# Evidencing the influence of temperature and mineralogy on microbial competition for hydrogen consumption: Implications for underground hydrogen storage (UHS)

Elodie Muller, Julia Guélard, Olivier Sissmann, Ambre Tafit, Simon Poirier

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Elodie Muller, Julia Guélard, Olivier Sissmann, Ambre Tafit, Simon Poirier. Evidencing the influence of temperature and mineralogy on microbial competition for hydrogen consumption: Implications for underground hydrogen storage (UHS). International Journal of Hydrogen Energy, 2024, 82, pp.1101-1113. 10.1016/j.ijhydene.2024.08.024. hal-04694763

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# Evidencing the influence of temperature and mineralogy on microbial competition for hydrogen consumption: Implications for underground hydrogen storage (UHS)

Elodie Muller*, Julia Guélard, Olivier Sissmann, Ambre Tafit, Simon Poirier

IFP Energies Nouvelles, 1- 4 Avenue Du Bois Préau, 92852, Rueil-Malmaison, France

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

Handling editor: M Mahdi Najafpour

**Keywords:**

Hydrogen
underground gas storage
Microbiome
Porous media
Metataxonomics

## ABSTRACT

Understanding the environmental drivers of microbial H₂ metabolisms is crucial for evaluating the risks for geological hydrogen storage. This study investigates how mineralogy and temperature affect H₂ consumption kinetics and metabolic activity of a microbial consortium containing methanogens, homoacetogens and sulfate reducers, incubated at 25, 34 and 40 °C under H₂/CO₂ (80/20, v/v; 2 bars) with different rock powders (basalt, calcite, gypsum, and sandstone). The presence of gypsum favors sulfate reduction over methanogenesis and homoacetogenesis, especially at 25 °C. Methanogenesis is dominant at 34 and 40 °C with all sulfate-free mineralogies. At 25 °C, homoacetogenic bacteria are favored over methanogens and acetate production varies with the mineralogy. This suggests interactions of the microbial community with the rock powders, which may serve as a surface to form biofilms. These metabolic shifts are associated with radical changes in microbial populations, highlighting that ecosystems plasticity towards H₂ also depends on the mineralogical composition of the reservoir.

## 1. Introduction

Hydrogen (H₂) is considered as a key energy carrier for the energy transition from fossil fuel to renewable energies (e.g. Ref. [1], and references therein). Although the energy density of hydrogen is the highest in mass (33.3 kWh/kg), the volumetric calorific values is the lowest (530 kWh/m³) compared with other fuels [2]. Consequently, greater volumetric storage capacities will be required to deliver the same energy output as natural gas. Thus, large-scale and long-term hydrogen storage has attracted global attention to support the hydrogen economy supply chain (e.g., Ref. [3,4]).

Underground hydrogen storage (UHS) offers many advantages over surface storage, including: a huge storage space (483 billion cubic meters in 2019 [5]), and high availability in existing underground storage sites [6]. Salt caverns are considered as the most suitable geological structures for UHS in terms of caprock integrity [7,8]. However, given that they are geographically constrained to the presence of evaporitic formations with suitable thickness and extent, deep saline aquifers and depleted hydrocarbon reservoirs in particular gas reservoirs have also been widely considered for large-scale and long-term hydrogen storage [4,9].

Many studies were conducted to identify the abiotic geochemical reactions that may occur between hydrogen and various subsurface minerals and fluids present in these geological structures. These reactions may lead to (i) significant loss of hydrogen; (ii) contamination of the stored hydrogen by the production of other gases (e.g. H₂S); (iii) mineral precipitation leading to reduced injectivity; (iv) mineral dissolution leading to enhanced opening of migration pathways through the caprock, and impacting the mechanical properties of the reservoir and the caprock [10]. For instance, Flesch et al. [11] showed that carbonate and sulfate cements tend to dissolve when widely present in H₂ storage under high pressure and high temperature and thus alter the formation's porosity and permeability, posing a risk to underground storage. In this case, there are changes in the composition of the formation water, which promote salt precipitation and lead to the formation of hydrogen sulfide (H₂S), decreasing the quality of the stored hydrogen gas and compromising the infrastructure due to its corrosive, flammable and toxic nature [2]. Moreover, reactions of hydrogen with dissolved sulfur species or sulfur-bearing minerals (e.g. pyrite, anhydrite, gypsum) are particularly expected to occur in salt caverns and some deep saline aquifers [12]. In contrast, framework minerals, such as quartz and feldspar, appeared to be unaffected by hydrogen exposure [10]. Despite these

<footnote>* Corresponding author.
E-mail address: elodie.muller@ifpen.fr (E. Muller).</footnote>
recent results, there is yet no consensus on the significance [2,13] or insignificance [14–16] of these geochemical reactions on storage operations.

On the other hand, recent models that take into account microorganisms have shown that microorganisms are likely to have a strong impact on the evolution of the storage sites [17–22]. Indeed, all of these subsurface structures are mostly not life-free (sterile) environments [23–27]. They harbor a great diversity of microorganisms, including methanogens (e.g., *Methanobacterium* genus), sulfate-reducers (e.g., *Desulfovibrio*, *Desulfotomaculum*), homoacetogens (e.g., *Acetobacterium*) and metal reducers (e.g., *Geobacter*, *Shewanella*), which are known as major hydrogen consumers [27,28]. Artificially increasing the H₂ concentration in the subsurface may stimulate the growth of these hydrogenotrophic microorganisms [3, 21, 29, 30] and have major consequences for UHS and surrounding environments: permanent loss of hydrogen due to its conversion into products like CH₄ or H₂S, mineral precipitates, and even tubing corrosion and clogging [18]. For example, previous hydrogen-enriched gas storage operations in deep saline sandstone aquifers reported various hydrogen consumption from 3% in Austria (Underground Sun Storage project [31]) to 17% in Czech Republic (Lobodice storage; [32,33]) due to the conversion into methane by methanogenesis [34]. Other reservoirs such as the calcareous formations of the Paris Basin harbor other hydrogen-utilizing autotrophic bacteria, including sulfate reducers (e.g. *Desulfovibrio aespoensis*) and homoacetogens (e.g. *Acetobacterium carbinolicum*), resulting in different H₂ consumption dynamics [28]. These variations are linked to the composition of the microbial ecosystems, which may vary depending on several parameters such as temperature, pressure, salt concentration, pH, and nutrient and substrate supply (e.g., organic matter, sulfate and ferric iron) [21,23,24,26–28]. It is therefore very important to understand the control mechanisms of microbial H₂ metabolisms.

In 2023, Dohrmann and Krüger performed microbial H₂ consumption experiments with a natural gas field consortium including sulfate-reducing prokaryotes (SRP) at near *in situ* pressure (100 bar) and temperature conditions (30 °C and 60 °C). Their findings revealed that temperature strongly influence their activity, which is higher at 30 °C. In addition, most microorganisms in nature grow in biofilms attached to surfaces (communities of aggregated microbial cells embedded in a secreted matrix of extracellular polymeric substances (EPS) [35]). Recent work by Khajooie et al. [36] demonstrated, through batch experiments, that the microbial activity of methanogens is influenced by the surface area available for microorganism colonization. These authors showed that methanogenetic activity may increase up to 8–10 times in the presence of mineral substrates. Moreover, Mura et al. [37] and Ranchou-Peyruse et al. [38] highlighted that the pH increase initiated by lithoautotrophic microbial metabolisms could considerably curb microbial consumption of H₂ by mineralizing CO₂ dissolved in carbonates and thus making this CO₂ inaccessible to autotrophic microorganisms. Thaysen et al. [21] presented the growth conditions of 518 cultivated strains from the three major groups of H₂-oxidizing microorganisms (methanogens, homoacetogens and sulfate reducers) to predict where microbial growth can be expected in a future H₂ storage scenario. These authors estimated that microbial growth and H₂ consumption may increase during repeated storage cycles, giving replenishment of nutrients by decaying microbial cells, inflowing water and mineral weathering. These findings collectively underline the significant influence of reservoir characteristics on the biodegradation potential of gas storage by deep ecosystems. Yet, an assessment of the impact of mineralogy on H₂ consumption by hydrogenotrophic microbial communities is lacking.

