Real time monitoring of hydrogenotrophic methanogenesis under deep saline aquifers conditions

Real time monitoring of hydrogenotrophic methanogenesis under deep saline aquifers conditions | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Real time monitoring of hydrogenotrophic methanogenesis under deep saline aquifers conditions Emeline Vidal , View ORCID Profile Anaïs Cario , Mathilda Jouvin , Maïder Abadie , Olivier Nguyen , View ORCID Profile Arnaud Erriguible , View ORCID Profile Anthony Ranchou-Peyruse , View ORCID Profile Samuel Marre doi: https://doi.org/10.1101/2025.05.05.652176 Emeline Vidal 1 CNRS, Univ. Bordeaux, Bordeaux INP, ICMCB , F-33600, Pessac Cedex, France 2 Marine Biological Laboratory, Ecosystems Center and J Bay Paul Center for Comparative Molecular Biology and Evolution , Woods Hole, MA 02543, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anaïs Cario 1 CNRS, Univ. Bordeaux, Bordeaux INP, ICMCB , F-33600, Pessac Cedex, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anaïs Cario Mathilda Jouvin 1 CNRS, Univ. Bordeaux, Bordeaux INP, ICMCB , F-33600, Pessac Cedex, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maïder Abadie 1 CNRS, Univ. Bordeaux, Bordeaux INP, ICMCB , F-33600, Pessac Cedex, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Olivier Nguyen 1 CNRS, Univ. Bordeaux, Bordeaux INP, ICMCB , F-33600, Pessac Cedex, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Arnaud Erriguible 1 CNRS, Univ. Bordeaux, Bordeaux INP, ICMCB , F-33600, Pessac Cedex, France 3 CNRS, Univ. Bordeaux, Bordeaux INP, I2M, site ENSCPB , 16 avenue Pey-Berland, Pessac Cedex, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Arnaud Erriguible Anthony Ranchou-Peyruse 4 Universite de Pau et Pays de l’Adour, E2S UPPA, CNRS, IPREM , Pau, 64000, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anthony Ranchou-Peyruse For correspondence: samuel.marre{at}icmcb.cnrs.fr anthony.ranchou-peyruse{at}univ-pau.fr Samuel Marre 1 CNRS, Univ. Bordeaux, Bordeaux INP, ICMCB , F-33600, Pessac Cedex, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Samuel Marre For correspondence: samuel.marre{at}icmcb.cnrs.fr anthony.ranchou-peyruse{at}univ-pau.fr Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract To investigate the microbial response to deep underground gas injection, specifically CO 2 and H 2 , a new optically transparent high-pressure reactor was developed to monitor autotrophic microbial growth via in situ and ex situ characterization techniques. The main advantages rely on avoiding any decompression phases during the entire process, thanks to direct optical access. Here, we monitored the growth of the model methanogenic strain Methanothermococcus thermolithotrophicus by applying different H 2 /CO 2 partial pressures at a total pressure of 100 bar, which is representative of the deep underground storage environment. Additionally, we measured the methane production of the strain at the end of the incubation, which resulted in an increase in methane production with increasing CO 2 and H 2 partial pressures until a certain point. These reactors can be used to investigate deep microbial strains under pressure conditions close to their natural environments, eliminating decompression biases. Introduction Since the early twentieth century, the industry has utilized deep geological underground formations for gas storage in various forms, including salt caverns, depleted hydrocarbon reservoirs, and deep aquifers, often alongside indigenous microbial communities 1 , 2 . Among these options, porous reservoirs (depleted reservoirs and aquifers) provide the highest storage capacity and host a significant diversity of microbial taxa 3 . Historically, these storage facilities were designed primarily for methane and natural gases 4 . As we entered the 21 st century, the substantial rise in anthropogenic CO 2 emissions and the challenges posed by climate change prompted serious consideration of carbon capture and storage (CCS) strategies, which appear to offer a long-term solution for CO 2 sequestration in deep geological formations (Carbon Geological Storage – CGS) 5 . More recently, geological storage facilities have been recognized as essential for the emerging dihydrogen (H 2 ) sector, presenting a robust, massive and secure storage solution for this energy carrier 6 – 8 . Operators and models developers require physico-chemical and microbiological data to ensure these potential future storage sites, which are critical to future energy networks. While studies have demonstrated the ability of methanogenic archaea to convert CO 2 into methane during enhanced oil recovery (EOR) or CO 2 geological storage, this phenomenon has generally not been perceived as a concern; quite the opposite 9 . In the context of underground H 2 storage (UHS), the sustainability of such systems is important. Lithoautotrophic prokaryotes capable of utilizing H 2 and CO 2 ( i.e. , sulfate reducers, methanogens and homoacetogens) exist at storage sites, although their concentrations and activities vary widely 10 – 16 . Depleted hydrocarbon reservoirs seem particularly suitable for developing underground methanation reactors (UMRs) 17 – 19 , although some do not display lithoautotrophic properties 20 . While some deep aquifers show significant potential for biomethanation, others do not, suggesting site-specific variability that requires further investigation 2 , 14 – 16 . In the UMR context, CO 2 can originate from several sources: (i) the