum quantifiable limit and were thus removed from additional analyses. To maintain consistency and ensure comparability across the Mars gas treatment, we adjusted the other Mars gas treatments (30 °C/1500 mbar/Mars gas and 0 °C/1500 mbar/Mars gas) to four replicates as well. This approach provided uniform sample sizes across these conditions, simplifying statistical comparisons and reducing the potential for bias due to unequal sample sizes. Data were log-transformed to induce homogeneity of treatment variances. Transformed data were subjected to ANOVA (PROC GLM) and protected least-squares mean (LSM) separation tests (P ≤ 0.01; n = 6 replicates for the first treatment [80:20 H₂:CO₂] and n = 4 replicates for the second treatment [Mars gas mix]). Data in Fig. 1D are presented as untransformed values. Both within-group and between-group statistical differences in methane production were assessed with respect to atmospheric gas mix condition (e.g., H₂:CO₂ across assayed temperatures and pressures, and H₂:CO₂ vs. Mars gas mix for a given temperature and pressure condition).

Culture preservation for downstream transcriptomics analyses

After collecting gas samples from individual MAC units, the units were repressurized with the headspace gases described above for each experiment. As soon as the MAC units and the bulk Mars chamber headspace were equilibrated to 1015 mbar, the PAC system was opened, the MAC tops unbolted, and 10 mL of RNAlater™ (Invitrogen, Thermo-Fisher Scientific, Waltham, MA USA) were immediately added to each culture. The process was completed as quickly as possible to faithfully capture gene expression profiles at the ends of both experiments, with timed extractions accomplished within 12–15 min from the initial re-pressurization step. The liquid nitrogen cold plate, and thus, the M. barkeri cultures, were maintained at 0 °C during the extraction process.

Once the RNAlater™ was injected into the M. barkeri cultures, the cells were transferred to sterile 15 cc Falcon tubes (Corning Inc., Corning, NY USA), pelleted by centrifugation, decanted, and refreshed with pre-chilled RNAlater™ added to the tubes. The cells were washed thrice with RNAlater™ while holding the temperature at 0 °C. Cultures were stored at −80 °C and shipped overnight on dry ice to Harvard University, where they were maintained at −80 °C until further processing.

RNA isolation, library prep, and sequencing

RNA was isolated and purified as previously described³⁹ with parallel extraction blanks and thorough RNAse decontamination at each step to ensure cleanliness of the extraction procedure. An aliquot of RNA from each sample was quantified using a Qubit hs RNA assay kit coupled to a Qubit 2.0 fluoremeter (ThermoFisher Scientific). A two-directional library preparation was performed for each experiment using the Nextera XT DNA Library Prep Kit according to the manufacturer’s instructions (Illumina, Inc., San Diego, CA USA). The resulting libraries were quality checked via TapeStation as described above, and final qPCR of pooled libraries was performed on a Bio-Rad CFX96 system (Bio-Rad Laboratories, Hercules, CA USA) following the standard Illumina qPCR protocol (Illumina, Inc.). Two-directional (2 × 150 bp) sequencing was performed on one lane of a NovaSeq S4 flowcell (Illumina) at the Harvard University Bauer Core Facility.

Bioinformatic and statistical pipeline

Quality filtering, mapping, and annotation of paired-end reads to M. barkeri coding sequences (CDS) were performed as previously described³⁹. Single variable differential expression analyses were performed using the ‘DESeq2’ package in R⁸⁷, considering 30 °C as the reference temperature condition, 1500 mbar as the reference pressure condition, and 80:20 H₂:CO₂ as the reference atmospheric condition. We observed an overabundance of zero counts (i.e., zero inflation) in several transcriptomic libraries of Mars gas mix-incubated samples. Zero inflation in transcriptomic libraries may be attributable to biological factors, such as the true absence of transcripts, as well as technical artifacts from low sequencing depth. Because of the significant reduction in library size for the Mars gas mix-incubated samples, a highly conservative statistical approach was taken to minimize the risk of false discovery rates.

Transcripts were indexed by gene assignment into a count table and library size factors were estimated using the ‘poscounts’ option to address instances of zero inflation in low-biomass libraries. Fragments per million mapped reads (FPM) were calculated using the robust median ratio method to account for sequencing differences in library size and RNA composition of samples. Gene-wise dispersion and mean-dispersion relationships were estimated using negative binomial generalized linear model with a local fit type and Wald significance tests. Resulting log₂-fold changes in expression (LFC) were then shrunk using the ‘apeglm’ shrinkage estimator⁸⁸. Resulting model count data were then variance-stabilized using a regularized log transformation to minimize differences between samples containing genes with zero and near-zero transcript counts. The resulting dataset was scaled and centered. A principal component analysis (PCA) was conducted on the regularized, log-transformed read counts across the 28 sequenced libraries using the ‘plotPCA’ function in the ‘DESeq2’ package⁸⁷ to identify the primary sources of variance within the dataset. A two-dimensional non-metric multiscale dimensional analysis (NMDS) solution was determined using a Bray-Curtis dissimilarity index iterated over 2000 random starts using function ‘metaMDS’ in the ‘vegan’ package⁸⁹.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

RLH and ACS conceived the study. ACS grew M. barkeri and conducted methane evolution experiments at the University of Florida. RLH performed RNA extractions, cDNA generation, and library prep for sequencing efforts at Harvard University and carried out all bioinformatic and statistical analyses on resulting transcriptomic libraries. RLH and ACS both contributed to the writing and editing of the manuscript.

Data availability

Raw sequencing data were deposited to NCBI Genbank under BioProject PRJNA998042. The complete genome of Methanosarcina barkeri MS is available at NCBI under accession GCA_000970025.1. The protein table for M. barkeri MS is available at https://rest.uniprot.org/uniprotkb/stream? compressed=true&format=fasta&query=%28%28taxonomy_id%3A1434108%29%29. The KEGG pathway module for methane metabolism in M. barkeri MS can be found at https://www.genome.jp/pathway/mby00680. All code written for analyses presented in this manuscript are available at https://github.com/rachel-l-harris/Harris2024_MarsLowP.

Competing interests

The authors declare no competing interests.