In this study, we designed experiments to investigate the influence of temperature and mineralogy on the activity of an H₂-specialized anaerobic microbial consortium harboring several hydrogenotrophic metabolisms usually found in subsurface environments (i.e., methanogenesis, sulfate reduction and homo-acetogenesis) and under high hydrogen concentration. The H₂-specialized anaerobic consortium was

incubated with different rock powders (basalt, calcite, gypsum, and sandstone) at 25, 34 et 40 °C in anaerobic flasks under constant atmosphere. An integrative analytical approach combining geochemical measurements (gas composition, pH, dissolved sulfur species and volatile organic compounds) with metataxonomics was conducted to monitor hydrogen conversion kinetics and pathways as well as microbial response to different geochemical conditions.

## 2. Material and methods

### 2.1. Experimental set-up

**Culture conditions.** Culture medium was composed of NaCl (1.0 g/L), MgCl₂·6H₂O (0.4 g/L), CaCl₂·2H₂O (0.08 g/L), KCl (0.5 g/L), NH₄Cl (0.25 g/L), Na₂SO₄ (0.3 g/L), and KH₂PO₄ (0.2 g/L) and 1 g/L of yeast extract. It was supplemented with 5 mL of trace-element solution and 10 mL of vitamin solution (Table S1). After filtration at 0.22 µm, the medium was flushed with N₂/CO₂ (80:20, v/v; 1 bar), prior to the addition of sodium bicarbonate solution from a sterile stock solution to adjust the pH at 6.8–7.0 (3.4 g/L). The medium was then reduced with sodium sulfide (Na₂S, 9H₂O) from a sterile stock solution at a desired final concentration of 0.1 g/L and 20 mg of dithionite (Na₂S₂SO₄). Sodium resazurin (0.5 mg/L), a phenoxazine dye, was used as an indicator of the redox potential of the prepared medium (below –110 mV).

**Source of organisms.** The inoculum derives from an industrial anaerobic digester operating on a wastewater treatment plant (Achères, France). This anaerobic consortium was specialized in flasks with the standard culture medium (see above) and under H₂/CO₂ (80:20, v/v) during several months prior to the experiments to select the hydrogenotrophic metabolisms that may interact with gas storage in deep environments (e.g., methanogenesis, sulfate reduction, homoacetogenesis). The inoculum (0.5 mL) was sampled from a 1 L flask incubated at 34 °C with H₂/CO₂ (80:20, v/v; 2 bar) as the gas phase.

**H₂ consumption experiments.** For the experiments performed with minerals, various rocks were selected to encompass the lithologies likely to be encountered in an underground gas storage [9]: basalt from Iceland [39], Messinian gypsum (CaSO₄·2H₂O) from the Mediterranean Sea [40], calcium carbonate (CaCO₃) from the Lavoux formation [41] and sandstone (SiO₂) from Fontainebleau. Several kilograms of rocks were crushed with a mechanical grinder and sieve to 100–200 µm. Then, 1 g of powder was placed in 155 mL flasks prior autoclaving at 120 °C for 30 min.

The anoxic culture medium (15 mL) was transferred under nitrogen flow into sterile flasks (with and without powders). The flasks were sealed with a screw cap and butyl rubber septa (Fig. S1). Headspaces were flushed several times and pressurized (2 bars at 20 °C) with H₂/CO₂ (80/20, v/v). Experiments were carried out in agitated conditions (110 rpm) at 25, 34 and 40 °C until complete consumption of H₂ and/or CO₂ from the headspace gas. All incubations were performed in triplicate. Abiotic control experiments were also conducted with powders at each temperature to identify any abiotic geochemical reaction between powders and fluid phases. Thereby, variations of the aqueous and gas phase composition were measured at time zero (T₀) and after 19 days (i.e., maximum incubation period).

### 2.2. Microbial activity analyses

Measurements and sampling. Pressure measurements, gas analysis and liquid sampling were performed at regular intervals during the incubation (Table 1). Pressure variations in the flasks were measured using a differential manometer (Digitron 2082P) before and after each sampling session. When the pressure fell below 50 mbar, the flasks were re-pressurized with N₂. The composition of the headspace gas was measured by micro gas chromatography (Micro GC Fusion, Inficon). The volume fractions (in %) of each species determined by gas chromatography were then used to calculate their respective amounts applying the
<caption>**Table 1**</caption>

Monitoring of gas and fluid composition during incubations with and without powders at 25, 34 and 40 °C. Values are given as averages and standard deviations of triplicate experiments.