degradation of organic matter in the reservoir; (ii) the dissolution of carbonate minerals or emissions from deeper geological strata; and (iii) the intentional coinjection of CO 2 to produce biomethane 9 , 14 , 21 . The future of these storage facilities has generated considerable interest among modelers, yet they work with limited data 22 , 23 . Experimental data gaps must be filled through multiscale studies from the pore scale 24 to the pilot scale, high-pressure simulation experiments, and assessments of model microbial strains to establish growth limits and yields, particularly for methanogens 13 , 25 . Various pathways exist for methanogenesis, including the hydrogenotrophic pathway, which converts H 2 and CO 2 into methane 26 . Research suggests that when H 2 is injected into deep aquifers, hydrogenotrophic methanogenic populations can develop rapidly, producing substantial amounts of methane. For instance, Amigáň et al . 27 observed 30% consumption of H 2 within seven months in a deep aquifer used to store town gas (composed of 54% H 2 , 22% CH 4 , and 12% CO 2 at 40 bar and 25–45°C). Similarly, Haddad and colleagues 14 simulated 10% H 2 storage in an aquifer used to store natural gas (99% CH 4 , 1% CO 2 at 95 bar and 47°C), resulting in a 40% reduction in H 2 over three months, largely attributed to methanogenesis. Currently, most models rely on the Monod equation, which considers theoretical yields for H 2 and CO 2 consumption and methane production 23 . Additionally, the majority of available data in the literature have been gathered at atmospheric pressure and may not accurately reflect the conditions necessary for high-pressure (HP) applications 13 , 28 , 29 . The study of deep microorganisms that grow autotrophically, such as hydrogenotrophic methanogens, poses challenges for HP culture investigations, as these organisms require both aqueous and gaseous (H 2 /CO 2 ) phases for growth. Specific cultivation methods have been developed over the last few decades 30 – 33 , but these methods face several limitations, including (i) the availability of specialized equipment, (ii) the expertise required to utilize this equipment effectively, and (iii) restricted optical access for monitoring growth. Consequently, very few methanogens have been characterized for their piezophilic capabilities 31 , 33 , 34 . To cultivate (hyper)thermophilic piezophilic autotrophic methanogens, several strategies have been suggested: (i) use gas-tight syringes with a 2 bar pressure of H 2 /CO 2 (4:1) in pressurized static pressure vessels 30 ; (ii) employ pressure cell reactors with sapphire windows placed in an oven filled with 7.8 bar of H 2 /CO 2 (4:1) supplemented with helium at the required experimental pressure 31 ; and (iii) utilize autoclaves containing cultures in nickel tubes with 2 bar of H 2 /CO 2 (4:1) 35 . Nevertheless, the decompression step - required for counting cells - can be detrimental and could lead to biases. 36 Hence, transparent approaches, including high-pressure microfluidics, have been proven to overcome this limitation in monitoring cell growth in real time. 37 Researchers have generally observed a trend when incubating at very low H 2 /CO 2 partial pressures while increasing the total pressure of the system, to evaluate the strain tolerance limits to hydrostatic pressure. To assess microbial responses to gas injections effectively in their environment, it is essential to recreate laboratory-scale conditions that mimic those found within the natural ecosystem. The interaction between microorganisms and stored gases is critically important, as it can result in variations in gas quality (including H 2 consumption and the production of sulfide and/or methane), deterioration of infrastructures (due to biocorrosion), and shifts in physicochemical conditions (affecting water quality and the dissolution or precipitation of biominerals, bioclogging). In this study, we focus on simulating the physicochemical conditions of a one-kilometer-deep saline aquifer used as either a UHS or a UMR within high-pressure transparent reactors (HPTRs) 5 , 38 – 40 . The impact of increasing H 2 /CO 2 pressure on hydrogenotrophic methanogens is a relatively unexplored area of study. However, given the importance of geological gas storage and the prevalence of these components in the environment, investigating this issue is warranted. One of the novelties of our approach lies in the creation of transparent sapphire cultivation cells that feature a fiber optic system to enable real-time monitoring of microbial growth under conditions simulating various gas injection scenarios. Specifically, this study aims to achieve two objectives: (i) to assess how increases in pressure affect both the growth and metabolism of autotrophic methanogens and (ii) to evaluate the feasibility of biomethane production under conditions that mimic gas injection scenarios by monitoring the kinetics of methane production. 1. Materials and methods 1.1. Strain and Growth Medium Methanothermococcus thermolithotrophicus strain SN-1 (DSMZ 2095) was used as a model lithoautotrophic thermophilic methanogen 35 . This strain is known for its ability to grow under piezophilic conditions. The strain was cultivated in modified artificial ground water 41 (AGW) containing NaCl (25.84 g.L -1 ), KCl (0.14 g.L -1 ), MgCl 2 ·6H 2 O (1.42 g.L -1 ), CaSO 4 ·2H 2 O (1.37 g.L -1 ), CaCl 2 ·2H 2 O (0.73 g.L -1 ), NH 4 Cl (0.02 g.L -1 ), yeast extract (1 g.L -1 ) and resazurin (1 mg.L -1 ). To prevent potential acidification during high-pressure experiments, the medium was supplemented with 120 mM HEPES buffer. After autoclaving, the medium was further supplemented with 250 µL of 30 mM K 2 HPO 4 and 1 mL of SL10 Widdel trace elements 42 . The liquid phase was reduced with 1% (v/v) Na S 2 9H 2 O (25 g.L -1 ). The gas phase was replaced with a mixture of H 2 /CO 2 at a ratio of 4:1 (Messer) by 15 min of sparging at 1.5 bar. Finally, the pH of the medium was adjusted to 6.8. 1.2. High-Pressure Reactors The high-pressure transparent reactors (HPTRs) utilized in this study are composed of transparent sapphire tubes, each 12 cm long with an internal diameter of 8 mm, providing a total volume of 5 mL ( Figure 1-a-b ) 43 . Each tube is fitted with titanium alloy (Ta6V) plugs, sealed with Viton® O-rings, and secured by metal clamps. For connectivity, the plugs are machined to accommodate standard commercial VALCO connectors for 1/16’’ tubing (standard 6/32 port). These reactors can withstand pressures of up to 500 bar and temperatures of up to 200°C. The chemical and biological inertness, along with the full transparency of these reactors, allows in situ optical measurements such as optical density (OD) ( Figure 1-c ), facilitating the monitoring of microbial growth without the need to subsample the system under pressure. In certain experiments, internal mixing was achieved by placing a stirring magnet within the growth media and positioning the reactor on a magnetic stirrer. Download figure Open in new tab Figure 1: (A) Schematic of the high-pressure transparent reactors (HPTRs) used in this study; (B) photograph of the reactor; and (C) millifluidic sapphire reactor equipped with optical fibers for in situ and real-time O.D. measurements. 1.3. High-pressure setup and culture The archaeal strain was initially cultivated in vials at atmospheric pressure (1.5 bar) and then reinoculated at approximately 2.10 6 cells.mL -1 in fresh medium during the growth phase. Next, 1.5 mL of the inoculated medium was transferred into the HPTRs within an anaerobic chamber (glove box, MB-LABstar, MBraun) under a pure nitrogen atmosphere. The reactors were sealed, removed from the glove box, and immediately incubated at 65°C, the optimum temperature for the strain. The HPTRs were connected to a high-pressure Teledyne ISCO pump (260 HP) filled with a H 2 /CO 2 mixture (4:1 molar ratio; Messer), adjusted to the desired partial pressure, and supplemented with nitrogen to achieve a total pressure of 100 bar, corresponding to conditions relevant to deep aquifer storage ( Figure 2 ). All the tubing was purged with nitrogen before any injection. Experiments have examined the effects of varying H 2 /CO 2 partial (p(H 2 /CO 2 )) pressures (5, 15, 20, 30, and 50 bar) on strain growth. Some tests included stirring at 150 rpm via a heat-resistant magnetic stirring device inside an oven (magnetic emotion, MIXdrive 1 eco HT model). All experiments were conducted in triplicate, including a control setup. Download figure Open in new tab Figure 2. Scheme of the setup used for this study. 1.4. Cell Growth Measurements Optical density (OD) was monitored in real time via optical fibers (IDIL Fibres Optiques, France) connected to the HPTR ( Figure 1-c and 2 ). A spectrometer (Maya 2000 Pro, OceanView software, Ocean Optics) recorded the OD values every second at wavelengths between 600 and 605 nm. Prior to the experiments, validations of the optical system at atmospheric pressure and room temperature confirmed its accuracy; this involved testing with dilutions of stationary phase cultures of M. thermolithotrophicus . The initial and final cell concentrations were verified via direct cell counting via a Thoma chamber (Preciss, France; surface area: 0.0025 mm 2 ; depth: 0.1 mm) under a DM2000 LED phase contrast optical microscope (Leica Microsystems CMS GmbH, Germany). The absorbance of the dilutions was measured at two different wavelengths ( i.e., λ = 500 and 600 nm), and the results were similar. The results obtained from the measured values show a linear trend, which validates the system. The validated optic system allowed cell concentrations to be inferred from the recorded optical density (OD) data. 1.5. Gas Sampling and Analysis After the experiment, the methane (CH 4 ), dihydrogen (H 2 ), carbon dioxide (CO 2 ), and nitrogen (N 2 ) contents of the gas samples from the HPTRs were analyzed via microgas chromatography (VARIAN CP-4900 PRO Micro-GC) instrument equipped with a thermal conductivity detector and a CP-5A column, with argon used as the carrier gas. A 2 L Tedlar® sampling bag (Supelco) was used to minimize decompression-related artifacts, sampling through a system designed to reduce the pressure drop to 5 bar. A 1/16” 20 cm long tube, with an internal diameter of 500 µm and equipped with two HP valves on both sides, was connected between the HPTRs and the sampling bag. A vacuum-prepared setup enabled sampling of approximately 40 µL of the gas phase at 100 bar, expanding to approximately 4 mL at 1 bar, for analysis. Once recovered, the Tedlar® sampling bag was connected to the micro-GC to be analyzed. 