<table><thead><tr><th rowspan="3">T (°C)</th><th rowspan="3">Mineralogy</th><th rowspan="3">Time (day)</th><th colspan="8">Gas composition (µmol)</th><th colspan="8">Fluid composition</th></tr><tr><th colspan="2">H2</th><th colspan="2">CO2</th><th colspan="2">CH4</th><th colspan="2">H2S</th><th colspan="2">pH</th><th colspan="2">Sulfide (mM)</th><th colspan="2">Sulfate (mM)</th><th colspan="2">Acetate (g/l)</th><th colspan="2">Formate (g/l)</th></tr><tr><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th></tr></thead><tbody><tr><td>25</td><td>Sandstone</td><td>0.0</td><td>9096</td><td>84</td><td>2459</td><td>23</td><td>0</td><td>0</td><td>1</td><td>0</td><td>6.7</td><td>0.1</td><td>0.0</td><td>0.0</td><td>5.1</td><td>0.2</td><td>1.1</td><td>0.2</td><td>2.1</td><td>0.3</td></tr><tr><td>25</td><td>Sandstone</td><td>1.1</td><td>8665</td><td>128</td><td>2357</td><td>41</td><td>0</td><td>0</td><td>1</td><td>0</td><td>7.0</td><td>0.1</td><td>0.0</td><td>0.1</td><td>5.0</td><td>1.1</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Sandstone</td><td>5.0</td><td>7915</td><td>163</td><td>2193</td><td>70</td><td>29</td><td>7</td><td>46</td><td>1</td><td>7.0</td><td>0.0</td><td>2.7</td><td>0.1</td><td>2.3</td><td>0.2</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Sandstone</td><td>6.0</td><td>7355</td><td>174</td><td>2024</td><td>76</td><td>83</td><td>20</td><td>56</td><td>2</td><td>7.2</td><td>0.0</td><td>3.7</td><td>0.1</td><td>1.3</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Sandstone</td><td>7.0</td><td>6527</td><td>317</td><td>1753</td><td>121</td><td>246</td><td>58</td><td>53</td><td>3</td><td>7.2</td><td>0.0</td><td>3.5</td><td>0.1</td><td>2.0</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Sandstone</td><td>8.0</td><td>3165</td><td>1194</td><td>752</td><td>357</td><td>700</td><td>168</td><td>40</td><td>6</td><td>7.6</td><td>0.1</td><td>4.8</td><td>0.3</td><td>1.6</td><td>0.5</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Sandstone</td><td>9.0</td><td>75</td><td>84</td><td>12</td><td>12</td><td>1155</td><td>58</td><td>4</td><td>3</td><td>9.1</td><td>0.3</td><td>10.5</td><td>0.7</td><td>1.5</td><td>0.3</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Sandstone</td><td>12.0</td><td>1</td><td>0</td><td>10</td><td>5</td><td>1138</td><td>53</td><td>3</td><td>2</td><td>9.1</td><td>0.2</td><td>10.0</td><td>0.4</td><td>2.0</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Sandstone</td><td>13.9</td><td>1</td><td>0</td><td>12</td><td>6</td><td>1123</td><td>52</td><td>3</td><td>2</td><td>8.9</td><td>0.2</td><td>9.5</td><td>0.7</td><td>2.2</td><td>0.2</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Sandstone</td><td>15.9</td><td>1</td><td>0</td><td>13</td><td>7</td><td>1103</td><td>60</td><td>4</td><td>2</td><td>8.9</td><td>0.2</td><td>9.5</td><td>1.0</td><td>2.4</td><td>0.0</td><td>6.4</td><td>2.1</td><td>0.0</td><td>0.0</td></tr><tr><td>25</td><td>Sandstone</td><td>19.0</td><td>3</td><td>1</td><td>14</td><td>7</td><td>1065</td><td>52</td><td>4</td><td>2</td><td>8.8</td><td>0.2</td><td>9.1</td><td>0.7</td><td>2.2</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Calcite</td><td>0.0</td><td>8988</td><td>157</td><td>2436</td><td>43</td><td>0</td><td>0</td><td>1</td><td>0</td><td>6.8</td><td>0.1</td><td>0.1</td><td>0.1</td><td>5.5</td><td>0.9</td><td>1.2</td><td>0.2</td><td>2.2</td><td>0.4</td></tr><tr><td>25</td><td>Calcite</td><td>1.1</td><td>8324</td><td>468</td><td>2263</td><td>124</td><td>0</td><td>0</td><td>1</td><td>0</td><td>6.9</td><td>0.0</td><td>0.1</td><td>0.0</td><td>4.3</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Calcite</td><td>5.0</td><td>7507</td><td>485</td><td>2060</td><td>150</td><td>25</td><td>8</td><td>67</td><td>8</td><td>7.1</td><td>0.1</td><td>4.4</td><td>0.8</td><td>2.3</td><td>0.8</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Calcite</td><td>6.0</td><td>7091</td><td>476</td><td>1948</td><td>145</td><td>55</td><td>14</td><td>63</td><td>7</td><td>7.2</td><td>0.1</td><td>4.5</td><td>0.8</td><td>1.5</td><td>0.7</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Calcite</td><td>7.0</td><td>6652</td><td>560</td><td>1764</td><td>168</td><td>118</td><td>43</td><td>58</td><td>7</td><td>7.1</td><td>0.0</td><td>4.5</td><td>0.8</td><td>1.4</td><td>0.8</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Calcite</td><td>8.0</td><td>4482</td><td>1881</td><td>1102</td><td>590</td><td>254</td><td>106</td><td>53</td><td>6</td><td>7.3</td><td>0.3</td><td>4.4</td><td>1.8</td><td>1.5</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Calcite</td><td>9.0</td><td>1741</td><td>1764</td><td>312</td><td>514</td><td>445</td><td>174</td><td>34</td><td>20</td><td>8.0</td><td>0.5</td><td>6.9</td><td>3.9</td><td>1.5</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Calcite</td><td>12.0</td><td>472</td><td>403</td><td>2</td><td>1</td><td>531</td><td>147</td><td>11</td><td>9</td><td>8.7</td><td>0.7</td><td>9.8</td><td>2.3</td><td>1.3</td><td>0.3</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Calcite</td><td>13.9</td><td>436</td><td>397</td><td>1</td><td>0</td><td>528</td><td>140</td><td>9</td><td>7</td><td>8.7</td><td>0.5</td><td>9.9</td><td>1.8</td><td>2.1</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Calcite</td><td>15.9</td><td>411</td><td>384</td><td>1</td><td>0</td><td>519</td><td>133</td><td>8</td><td>6</td><td>8.7</td><td>0.5</td><td>10.3</td><td>2.2</td><td>1.5</td><td>0.7</td><td>8.0</td><td>0.3</td><td>0.0</td><td>0.0</td></tr><tr><td>25</td><td>Calcite</td><td>19.0</td><td>388</td><td>372</td><td>1</td><td>0</td><td>505</td><td>126</td><td>7</td><td>5</td><td>8.7</td><td>0.5</td><td>9.9</td><td>1.5</td><td>2.6</td><td>0.8</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Basalt</td><td>0.0</td><td>9074</td><td>61</td><td>2464</td><td>17</td><td>0</td><td>0</td><td>1</td><td>0</td><td>6.7</td><td>0.1</td><td>0.0</td><td>0.1</td><td>5.7</td><td>0.9</td><td>1.2</td><td>0.1</td><td>2.1</td><td>0.1</td></tr><tr><td>25</td><td>Basalt</td><td>1.1</td><td>8655</td><td>90</td><td>2338</td><td>19</td><td>0</td><td>0</td><td>0</td><td>0</td><td>6.9</td><td>0.1</td><td>0.0</td><td>0.0</td><td>5.7</td><td>1.5</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Basalt</td><td>5.0</td><td>8016</td><td>276</td><td>2203</td><td>97</td><td>12</td><td>6</td><td>20</td><td>25</td><td>7.0</td><td>0.0</td><td>1.2</td><td>1.3</td><td>3.1</td><td>1.4</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Basalt</td><td>6.0</td><td>7603</td><td>222</td><td>2086</td><td>77</td><td>25</td><td>9</td><td>35</td><td>10</td><td>7.2</td><td>0.1</td><td>2.3</td><td>0.7</td><td>2.8</td><td>0.5</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Basalt</td><td>7.0</td><td>7362</td><td>178</td><td>1949</td><td>59</td><td>45</td><td>11</td><td>54</td><td>11</td><td>7.1</td><td>0.0</td><td>3.5</td><td>0.7</td><td>2.2</td><td>0.2</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Basalt</td><td>8.0</td><td>6946</td><td>326</td><td>1820</td><td>126</td><td>66</td><td>12</td><td>50</td><td>11</td><td>7.2</td><td>0.1</td><td>3.3</td><td>0.6</td><td>2.4</td><td>1.3</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Basalt</td><td>9.0</td><td>5560</td><td>2296</td><td>1407</td><td>725</td><td>89</td><td>14</td><td>49</td><td>8</td><td>7.2</td><td>0.4</td><td>2.7</td><td>1.1</td><td>2.2</td><td>1.2</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Basalt</td><td>12.0</td><td>3728</td><td>1847</td><td>766</td><td>644</td><td>141</td><td>33</td><td>41</td><td>8</td><td>6.7</td><td>1.1</td><td>2.4</td><td>1.5</td><td>1.9</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Basalt</td><td>13.9</td><td>2128</td><td>429</td><td>280</td><td>208</td><td>173</td><td>64</td><td>42</td><td>13</td><td>6.0</td><td>0.4</td><td>1.2</td><td>0.5</td><td>1.3</td><td>0.3</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Basalt</td><td>15.9</td><td>1808</td><td>202</td><td>189</td><td>128</td><td>201</td><td>93</td><td>35</td><td>12</td><td>5.9</td><td>0.3</td><td>1.3</td><td>0.4</td><td>1.2</td><td>0.3</td><td>8.8</td><td>0.8</td><td>0.0</td><td>0.0</td></tr><tr><td>25</td><td>Basalt</td><td>19.0</td><td>1409</td><td>225</td><td>80</td><td>52</td><td>255</td><td>160</td><td>30</td><td>11</td><td>5.9</td><td>0.2</td><td>0.9</td><td>0.3</td><td>1.1</td><td>0.4</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Gypsum</td><td>0.0</td><td>9110</td><td>139</td><td>2482</td><td>38</td><td>0</td><td>0</td><td>1</td><td>0</td><td>7.