1.6. H 2 and CO 2 solubility and diffusion modeling Gaseous substrate solubilities in the liquid phase, including medium salinity effects, were calculated via Crozier and Yamamoto’s model for H 2 44 and Duan and Sun’s model for CO 2 45 , with fugacity computed via their 2006 improvements 46 . For ease of rationalizing the influence of gas penetration in the liquid medium when the system relies only on diffusivity (no stirring, which is not fully accurate since convection occurs in sapphire reactors), we propose analytically estimating the evolution of CO 2 and H 2 in the aqueous phase. We assume that the concentration evolves slowly in only one direction (depth of the reactor) under the assumption that convection is negligible. Therefore, the evolution of the concentration is given by the unsteady state mass conservation equation according to the classical Fick’s law for diffusion: where c i is the concentration of CO 2 or H 2 , z is the position and D i is the diffusion coefficient of CO 2 or H 2 in the water phase via the Stokes--Einstein relation at T = 65°C, p tot = 100 bar ( D co2 = 2.37·10 −9 m 2 .s -1 and D h2 = 8·10 −10 m 2 .s -1 ). Assuming one-dimensional diffusion in a semi-infinite medium, which is an acceptable hypothesis due to the very low diffusion in water, the well-known analytical solution 47 is given by: where c sat and c 0 are the initial concentration of CO 2 or H 2 in the liquid phase ( c 0 =0) and the saturation concentration of CO 2 or H 2 in the liquid phase, i.e. , the concentration values imposed at the boundary conditions z = 0, respectively; z is the distance (m); D i is the diffusion coefficient of molecule i in water; and t is the time (s). The saturation concentration is given by Henry’s law, which is directly calculated from the partial pressure of the gas in the reactor. 1.7. Growth Curves Analysis The growth curves were analyzed via the R package Growthcurver 48 . Initially, the data were plotted as the average log cell density (LCD). The data were subsequently normalized for each replicate, and the growth rate (R) was subsequently calculated via the following formula: where (N(t)) represents the cell concentration over time, (K) represents the maximum cell population, (N0) represents the initial cell population, (r) represents the growth rate, and (t) represents the time in hours. All raw data are provided in the supplemental material (ESI-1). 2. Results and Discussion Initially, high-pressure transparent reactors (HPTRs) were stirred at 150 rpm to facilitate thermodynamic equilibrium between the gas and liquid phases across a water/liquid interface of 50 mm 2 and to eliminate concentration gradients. The primary objective of this step was to assess the intrinsic growth potential and production yield of Methanothermococcus thermolithotrophicus under varying H 2 and CO 2 partial pressures (p(H 2 /CO 2 )). In the second phase, stirring was removed to simulate better environmental conditions, relying solely on diffusivity and slow convective mixing, which is the major transfer mechanism in porous environments. 2.1. Effects of p(H 2 /CO 2 ) on growth and metabolism 2.1.1. Cell development In the stirred system, growth kinetics at different partial pressures were monitored via an optical fiber system for in situ optical density (OD) measurements. As illustrated in Figure 3 , cell growth was observed for p(H 2 /CO 2 ) values ranging from 5 to 20 bar, with the highest cell density (1·10 8 cell.mL -1 ) achieved at 15 bar. No growth was detected at p(H 2 /CO 2 ) > 30 bar within the 24-h cultivation period. Comparatively, experiments by Haddad and colleagues 14 simulated the physicochemical conditions of underground gas storage (UGS) in a deep aquifer with low agitation (20 rpm). The tested H 2 concentration of approximately 10 bar closely aligned with the optimal condition, from the methanogenesis point of view, established in the current study (p H2 = 12 bar). These results warrant further investigation over extended incubation periods ( e.g. , 5 to 7 days), as higher p(H 2 /CO 2 ) levels (30 and 50 bar) may result in longer lag phases for the strain. Download figure Open in new tab Figure 3. Methanothermococcus thermolithotrophicus growth in response to applied p(H 2 /CO 2 ) (80/20 mol%) at a total pressure of 100 bar over 24 hours of incubation. Each curve represents the average of two in situ OD measurements (Log (N(t))). The specific growth rates for each condition are displayed in Figure 4 . Interestingly, the highest growth rate (0.84 ± 0.27 h -1 ) was observed at 20 bar, not at 5 bar (0.57 ± 0.34 h -1 ) or 15 bar (0.60 ± 0.14 h -1 ), despite 15 bar yielding the highest cell density. This suggests that while higher p(H 2 /CO 2 ) may accelerate cell growth and production, they do not necessarily correlate with maximum density or methane production. Although an increase in substrates (H 2 and CO 2 ) could imply enhanced growth and production, the optimal cell density at p(H 2 /CO 2 ) = 15 bar may indicate potential toxicity from one or both gases. Download figure Open in new tab Figure 4. (a-e) Specific growth rates (R) calculated for each replicate across various p(H 2 /CO 2 ) conditions (only the average growth rate is plotted). No growth (NG) was observed at p(H 2 /CO 2 ) = 30 or 50 bar. (f) Mean growth rates of M. thermolithotrophicus under applied p(H 2 /CO 2 ) (80/20 mol%) at a total pressure of 100 bar. To better simulate conditions in deeper underground environments, stirring was removed, leading to a noticeable change in the strain behavior. The typical growth curves could no longer be measured via optical fibers, as a significant biofilm structure developed on the tube wall ( Figure 5 ). This biofilm formation hindered the correlation of OD variation with cell density. Although planktonic cells were