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>25.7</td><td>3.0</td><td>1.1</td><td>0.0</td><td>2.0</td><td>0.1</td></tr><tr><td>25</td><td>Gypsum</td><td>1.1</td><td>8286</td><td>759</td><td>2277</td><td>196</td><td>0</td><td>0</td><td>1</td><td>0</td><td>6.9</td><td>0.0</td><td>0.1</td><td>0.0</td><td>29.0</td><td>2.6</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Gypsum</td><td>5.0</td><td>7365</td><td>851</td><td>2064</td><td>224</td><td>19</td><td>7</td><td>85</td><td>24</td><td>7.0</td><td>0.1</td><td>5.5</td><td>1.8</td><td>27.3</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Gypsum</td><td>6.0</td><td>6116</td><td>993</td><td>1708</td><td>144</td><td>40</td><td>15</td><td>211</td><td>63</td><td>7.2</td><td>0.1</td><td>15.4</td><td>3.4</td><td>28.3</td><td>3.2</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Gypsum</td><td>7.0</td><td>4665</td><td>1043</td><td>1374</td><td>239</td><td>59</td><td>19</td><td>403</td><td>64</td><td>7.1</td><td>0.0</td><td>28.0</td><td>5.3</td><td>30.8</td><td>1.2</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Gypsum</td><td>8.0</td><td>3294</td><td>957</td><td>1061</td><td>215</td><td>70</td><td>21</td><td>592</td><td>45</td><td>7.1</td><td>0.1</td><td>46.5</td><td>4.4</td><td>31.9</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Gypsum</td><td>9.0</td><td>2211</td><td>879</td><td>805</td><td>188</td><td>72</td><td>22</td><td>706</td><td>27</td><td>7.3</td><td>0.1</td><td>58.4</td><td>4.4</td><td>32.4</td><td>0.8</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Gypsum</td><td>12.0</td><td>677</td><td>777</td><td>479</td><td>152</td><td>77</td><td>25</td><td>893</td><td>32</td><td>7.3</td><td>0.1</td><td>66.0</td><td>9.7</td><td>32.6</td><td>1.3</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Gypsum</td><td>13.9</td><td>382</td><td>603</td><td>415</td><td>116</td><td>76</td><td>24</td><td>910</td><td>57</td><td>7.3</td><td>0.0</td><td>80.6</td><td>2.6</td><td>32.9</td><td>1.7</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>Gypsum</td><td>15.9</td><td>273</td><td>472</td><td>381</td><td>88</td><td>74</td><td>23</td><td>896</td><td>74</td><td>7.3</td><td>0.0</td><td>85.6</td><td>8.0</td><td>35.7</td><td>4.7</td><td>1.1</td><td>0.2</td><td>0.0</td><td>0.0</td></tr><tr><td>25</td><td>Gypsum</td><td>19.0</td><td>204</td><td>353</td><td>368</td><td>65</td><td>71</td><td>22</td><td>907</td><td>93</td><td>7.3</td><td>0.0</td><td>76.1</td><td>6.9</td><td>34.7</td><td>0.7</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>No powder</td><td>0.0</td><td>9189</td><td>97</td><td>2496</td><td>26</td><td>0</td><td>0</td><td>1</td><td>0</td><td>6.7</td><td>0.0</td><td>0.0</td><td>0.0</td><td>5.3</td><td>1.0</td><td>1.0</td><td>0.0</td><td>2.0</td><td>0.0</td></tr><tr><td>25</td><td>No powder</td><td>1.1</td><td>8787</td><td>137</td><td>2387</td><td>36</td><td>0</td><td>0</td><td>1</td><td>0</td><td>7.0</td><td>0.1</td><td>0.1</td><td>0.0</td><td>4.1</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>No powder</td><td>5.0</td><td>7917</td><td>241</td><td>2175</td><td>61</td><td>42</td><td>13</td><td>61</td><td>1</td><td>7.1</td><td>0.0</td><td>3.8</td><td>0.2</td><td>2.0</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>No powder</td><td>6.0</td><td>7182</td><td>350</td><td>1996</td><td>101</td><td>130</td><td>45</td><td>58</td><td>2</td><td>7.5</td><td>0.1</td><td>3.9</td><td>0.0</td><td>1.6</td><td>0.3</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>No powder</td><td>7.0</td><td>5876</td><td>784</td><td>1520</td><td>222</td><td>366</td><td>95</td><td>50</td><td>4</td><td>7.4</td><td>0.2</td><td>4.1</td><td>0.1</td><td>1.4</td><td>0.5</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>No powder</td><td>8.0</td><td>4479</td><td>890</td><td>1099</td><td>238</td><td>620</td><td>120</td><td>41</td><td>5</td><td>7.6</td><td>0.1</td><td>4.7</td><td>0.1</td><td>1.4</td><td>0.4</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>No powder</td><td>9.0</td><td>3086</td><td>927</td><td>723</td><td>241</td><td>828</td><td>137</td><td>33</td><td>6</td><td>8.0</td><td>0.1</td><td>5.1</td><td>0.6</td><td>1.5</td><td>0.4</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>No powder</td><td>12.0</td><td>849</td><td>1068</td><td>177</td><td>229</td><td>1266</td><td>146</td><td>12</td><td>13</td><td>8.6</td><td>0.4</td><td>7.7</td><td>1.5</td><td>1.6</td><td>0.3</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>No powder</td><td>13.9</td><td>432</td><td>744</td><td>96</td><td>128</td><td>1318</td><td>81</td><td>8</td><td>9</td><td>8.7</td><td>0.5</td><td>8.5</td><td>0.8</td><td>1.8</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>25</td><td>No powder</td><td>15.9</td><td>297</td><td>511</td><td>63</td><td>71</td><td>1311</td><td>28</td><td>6</td><td>6</td><td>8.8</td><td>0.4</td><td>9.0</td><td>1.1</td><td>1.9</td><td>0.2</td><td>2.9</td><td>0.2</td><td>0.0</td><td>0.0</td></tr><tr><td>25</td><td>No powder</td><td>19.0</td><td>186</td><td>319</td><td>42</td><td>32</td><td>1280</td><td>27</td><td>4</td><td>3</td><td>8.8</td><td>0.2</td><td>9.3</td><td>0.1</td><td>1.7</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Sandstone</td><td>0.0</td><td>9394</td><td>224</td><td>2260</td><td>54</td><td>0</td><td>0</td><td>1</td><td>0</td><td>6.9</td><td>0.0</td><td>0.1</td><td>0.0</td><td>3.2</td><td></td><td>1.1</td><td>0.2</td><td>2.1</td><td></td></tr><tr><td>34</td><td>Sandstone</td><td>0.9</td><td>8970</td><td>247</td><td>2184</td><td>92</td><td>25</td><td>11</td><td>26</td><td>3</td><td>7.1</td><td>0.0</td><td>1.4</td><td>0.2</td><td>1.6</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Sandstone</td><td>1.9</td><td>6816</td><td>760</td><td>1705</td><td>178</td><td>451</td><td>205</td><td>61</td><td>9</td><td>7.3</td><td>0.1</td><td>4.2</td><td>0.8</td><td>0.2</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Sandstone</td><td>2.9</td><td>111</td><td>190</td><td>166</td><td>49</td><td>1879</td><td>40</td><td>31</td><td>24</td><td>8.3</td><td>0.1</td><td>9.1</td><td>1.3</td><td>0.1</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Sandstone</td><td>3.9</td><td>1</td><td>0</td><td>137</td><td>14</td><td>1831</td><td>82</td><td>13</td><td>2</td><td>8.4</td><td>0.0</td><td>9.7</td><td>1.0</td><td>0.1</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Sandstone</td><td>6.9</td><td>1</td><td>0</td><td>106</td><td>13</td><td>1773</td><td>68</td><td>8</td><td>1</td><td>8.8</td><td>0.1</td><td>10.3</td><td>0.9</td><td>0.4</td><td>0.0</td><td>1.7</td><td>0.0</td><td>0.0</td><td>0.0</td></tr><tr><td>34</td><td>Calcite</td><td>0.0</td><td>9302</td><td>21</td><td>2196</td><td>5</td><td>0</td><td>0</td><td>1</td><td>0</td><td>7.0</td><td>0.0</td><td>0.1</td><td>0.0</td><td>3.0</td><td></td><td>0.9</td><td>0.2</td><td>1.9</td><td></td></tr><tr><td>34</td><td>Calcite</td><td>0.9</td><td>8773</td><td>33</td><td>2173</td><td>15</td><td>31</td><td>9</td><td>44</td><td>2</td><td>7.2</td><td>0.1</td><td>3.0</td><td>0.4</td><td>1.3</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Calcite</td><td>1.9</td><td>6011</td><td>579</td><td>1511</td><td>142</td><td>576</td><td>150</td><td>53</td><td>7</td><td>7.4</td><td>0.0</td><td>4.3</td><td>0.6</td><td>0.2</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Calcite</td><td>2.9</td><td>2</td><td>2</td><td>143</td><td>14</td><td>1900</td><td>28</td><td>13</td><td>2</td><td>8.3</td><td>0.0</td><td>10.1</td><td>0.6</td><td>0.1</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Calcite</td><td>3.9</td><td>4</td><td>3</td><td>140</td><td>11</td><td>1870</td><td>27</td><td>13</td><td>2</td><td>8.4</td><td>0.1</td><td>9.3</td><td>0.8</td><td>0.1</td><td>0.0</td><td></td><td></td><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>Table 1 (continued)</caption><thead><tr><th rowspan="3">T (°C)</th><th rowspan="3">Mineralogy</th><th rowspan="3">Time (day)</th><th colspan="8">Gas composition (µmol)</th><th colspan="8">Fluid composition</th></tr><tr><th colspan="2">H2</th><th colspan="2">CO2</th><th colspan="2">CH4</th><th colspan="2">H2S</th><th colspan="2">pH</th><th colspan="2">Sulfide (mM)</th><th colspan="2">Sulfate (mM)</th><th colspan="2">Acetate (g/l)</th><th colspan="2">Formate (g/l)</th></tr><tr><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th></tr></thead><tbody><tr><td>34</td><td>Calcite</td><td>6.9</td><td>1</td><td>0</td><td>102</td><td>13</td><td>1780</td><td>35</td><td>7</td><td>2</td><td>8.7</td><td>0.0</td><td>10.3</td><td>0.9</td><td>0.3</td><td>0.0</td><td>1.6</td><td>0.0</td><td>0.0</td><td>0.0</td></tr><tr><td>34</td><td>Basalt</td><td>0.0</td><td>9212</td><td>172</td><td>2229</td><td>42</td><td>0</td><td>0</td><td>1</td><td>0</td><td>7.0</td><td>0.0</td><td>0.1</td><td>0.0</td><td>2.8</td><td>0.0</td><td>0.9</td><td>0.2</td><td>1.9</td><td></td></tr><tr><td>34</td><td>Basalt</td><td>0.9</td><td>8660</td><td>209</td><td>2146</td><td>46</td><td>47</td><td>34</td><td>32</td><td>13</td><td>7.3</td><td>0.0</td><td>1.9</td><td>1.0</td><td>1.1</td><td>0.5</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Basalt</td><td>1.9</td><td>4786</td><td>1905</td><td>1221</td><td>445</td><td>650</td><td>391</td><td>38</td><td>10</td><td>7.5</td><td>0.1</td><td>3.5</td><td>0.7</td><td>0.2</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Basalt</td><td>2.9</td><td>308</td><td>533</td><td>209</td><td>105</td><td>1636</td><td>220</td><td>14</td><td>6</td><td>8.2</td><td>0.2</td><td>7.0</td><td>0.6</td><td>0.2</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Basalt</td><td>3.9</td><td>2</td><td>3</td><td>138</td><td>10</td><td>1687</td><td>304</td><td>9</td><td>1</td><td>8.4</td><td>0.1</td><td>7.2</td><td>1.3</td><td>0.1</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Basalt</td><td>6.9</td><td>2</td><td>3</td><td>105</td><td>5</td><td>1634</td><td>273</td><td>6</td><td>1</td><td>8.7</td><td>0.0</td><td>8.2</td><td>1.0</td><td>0.4</td><td>0.0</td><td>1.6</td><td>0.0</td><td>0.0</td><td>0.0</td></tr><tr><td>34</td><td>Gypsum</td><td>0.0</td><td>9225</td><td>155</td><td>2227</td><td>37</td><td>0</td><td>0</td><td>1</td><td>0</td><td>7.0</td><td>0.0</td><td>0.1</td><td>0.0</td><td>13.5</td><td>0.0</td><td>1.1</td><td>0.2</td><td>2.2</td><td></td></tr><tr><td>34</td><td>Gypsum</td><td>0.9</td><td>8891</td><td>172</td><td>2249</td><td>46</td><td>8</td><td>1</td><td>21</td><td>1</td><td>7.1</td><td>0.0</td><td>1.5</td><td>0.2</td><td>30.1</td><td>2.7</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Gypsum</td><td>1.9</td><td>7347</td><td>118</td><td>1933</td><td>27</td><td>80</td><td>22</td><td>146</td><td>12</td><td>7.2</td><td>0.0</td><td>8.2</td><td>1.1</td><td>28.8</td><td>2.5</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Gypsum</td><td>2.9</td><td>3755</td><td>308</td><td>1179</td><td>55</td><td>351</td><td>108</td><td>456</td><td>25</td><td>7.2</td><td>0.0</td><td>35.0</td><td>2.0</td><td>32.1</td><td>2.2</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Gypsum</td><td>3.9</td><td>1013</td><td>437</td><td>643</td><td>72</td><td>599</td><td>179</td><td>691</td><td>33</td><td>7.3</td><td>0.0</td><td>53.2</td><td>1.9</td><td>32.6</td><td>1.5</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>Gypsum</td><td>6.9</td><td>1</td><td>0</td><td>470</td><td>33</td><td>696</td><td>154</td><td>724</td><td>47</td><td>7.6</td><td>0.0</td><td>65.8</td><td>0.7</td><td>40.8</td><td>1.3</td><td>1.0</td><td>0.2</td><td>0.4</td><td>0.2</td></tr><tr><td>34</td><td>No powder</td><td>0.0</td><td>9426</td><td>149</td><td>2263</td><td>36</td><td>0</td><td>0</td><td>1</td><td>1</td><td>7.0</td><td>0.0</td><td>0.1</td><td>0.0</td><td>1.5</td><td>0.0</td><td>1.1</td><td>0.2</td><td>2.0</td><td></td></tr><tr><td>34</td><td>No powder</td><td>0.9</td><td>8888</td><td>218</td><td>2187</td><td>51</td><td>48</td><td>9</td><td>30</td><td>26</td><td>7.2</td><td>0.0</td><td>3.0</td><td>0.6</td><td>1.0</td><td>0.2</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>No powder</td><td>1.9</td><td>5529</td><td>231</td><td>1397</td><td>62</td><td>726</td><td>40</td><td>41</td><td>18</td><td>7.4</td><td>0.0</td><td>4.1</td><td>0.2</td><td>0.1</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>No powder</td><td>2.9</td><td>2110</td><td>172</td><td>586</td><td>54</td><td>1430</td><td>81</td><td>21</td><td>18</td><td>7.8</td><td>0.1</td><td>5.9</td><td>0.6</td><td>0.1</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>No powder</td><td>3.9</td><td>102</td><td>50</td><td>147</td><td>24</td><td>1903</td><td>75</td><td>8</td><td>8</td><td>8.4</td><td>0.1</td><td>8.8</td><td>0.4</td><td>0.1</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>34</td><td>No powder</td><td>6.9</td><td>1</td><td>1</td><td>98</td><td>13</td><td>1855</td><td>46</td><td>5</td><td>4</td><td>8.7</td><td>0.1</td><td>9.7</td><td>0.7</td><td>0.3</td><td>0.2</td><td>1.3</td><td>0.2</td><td>0.3</td><td>0.2</td></tr><tr><td>40</td><td>Sandstone</td><td>0.0</td><td>9053</td><td>248</td><td>2537</td><td>70</td><td>1</td><td>0</td><td>2</td><td>0</td><td>7.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>4.5</td><td>0.0</td><td>1.1</td><td>0.2</td><td>2.1</td><td></td></tr><tr><td>40</td><td>Sandstone</td><td>1.1</td><td>8047</td><td>165</td><td>2252</td><td>41</td><td>13</td><td>1</td><td>24</td><td>1</td><td>7.2</td><td>0.1</td><td>1.4</td><td>0.1</td><td>2.6</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Sandstone</td><td>2.0</td><td>6408</td><td>123</td><td>1789</td><td>43</td><td>398</td><td>48</td><td>27</td><td>23</td><td>7.7</td><td>0.0</td><td>2.7</td><td>0.2</td><td>1.8</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Sandstone</td><td>3.0</td><td>20</td><td>6</td><td>76</td><td>7</td><td>1586</td><td>78</td><td>5</td><td>1</td><td>8.5</td><td>0.1</td><td>4.6</td><td>1.0</td><td>1.3</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Sandstone</td><td>4.0</td><td>2</td><td>1</td><td>85</td><td>8</td><td>1615</td><td>97</td><td>6</td><td>1</td><td>8.6</td><td>0.1</td><td>7.1</td><td>0.1</td><td>1.9</td><td>0.3</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Sandstone</td><td>4.3</td><td>2</td><td>1</td><td>86</td><td>9</td><td>1603</td><td>92</td><td>6</td><td>1</td><td>8.6</td><td>0.1</td><td>8.0</td><td>0.5</td><td>1.5</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Sandstone</td><td>7.0</td><td>3</td><td>0</td><td>88</td><td>11</td><td>1549</td><td>98</td><td>6</td><td>1</td><td>8.5</td><td>0.1</td><td>6.2</td><td>0.5</td><td>1.9</td><td>0.7</td><td>1.3</td><td>0.0</td><td>1.8</td><td>0.1</td></tr><tr><td>40</td><td>Sandstone</td><td>8.2</td><td>2</td><td>1</td><td>82</td><td>10</td><td>1457</td><td>87</td><td>5</td><td>1</td><td>8.5</td><td>0.1</td><td>7.4</td><td>0.1</td><td>2.0</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Sandstone</td><td>9.0</td><td>4</td><td>2</td><td>91</td><td>9</td><td>1504</td><td>91</td><td>6</td><td>1</td><td>8.6</td><td>0.0</td><td>5.8</td><td>0.8</td><td>2.6</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Sandstone</td><td>11.1</td><td>5</td><td>3</td><td>87</td><td>8</td><td>1423</td><td>90</td><td>6</td><td>1</td><td>8.6</td><td>0.0</td><td></td><td>0.0</td><td>1.7</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Calcite</td><td>0.0</td><td>9084</td><td>205</td><td>2538</td><td>57</td><td>1</td><td>0</td><td>1</td><td>0</td><td>7.0</td><td>0.0</td><td>0.0</td><td>0.1</td><td>4.2</td><td>0.0</td><td>1.1</td><td>0.2</td><td>2.1</td><td></td></tr><tr><td>40</td><td>Calcite</td><td>1.1</td><td>8318</td><td>199</td><td>2311</td><td>70</td><td>5</td><td>4</td><td>26</td><td>13</td><td>7.1</td><td>0.1</td><td>0.9</td><td>0.1</td><td>3.4</td><td>0.5</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Calcite</td><td>2.0</td><td>7388</td><td>625</td><td>2028</td><td>178</td><td>137</td><td>107</td><td>63</td><td>9</td><td>7.6</td><td>0.1</td><td>4.3</td><td>0.2</td><td>2.0</td><td>1.6</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Calcite</td><td>3.0</td><td>1022</td><td>663</td><td>290</td><td>159</td><td>1383</td><td>116</td><td>25</td><td>14</td><td>8.1</td><td>0.3</td><td>4.1</td><td>0.2</td><td>1.1</td><td>0.4</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Calcite</td><td>4.0</td><td>1</td><td>0</td><td>73</td><td>11</td><td>1693</td><td>74</td><td>7</td><td>1</td><td>8.7</td><td>0.0</td><td>12.2</td><td>0.8</td><td>2.1</td><td>0.2</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Calcite</td><td>4.3</td><td>1</td><td>0</td><td>72</td><td>10</td><td>1663</td><td>74</td><td>7</td><td>1</td><td>8.7</td><td>0.0</td><td>13.2</td><td>1.1</td><td>1.7</td><td>0.3</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Calcite</td><td>7.0</td><td>3</td><td>0</td><td>69</td><td>9</td><td>1635</td><td>83</td><td>6</td><td>1</td><td>8.7</td><td>0.0</td><td>11.0</td><td>1.6</td><td>2.4</td><td>0.2</td><td>1.2</td><td>0.0</td><td>0.9</td><td