detected in the liquid phase, their concentration never exceeded 1·10 7 cells.mL -1 (approximately an OD of 0.53). Consequently, estimating cell development under these conditions has become challenging. Notably, no biofilms were observed when the system was stirred, indicating that the shear forces generated during stirring prevented cell attachment and aggregation. Instead, biofilm formation was likely driven by local gas concentrations due to the concentration gradients created by slow gas diffusion and potential toxicity effects. Biofilm formation was evident across all the tested partial pressures, with the distance from the biofilm to the gas⍰liquid interface ranging from 1 ± 0.5 mm to 17 ± 2 mm (p(H 2 /CO 2 ) from 5 to 30 bar) and the thickness varying between 0.1 ± 0 mm and 2.3 ± 0.5 mm. Download figure Open in new tab Figure 5. Images of biofilms of M. thermolithotrophicus inside the HPTR at a total pressure of 100 bar and various p(H 2 /CO 2 ) ratios (80/20 mol%). At a pressure of 30 bar for p(H 2 /CO 2 ), the development of the biofilm can be distinctly observed in a halo pattern on the wall of the high-pressure reactor (HPTR). 2.12. Methane Production Methane production was measured under each p(H 2 /CO 2 ) condition, both with and without stirring ( Figure 6 ). A significant observation was that methane production varied by at least an order of magnitude, being 8 to almost 1000 times higher depending on the applied p(H 2 /CO 2 ) when stirring was absent. Under stirred conditions, methane production was limited to p(H 2 /CO 2 ) = 20 bar, with the highest production reaching 8.6·10 −7 ± 5.10 −7 mol at p(H 2 /CO 2 ) = 15 bar, which was correlated with the peak growth rate observed. Beyond p(H 2 /CO 2 ) ≥ 30 bar, neither methane production nor cell growth was detectable. Download figure Open in new tab Figure 6. Methane production by M. thermolithotrophicus over 24 h at 65°C, a total pressure of 100 bar, and various p(H 2 /CO 2 ) (80/20 mol%) with and without stirring (log scale). In contrast, methane production occurred across all the tested conditions when stirring was removed, reaching 2.07·10 −5 ± 5.60·10 −6 mol at p(H 2 /CO 2 ) = 30 bar before decreasing at higher pressures. This increase in production may be linked to biofilm formation, which tends to increase metabolic activity. The presence of biofilms has been associated with increased methane productivity in biomethanation studies 49 – 51 . Notably, biofilms of methanogenic communities are predominantly composed of hydrogenotrophic species, which exhibit increased methane production when only biofilm cells are present in the environment 49 . The authors reported that hydrogenotrophic species could constitute up to 37.9% of the community, highlighting their prominence 49 . Furthermore, Maegaard and colleagues 50 demonstrated that H 2 flux within biofilms surpassed equivalent liquid conditions in control tests, supporting the notion that biofilm structures can lead to increased methane production when a biofilm forms, which was confirmed in this study. The authors also reported that the production of methane varied from 7.2·10 −2 to 1.68·10 −1 L CH4 .L medium -1 .day -1 in their bioreactor, which contained a microbial community arranged into a biofilm, whereas the production of methane from 2·10 −2 to 5·10 −2 L CH4 .L medium -1 .day -1 was observed in the control. Consistent with these findings, our results indicate significantly greater CH 4 production when a biofilm formed, with levels reaching 3.9·10 −1 ± 9·10 −2 L CH4 .L medium -1 .day -1 for p(H 2 /CO 2 ) = 30 bar ( Figure 5 and Table 1 ). Moreover, biofilm formation appears to enhance the strain’s tolerance to elevated H 2 and CO 2 concentrations, as methane was produced at these concentrations in the presence of a biofilm, whereas no production occurred at p(H 2 /CO 2 ) above 20 bar ( i.e. , 30 and 50 bar) under stirring conditions when biofilms did not form ( Figure 6 ). View this table: View inline View popup Download powerpoint Table 1. Daily methane production per liter of medium by M. thermolithotrophicus at 65°C and a total pressure of 100 bar over 24 h, comparing conditions with and without stirring (biofilm formation). The differences in methane production can be attributed to the equilibrium maintained under stirred conditions, where H 2 and CO 2 concentrations are uniformly distributed in the liquid phase, potentially exerting toxic effects. In contrast, without stirring, the toxic effects develop more slowly, allowing the strain to expand in time for growth and metabolic activity. Notably, the most favorable pressure for metabolic activity increases from 15 bar with stirring to 30 bar without stirring, which aligns with the hypothesis regarding diffusion time. While biofilm formation prevented accurate measurements of the cell concentration and individual methane production per cell under nonstirred conditions, such data could be calculated during stirring. Therefore, under stirring conditions, the methane production per cell for p(H 2 /CO 2 ) = 5, 15 and 20 bar was estimated to be approximately 1.1·10 −5 nmol CH 4 .cell -1 , 1.5·10 −5 nmol CH 4 .cell -1 and 4.3·10 −5 nmol CH 4 .cell -1 , respectively This finding indicates that individual cellular production remains within a similar order of magnitude across all conditions, with a slight increase observed at p(H 2 /CO 2 ) = 20 bar. This pattern suggests that as the pressure of H 2 /CO 2 increases, the cells may begin to experience