>0.6</td></tr><tr><td>40</td><td>Calcite</td><td>8.2</td><td>1</td><td>0</td><td>60</td><td>7</td><td>1522</td><td>86</td><td>5</td><td>1</td><td>8.7</td><td>0.0</td><td>14.7</td><td>3.2</td><td>1.8</td><td>0.3</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Calcite</td><td>9.0</td><td>1</td><td>0</td><td>71</td><td>8</td><td>1593</td><td>90</td><td>6</td><td>1</td><td>8.8</td><td>0.0</td><td>10.6</td><td>1.4</td><td>2.6</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Calcite</td><td>11.1</td><td>0</td><td>0</td><td>66</td><td>8</td><td>1483</td><td>95</td><td>5</td><td>1</td><td>8.7</td><td>0.0</td><td></td><td>0.0</td><td>1.9</td><td>0.4</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Basalt</td><td>0.0</td><td>9092</td><td>186</td><td>2547</td><td>52</td><td>1</td><td>0</td><td>1</td><td>0</td><td>7.0</td><td>0.0</td><td>0.0</td><td>0.0</td><td>5.3</td><td>0.0</td><td>1.4</td><td>0.2</td><td>2.4</td><td></td></tr><tr><td>40</td><td>Basalt</td><td>1.1</td><td>8107</td><td>156</td><td>2250</td><td>33</td><td>3</td><td>2</td><td>10</td><td>5</td><td>7.2</td><td>0.2</td><td>1.6</td><td>0.8</td><td>3.6</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Basalt</td><td>2.0</td><td>7739</td><td>130</td><td>2126</td><td>43</td><td>51</td><td>35</td><td>56</td><td>4</td><td>7.5</td><td>0.0</td><td>3.7</td><td>0.2</td><td>2.4</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Basalt</td><td>3.0</td><td>5323</td><td>1678</td><td>1458</td><td>496</td><td>450</td><td>280</td><td>46</td><td>10</td><td>7.5</td><td>0.2</td><td>6.6</td><td>0.5</td><td>1.3</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Basalt</td><td>4.0</td><td>1014</td><td>1739</td><td>328</td><td>453</td><td>1365</td><td>403</td><td>17</td><td>19</td><td>8.3</td><td>0.6</td><td>8.2</td><td>2.0</td><td>1.8</td><td>0.2</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Basalt</td><td>4.3</td><td>317</td><td>545</td><td>144</td><td>136</td><td>1485</td><td>157</td><td>11</td><td>9</td><td>8.5</td><td>0.4</td><td>9.8</td><td>1.2</td><td>1.3</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Basalt</td><td>7.0</td><td>2</td><td>1</td><td>72</td><td>16</td><td>1511</td><td>113</td><td>6</td><td>2</td><td>8.7</td><td>0.1</td><td>8.8</td><td>1.8</td><td>1.9</td><td>0.3</td><td>1.2</td><td>0.1</td><td>1.5</td><td>0.1</td></tr><tr><td>40</td><td>Basalt</td><td>8.2</td><td>1</td><td>0</td><td>64</td><td>12</td><td>1393</td><td>101</td><td>5</td><td>2</td><td>8.6</td><td>0.1</td><td>9.5</td><td>0.9</td><td>1.2</td><td>0.6</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Basalt</td><td>9.0</td><td>1</td><td>0</td><td>74</td><td>12</td><td>1442</td><td>108</td><td>6</td><td>1</td><td>8.8</td><td>0.1</td><td>8.5</td><td>1.8</td><td>2.4</td><td>0.9</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Basalt</td><td>11.1</td><td>1</td><td>1</td><td>66</td><td>12</td><td>1320</td><td>130</td><td>5</td><td>2</td><td>8.7</td><td>0.1</td><td></td><td>0.0</td><td>1.6</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Gypsum</td><td>0.0</td><td>9023</td><td>243</td><td>2576</td><td>69</td><td>1</td><td>0</td><td>1</td><td>0</td><td>6.9</td><td>0.1</td><td>0.0</td><td>0.1</td><td>40.7</td><td>0.0</td><td>1.3</td><td>0.2</td><td>1.8</td><td></td></tr><tr><td>40</td><td>Gypsum</td><td>1.1</td><td>8159</td><td>162</td><td>2337</td><td>44</td><td>5</td><td>1</td><td>16</td><td>2</td><td>7.0</td><td>0.1</td><td>0.6</td><td>0.4</td><td>39.2</td><td>0.8</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Gypsum</td><td>2.0</td><td>7134</td><td>134</td><td>2091</td><td>37</td><td>74</td><td>16</td><td>147</td><td>8</td><td>7.1</td><td>0.0</td><td>7.3</td><td>0.1</td><td>35.4</td><td>0.2</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Gypsum</td><td>3.0</td><td>3610</td><td>326</td><td>1170</td><td>59</td><td>401</td><td>71</td><td>357</td><td>32</td><td>7.1</td><td>0.0</td><td>18.3</td><td>1.2</td><td>34.9</td><td>0.4</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Gypsum</td><td>4.0</td><td>1487</td><td>679</td><td>691</td><td>149</td><td>764</td><td>169</td><td>456</td><td>40</td><td>7.3</td><td>0.0</td><td>38.0</td><td>2.9</td><td>38.1</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Gypsum</td><td>4.3</td><td>1077</td><td>792</td><td>596</td><td>179</td><td>833</td><td>190</td><td>457</td><td>48</td><td>7.3</td><td>0.1</td><td>44.8</td><td>3.8</td><td>37.5</td><td>0.9</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Gypsum</td><td>7.0</td><td>175</td><td>302</td><td>375</td><td>75</td><td>959</td><td>105</td><td>441</td><td>70</td><td>7.4</td><td>0.1</td><td>37.1</td><td>4.3</td><td>40.1</td><td>2.5</td><td>1.0</td><td>0.2</td><td>1.7</td><td>0.2</td></tr><tr><td>40</td><td>Gypsum</td><td>8.2</td><td>89</td><td>152</td><td>317</td><td>39</td><td>884</td><td>80</td><td>399</td><td>59</td><td>7.4</td><td>0.1</td><td>40.4</td><td>2.6</td><td>38.7</td><td>1.0</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Gypsum</td><td>9.0</td><td>59</td><td>101</td><td>332</td><td>33</td><td>915</td><td>73</td><td>417</td><td>58</td><td>7.5</td><td>0.1</td><td>36.0</td><td>4.4</td><td>41.3</td><td>1.6</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>Gypsum</td><td>11.1</td><td>11</td><td>17</td><td>297</td><td>20</td><td>874</td><td>59</td><td>387</td><td>47</td><td>7.4</td><td>0.1</td><td></td><td>0.0</td><td>1.6</td><td>0.0</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>No powder</td><td>0.0</td><td>9055</td><td>273</td><td>2521</td><td>76</td><td>1</td><td>0</td><td>1</td><td>0</td><td>7.0</td><td>0.0</td><td>0.0</td><td>0.1</td><td>4.4</td><td>0.0</td><td>1.0</td><td>0.2</td><td>2.0</td><td></td></tr><tr><td>40</td><td>No powder</td><td>1.1</td><td>7945</td><td>164</td><td>2202</td><td>49</td><td>5</td><td>7</td><td>39</td><td>1</td><td>7.2</td><td>0.1</td><td>2.4</td><td>0.1</td><td>2.7</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>No powder</td><td>2.0</td><td>7332</td><td>713</td><td>2031</td><td>191</td><td>160</td><td>239</td><td>58</td><td>2</td><td>7.6</td><td>0.1</td><td>3.8</td><td>0.2</td><td>1.8</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>No powder</td><td>3.0</td><td>4549</td><td>2202</td><td>1253</td><td>597</td><td>682</td><td>489</td><td>46</td><td>9</td><td>7.6</td><td>0.3</td><td>8.2</td><td>0.7</td><td>1.4</td><td>0.2</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>No powder</td><td>4.0</td><td>2143</td><td>833</td><td>601</td><td>217</td><td>1179</td><td>245</td><td>32</td><td>6</td><td>7.9</td><td>0.2</td><td>7.1</td><td>1.0</td><td>1.9</td><td>0.1</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>No powder</td><td>4.3</td><td>1640</td><td>625</td><td>469</td><td>165</td><td>1278</td><td>160</td><td>27</td><td>6</td><td>8.0</td><td>0.1</td><td>8.0</td><td>0.8</td><td>1.2</td><td>0.7</td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>No powder</td><td>7.0</td><td>2</td><td>0</td><td>73</td><td>22</td><td>1659</td><td>57</td><td>6</td><td>2</td><td>8.8</td><td>0.1</td><td>9.2</td><td>0.9</td><td>1.9</td><td>0.0</td><td>1.3</td><td>0.1</td><td>0.5</td><td>0.3</td></tr><tr><td>40</td><td>No powder</td><td>8.2</td><td>5</td><td>4</td><td>69</td><td>17</td><td>1554</td><td>39</td><td>5</td><td>2</td><td>8.8</td><td>0.1</td><td>10.8</td><td>0.1</td><td>1.6</td><td>0.1</td><td></td><td></td><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 1 (continued)**</caption><thead><tr><th rowspan="3">T (°C)</th><th rowspan="3">Mineralogy</th><th rowspan="3">Time (day)</th><th colspan="6">Gas composition (µmol)</th><th colspan="6">Fluid composition</th></tr><tr><th colspan="2">H2</th><th colspan="2">CO2</th><th colspan="2">CH4</th><th colspan="2">H2S</th><th colspan="2">pH</th><th colspan="2">Sulfide (mM)</th><th colspan="2">Sulfate (mM)</th><th colspan="2">Acetate (g/l)</th><th colspan="2">Formate (g/l)</th></tr><tr><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th><th>X<sup>-</sup></th><th>σ</th></tr></thead><tbody><tr><td>40</td><td>No powder</td><td>9.0</td><td>4</td><td>2</td><td>73</td><td>20</td><td>1579</td><td>63</td><td>5</td><td>2</td><td>8.9</td><td>0.1</td><td>9.3</td><td>0.4</td><td>2.3</td><td>0.8</td><td></td><td></td><td></td></tr><tr><td>40</td><td>No powder</td><td>11.1</td><td>3</td><td>2</td><td>73</td><td>18</td><td>1537</td><td>60</td><td>5</td><td>2</td><td>8.8</td><td>0.1</td><td></td><td></td><td>2.0</td><td>0.1</td><td></td><td></td><td></td></tr></tbody></table>