stress conditions, favoring metabolic activity over cellular proliferation. Importantly, even with improved access to and concentration of substrates, these factors do not enhance the overall production capability of the cells. To further examine the behavior of methane production without stirring under optimal metabolic conditions ( i.e. , p(H 2 /CO 2 ) = 30 bar), production was monitored over a 24-hour period ( Figure 7 ). Methane production exhibited linear and stable accumulation over time (R 2 = 0.98). The continued increase in production (accumulation of methane in the reactor) beyond the 24-hour mark can be attributed to the constant connection of the reactor to a high-pressure ISCO pump (260 mL), which was kept filled with the gas mixture (p(H 2 /CO 2 ) (80/20 molar) = 30 bar, supplemented with nitrogen to achieve a total pressure of 100 bar). Consequently, the H 2 and CO 2 concentrations remained stable throughout the incubation period (approximately 260 mL of gas was added to the 1.5 mL of culture medium inside the HPTRs). This approach was designed to simulate underground hydrogen storage (UHS) and underground methane reactor (UMR) scenarios for storing millions of cubic meters of gas. The measurements obtained here are encouraging for stimulating methanogenic activity under a continuous supply of substrates ( i.e. , UMR). Hypothetically, lithoautotrophic methanogenic archaea should be capable of producing indefinitely with a constant influx of substrates in the natural environment if we abstract from all other limiting nutrients. Download figure Open in new tab Figure 7. Methane production by M. thermolithotrophicus over 24 h at 65°C, a total pressure of 100 bar, and p(H 2 /CO 2 ) (80/20 mol%) = 30 bar without stirring. Under these optimized conditions (at 65°C, a total pressure of 100 bar and p(H 2 /CO 2 ) = 30 bar), the methane production rate for M. thermolithotrophicus was found to be 1.85·10 −2 L CH4 .L medium -1 . hour -1 , equating to 4.4·10 −1 ± 9·10 −2 L CH4 .L medium -1 .day -1 . The kinetics of production also allowed the detection of biofilm formation, which began to appear on the tube walls between 2 and 4 hours of incubation. This formation period is notably rapid compared with similar observations reported under controlled laboratory conditions with model strains 52 , 53 . For example, compared with other model strains, Pseudomonas aeruginosa biofilm formation is often detected between 12 and 24 hours 52 but varies between 24 and 72 hours for certain Staphylococcus species 53 . These findings suggest that M. thermolithotrophicus has an exceptional ability to develop biofilm structures under specific conditions. 2.2. Gas Solubility and Diffusion Table 2 presents the concentrations of H 2 and CO 2 in the liquid phase, reflecting the experimental conditions. Although CO 2 is present in a lower proportion than H 2 in the gas mixture (80% H 2 to 20% CO 2 ), our calculations indicate that CO 2 concentrations in the liquid phase were higher, with a H 2 /CO 2 molar ratio largely in favor of CO 2 . View this table: View inline View popup Download powerpoint Table 2. Dissolved CO 2 and H 2 in the growth medium under the explored experimental conditions: As anticipated, both gases exhibited increased solubility with increasing partial pressures in the culture medium. However, CO 2 demonstrated higher solubility than H 2 . At the maximum p(H 2 /CO 2 ) of 50 bar, the concentration of CO 2 in the liquid phase reached 1.26·10 −1 mol.L -1 , whereas H 2 solubility was much lower at 2.05·10 −2 mol.L -1 . Owing to its small molecular size, H 2 is more volatile and challenging to dissolve. The influence of ions in saline solutions can complicate dissolution, as indicated in our calculation, which corroborates findings from previous studies 54 . The H 2 /CO 2 ratio in the liquid phase increased from 5.9·10 −2 at 5 bar to 1.63·10 −1 at 50 bar, displaying a rapid increase with increasing p(H 2 /CO 2 ) followed by stabilization above 20 bar. This suggests that H 2 solubility increases more significantly than CO 2 solubility with increasing partial pressure. Notably, the observed tolerance of M. thermolithotrophicus to elevated p(H 2 /CO 2 ), inferred from its growth data and methane production rates, peaked between 20 and 30 bar, which coincided with the stabilization of the H 2 /CO 2 ratio. Beyond p(H 2 /CO 2 ) = 30 bar, methane production decreased, indicating that the strain’s tolerance to H 2 and CO 2 may be exceeded even when biofilm formation occurs. Furthermore, the diffusion of gases in the medium over time is crucial and reveals distinct kinetic behaviors for each gas under purely diffusive scenarios ( i.e. , without stirring and neglecting convection). As shown in Figure 8 , H 2 diffused more rapidly than CO 2 across all the conditions examined. Even after 13 hours of incubation, the system had not achieved full equilibrium, indicating that both gases remained more concentrated near the gas/liquid interface. Our simulations indicate that the H 2 concentration at the biofilm level after one hour of incubation (at a depth of 17 ± 2 mm for p(H 2 /CO 2 ) = 30 bar) was 5·10 −4 mol.L -1 , whereas the CO 2 concentration was significantly greater at 1.75·10 −2 mol.L -1 . This localized concentration of H 2 and/or CO 2 (above p(H 2 /CO 2 ) = 20 bar). In contrast, under nonstirred conditions, biofilm formation appears to provide a protective mechanism, enabling the strain to withstand the harmful effects of H 2 