ideal gas law [42]:

$$n = PV/RT \qquad (1)$$

* n = amount of gas [mol]

* P = pressure [Pa]

* V = volume of headspace taken by the respective gas [m³]

* R = gas constant 8.314 462 [J/mol K]

* T = temperature [K]

Liquid samples (0.5 mL) were collected through the septum under N₂ flow. Ten to twenty µl were immediately used to quantify the dissolved sulfide (HS⁻) spectrophotometrically [43] and the rest was used for pH measurement. Then, liquid samples were centrifugated for 5 min at 14000×g and the supernatant was filtered at 0.2 µm. The pellets and supernatants thus obtained were stored separately at -20 °C for analysis of biomass and chemical indicators respectively.

**Analytical methods.** Dissolved sulfate concentration was measured by ionic chromatography (Dionex ICS-3000 with a column IonPac AG9-HC 4 mm × 250 mm) with a maximum precision of 5%; 1 g/L Na₂CO₃ mobile phase was used at a flow rate of 0.8 mL min⁻¹. The column temperature was set to 30 °C.

Volatile Fatty Acids (VFA) concentrations were measured by high-performance liquid chromatography (HPLC) (Aminex HPX-87H from Bio-Rad coupled to a Spectra System RI 150 refractometer and a Waters 2487 dual λ UV detector set at 210 nm) at time zero (T₀) and after >95% of H₂ consumption (day 16 at 25 °C and day 7 at 34 and 40 °C); 0.01 M sulfuric acid mobile phase was used at a flow rate of 0.6 mL min⁻¹. The column temperature was set to 60 °C. Acetate and formate were quantified with a precision better than 5%.

Chemical analyses for major elements (Ca, K, Mg, Na and Cl) are summarized in Table S2. They were carried out at the Laboratoire Géoscience Océan (UBO, Brest, France) using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The uncertainty on the concentration measurements is lower than ±5%.