and increasing methane production yields compared with those lower p(H 2 /CO 2 ) conditions ( Figure 6 ). In a stirred system, equilibrium conditions render the concentrations of H 2 and CO 2 uniform throughout the reactor, whereas nonstirred conditions facilitate varying concentrations and diffusion kinetics on the basis of depth from the interface. Download figure Open in new tab Figure 8. CO 2 and H 2 concentrations in the medium as a function of the distance (depth) from the gas–liquid interface over time for T = 65°C and total pressures of 100 bar (supplemented with nitrogen) and p(H 2 /CO 2 ) (80/20 mol%) = 30 bar. Our calculations confirm that CO 2 demonstrates higher overall solubility, although H 2 solubility increases more rapidly. CO 2 can affect cell metabolism through environmental acidification, leading to pH levels that may hinder cellular development 41 . However, pH measurements before and after each experiment, as presented in ESI-2, revealed a slight decrease at high partial pressures, stabilizing at approximately pH ≈ 5.7–5.9 (down from 6.5 prior to gas injection) due to the presence of HEPES buffer. These pH values remain within a favorable range for metabolic activity and cannot account for the observed growth variations, as previously reported 41 . Additionally, high concentrations of CO 2 can be detrimental to cells. For example, supercritical CO 2 is known to have sterilizing properties, disrupting membrane integrity and leading to cell death 55 . Nevertheless, in our experimental setup, CO 2 was diluted, among other gases, which mitigated its toxic effect. Previous studies evaluating M. thermolithotrophicus CO 2 tolerance revealed detrimental impacts on metabolic activity at elevated pressures, particularly in research conducted by Dupraz and colleagues 41 . They observed reduced metabolic function when CO 2 concentrations increased, with experiments conducted at pressures up to 10 bar and varying H 2 /CO 2 ratios, all using the same culture medium as our experiments, i.e. , AGW, with NaOH as a buffer. The calculated values of dissolved gases under their experimental conditions (see ESI-3) were 2.13·10 −4 < [CO 2 ] (mol.L -1 ) < 9.69·10 −2 and 6.02·10 −4 < [H 2 ] (mol.L -1 ) 30 bar, at which point the CO 2 concentration at equilibrium in the medium is 7.75·10 −2 mol.L -1 , which is quite close to the level reported by Dupraz and collaborators for p(H 2 /CO 2 ) = 10 bar. Another potential contributor to the observed effects may be the toxicity of H 2 itself. Prior investigations have shown that high concentrations of H 2 can inhibit cell growth in nonmethanogenic strains 56 , 57 , whereas biofilm formation in methanogens seems to improve when H 2 exposure in the liquid phase is minimized 49 . Jensen and colleagues 49 reported that reducing the H 2 retention time can significantly increase biofilm activity and methane production in methanogenic communities, resulting in up to 12.5 times greater effects at the lowest retention time than at the highest retention time. Similarly, research conducted in anaerobic digesters at 35°C and ambient pressure (up to 1.55 bar) has demonstrated that H 2 pressure beyond 0.8–0.9 bar negatively affects cumulative methane production, indicating a threshold above which methane-generating processes can be severely impaired. H 2 was consistently consumed during the reactions; however, at p(H 2 ) = 1.557 bar, methanogenesis was significantly hindered, and hydrolysis/acidogenesis ceased. These results of the deleterious effects of high H 2 partial pressure were notably supported by the fact that CO 2 was absent under most of the tested conditions and that when CO 2 was added to the system, metabolism was disrupted again. Indeed, for the same total pressure, i.e. , p(H 2 ) = 1.55 bar, but under a H 2 /CO 2 atmosphere, the methanogenic activity was greater than that under only H 2 conditions 58 . Despite the fact that all of these findings were made for ambient pressure conditions, the results that we obtained under much higher-pressure conditions seem to agree with the general trend observed in these studies: our system did present greater methane production when the strain exhibited biofilm behavior, as well as a decrease in metabolic activity and growth above a certain threshold. The potential negative impact of dihydrogen could then be assessed for methanogens in the frame of geological storage of this molecule 13 , even if its injection in underground formations could be favorable to other kinds of microorganisms, which could present greater tolerance. The fact that more H 2 is needed than CO 2 for methanogenesis leads to a faster decrease in its concentration in laboratory-controlled investigations; however, under high partial pressure, for example, in the case of UHS and UMR, it can be hypothesized that consumption is not fast enough to counteract the deleterious impact. The cells will then be quickly impacted; their metabolic activity will be reduced, leading to a longer accumulation of H 2 in the environment, leading to deleterious impacts on the cells and so on. One last hypothesis could be that both gases may reach toxic concentrations under elevated pressure conditions ( i.e. , above 30 bar), thereby diminishing growth and metabolic performance. A hypothetical synergistic toxicity could emerge from simultaneous exposure to both H 2 and CO 2 , even if neither is present at their maximum tolerable levels. This dual stress could demand excess energy for cellular maintenance and metabolism, consequently lowering the strain’s resilience as H 2 and CO 2 pressures increase. Finally, biofilm formation presents significant potential in the context of geological CO 2 storage, particularly for UMR, primarily through its effects on safety and production yields. The presence of biofilms within rock pores can increase sequestration safety by altering the permeability of the reservoir and sealing fractures, which could facilitate CO 2 leakage, a phenomenon that has been supported by the literature 59 – 61 . Additionally, the presence of biofilms is correlated with increased biovalorization and energy production. However, concerns regarding decreased injectivity due to bioclogging during fluid storage or withdrawal are commonly raised. In current UGSs, bioclogging near injection and production wells is deemed unlikely for two reasons: (i) direct contact with gases at such high pressures is a powerful biocide and (ii) massive gas injection over years, or even decades, has most certainly dried out the rock, making microbial difficult to settle. However, biofilm formation may still occur in surrounding environments, promoting beneficial processes that valorize stored CO 2 . Environmental biofilms may harbor fermenters capable of producing H 2 and enhancing the methanogenesis reaction through metabolic cooperation. 3. Conclusion The primary objective of this research was to determine how the partial pressures of H 2 and CO 2 influence the growth and yield of methanogenesis. To achieve this goal, Methanothermococcus thermolithotrophicus was cultivated in innovative sapphire-based high-pressure transparent reactors (HPTRs) that allow continuous monitoring of growth at a pressure of 100 bar, which is representative of underground gas storage (UGS) conditions where CO 2 , H 2 , or both can be injected. Notably, the absence of stirring in these reactors, which more accurately simulate natural underground conditions, resulted in the formation of biofilms that significantly increased methane production across the various investigated p(H 2 /CO 2 , 80/20 mol%) partial pressures. These findings underscore the critical role of biofilm dynamics in methanogenic processes. Furthermore, the complex interactions between H 2 and CO 2 at elevated pressures raise important considerations regarding the potential toxic effects and metabolic stress on microbial populations, challenging existing assumptions about gas interactions in high-pressure environments. The obtained results provide valuable insights into optimizing methanogenic pathways in the context of carbon capture and storage, as well as the sustainable management of underground gas reservoirs. Declarations Ethics approval and consent to participate Not Applicable Consent for publication Not Applicable Availability of data and materials The datasets generated during and/or analysed during the current study are available in the Electronic Supporting Information (ESI-1 – 3). Competing interests The authors declare that they have no competing interests. Funding This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under grant agreement No. 725100 (Project: BIG MAC) and from the French “Agence Nationale de la Recherche” for the funding of the project HOT DOG (ANR-22-CE02-0017). Authors’ contributions E.V., A.C., A.R-P and S.M. designed the work; E.V., A.C., M.J., O.N. participated in the acquisition of the data; E.V., A.C., A.E., A.R-P and S.M. interpreted the data, E.V. drafted the main text, E.V., A.C., O.N., A.E., A.R-P and S.M. made the figures, A.C., A.E., A.R-P and S.M. finalized and revised the work. Acknowledgments The authors would like to thank Marion Guignard (Université de Pau et des Pays de l’Adour, CNRS, IPREM) for providing trace element stock solutions for Archean cultures. Fabien Palencia (ICMCB, CNRS) is also acknowledged for the design and 3D printing fabrication of the optical fiber supports. Funder Information Declared European Research Council, https://ror.org/0472cxd90 , 725100 Agence Nationale de la Recherche , ANR-22-CE02-0017 References (1). ↵ Molíková , A. ; Vítězová , M. ; Vítěz , T. ; Buriánková , I. ; Huber , H. ; Dengler , L. ; Hanišáková , N. ; Onderka , V. ; Urbanová , I. 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Share Real time monitoring of hydrogenotrophic methanogenesis under deep saline aquifers conditions Emeline Vidal , Anaïs Cario , Mathilda Jouvin , Maïder Abadie , Olivier Nguyen , Arnaud Erriguible , Anthony Ranchou-Peyruse , Samuel Marre bioRxiv 2025.05.05.652176; doi: https://doi.org/10.1101/2025.05.05.652176 Share This Article: Copy Citation Tools Real time monitoring of hydrogenotrophic methanogenesis under deep saline aquifers conditions Emeline Vidal , Anaïs Cario , Mathilda Jouvin , Maïder Abadie , Olivier Nguyen , Arnaud Erriguible , Anthony Ranchou-Peyruse , Samuel Marre bioRxiv 2025.05.05.652176; doi: https://doi.org/10.1101/2025.05.05.652176 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Bioengineering Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13895) Bioinformatics (41953) Biophysics (21456) Cancer Biology (18595) Cell Biology (25521) Clinical Trials (138) Developmental Biology (13381) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24323) Genetics (15612) Genomics (22511) Immunology (17738) Microbiology (40401) Molecular Biology (17184) Neuroscience (88623) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7644) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)