The specific surface area (SSA) of the cleaned starting powder was determined by Kr adsorption analysis, according to the Bru-nauer–Emmett–Teller (BET) method [44] (Table S3).

**Modeling.** R CRAN software (version 3.1.2) was employed to fit the H₂ consumption data to a modified Gompertz three-parameter model (Eq. (2) [45]):

$$M(t) = P \times \exp\{-\exp[R_{\text{max}} \times e/P \times (\lambda - t) + 1]\} \qquad (2)$$

* M(t) is the cumulative H₂ consumption (µmol) at time t (d)

* P is the maximum H₂ consumption (µmol)

* Rmax is the maximum H₂ consumption rate (µmol/d)

* λ is the lag phase (d)

* e is the exponential.

## 2.3. Molecular analyses of microbial communities

**DNA extraction and PCR amplification.** DNA-based analyses were performed in the precultures, during the exponential phase of H₂ consumption (<60% of the hydrogen) and at the end of the incubation (H₂ consumption >95%). Total DNA was extracted from the pellet using the DNeasy Powersoil Pro kit (QIAGEN) according to the manufacturer's

instructions. Extracted DNA concentrations were quantified using a quantifying fluorescent dye assay (Qubit dsDNA HS Assay Kit, Invitrogen, Eugene) and Qubit 2.0 Fluorometer (Invitrogen, Life Technologies, Eugene). DNA were stored at -20 °C prior to sequencing. Bacterial and archaeal communities were characterized by targeting 16S V3–V5 and 16S V3–V4 regions, respectively. Amplicons libraries were constructed by Eurofins Genomics following two rounds of PCR amplification using an optimized protocol. Primers associated with amplification of bacterial populations were 341F 5'-CCTACGGNGGCWGCGAG-3' [46] and 926R 5'-CCGYCAATTYMTTTTRAGTTT-3' [47]. For Archaea, 5'-CCCTAYGGGGYGASCAG-3' [48] and 5'-GGACTACNVGGGTWTC-TAAT-3' [49] were used. Sequencing was performed by Eurofins Genomics with Illumina technology, MiSeq V3 (read mode 2x300 bp).

**Quality filtering, definition of OTUs, and taxonomic assignment.** The quality of the sequencing was first evaluated using FastQC [50]. Individual reports were merged into one with MultiQC [51]. Data were imported into the FROGS (Find Rapidly OTUs with Galaxy Solution) pipeline [52]. For each amplicon, paired-end sequences were merged into contigs with Vsearch (2.17.0) [53]. Adapters were trimmed with cutadapt v2.10 [54]. Sequences were dereplicated before being clustered using SWARM [55] with a local clustering threshold with a distance of 1. Chimeras were removed with vsearch 2.17.0. The resulting sequences were filtered for spurious OTUs likely arising from sequencing artifacts (OTUs with low abundance and low frequency) by keeping only those appearing more than 0.0005% in the whole dataset [56]. Taxonomic assignment of OTUs was performed using the EZBioCloud 16S database (release 52018) [57] as a reference database.

# 3. Results and discussion

## 3.1. H₂ consumption dynamics

All incubations were reproduced three times, each time showing the same patterns with good reproducibility (Table 1). Microbial consumption of H₂ and CO₂ occurred concomitantly in all incubations (Fig. 1a–f). Kinetic constants associated with H₂ consumption curves are presented in Table 2 and Fig. S2.

Control incubations performed without rock powder indicate that H₂ is more rapidly consumed at 34 °C and 40 °C than at 25 °C. Lag time (λ) and maximum H₂ consumption rate (Rmax) are similar at 34 and 40 °C, with λ = 1.1 ± 0.1 and 1.6 ± 0.5 days and Rmax = 3.5 ± 0.2 and 3.6 ± 0.7 mmol/day, respectively. By contrast, at 25 °C, the lag phase is four times longer (4.8 ± 0.1 days), and the rate of maximum H₂ consumption falls by 37% to 2.2 ± 0.2 mmol/day.

Interestingly, rock powder additions induced contrasted effects on H₂ consumption kinetics. Notably, the inclusion of sandstone, basalt, and calcite exhibits an extension of more than 35% of the lag phase at 25 °C, with durations of 6.7 ± 0.1, 6.8 ± 0.2, and 6.5 ± 1.0 days, respectively. Despite this initial delay, these rock powders appear to increase the maximum rate of H₂ consumption independently of temperature. The rate of H₂ consumption increases from 73 to 100% on average with sandstone, from 45 to 95% with calcite and from 23 to 68% with basalt compared with control incubations. Particularly, when adding basalt at 25 °C, approximately 15% of the initial hydrogen remains unconsumed after a 30-day experimental period.

Conversely, the introduction of gypsum does not yield significant
<figure><img src="image_4.png" /><figcaption>Fig. 1. Consumption of H₂ (A–C), CO₂ (D–F), and SO₄²⁻ (G–I) by an anaerobic microbial consortium harboring hydrogenotrophic metabolisms incubated at 25, 34 and 40 °C with different rock powders (sandstone, calcite, basalt, and gypsum; in colors) and in a powder-free negative control (in black). The data points and lines represent the mean concentrations in µmol obtained from triplicate experiments, and the reproducibility is indicated by the shaded envelopes. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

alterations in H₂ consumption kinetics across different temperatures when compared to control incubations. However, it is worth highlighting that between 15% and 20% of the initial CO₂ remains unconsumed in the presence of gypsum. Dissolved sulfate initially present in the culture medium (4 ± 1 mM) is consumed in all experiments (Fig. 1g–i). However, with gypsum powder, the concentration of dissolved sulfate is consistently 8 to 10 times higher than that in the standard medium (39 ± 3 mM), maintaining this level throughout the experiments at all temperatures. Similar high sulfate concentrations (9–10 times) are observed in abiotic controls with gypsum after 20 days at all tested temperatures indicating that gypsum dissolution occurs at thermodynamic equilibrium with the culture medium (Table 3).

In summary, H₂ consumption kinetics are overall faster at 34 and 40 °C than at 25 °C. They also vary in the presence of a mineral substrate, and depend on the mineralogy of the substrate, independently of

temperature.

## 3.2. H₂ conversion pathways

Methane and sulfides (H₂S and dissolved HS⁻) production kinetics are presented in Fig. 2. Fig. 3 indicates the production of acetate in all incubations. The contribution of the different hydrogenotrophic metabolisms to the H₂ consumption, i.e. methanogenesis, sulfate reduction and homoacetogenesis was calculated by mass balance based on the following reactions [58] (Fig. 4):

$$4H_2 + HCO_3^- + H^+ \Rightarrow CH_4 + 3H_2O$$

$$4H_2 + SO_4^{2-} + 2H^+ \Rightarrow H_2S + 4H_2O$$

$$4H_2 + 2HCO_3^- + 2H^+ \Rightarrow C_2H_4O_2 + 4H_2O$$
<caption>**Table 2**</caption>

Lag phase duration $\lambda$ (days) estimated by a Gompertz three-parameter model using R CRAN software and maximum H₂ consumption rate Rmax (mmol/d) calculated between 2 measured points. Values are averages on triplicate experiments.

<table><thead><tr><th rowspan="2"></th><th colspan="2">25 °C</th><th colspan="2">34 °C</th><th colspan="2">40 °C</th></tr><tr><th>λ (day)</th><th>Rmax (mmol/d)</th><th>λ (day)</th><th>Rmax (mmol/d)</th><th>λ (day)</th><th>Rmax (mmol/d)</th></tr></thead><tbody><tr><td>Control</td><td>4.8 ± 0.1</td><td>2.2 ± 0.2</td><td>1.1 ± 0.1</td><td>3.5 ± 0.2</td><td>1.6 ± 0.5</td><td>3.6 ± 0.7</td></tr><tr><td>Sandstone</td><td>6.7 ± 0.1</td><td>4.0 ± 0.3</td><td>1.8 ± 0.1</td><td>6.7 ± 0.7</td><td>1.9 ± 0.01</td><td>6.4 ± 0.1</td></tr><tr><td>Calcite</td><td>6.8 ± 0.2</td><td>3.9 ± 0.5</td><td>1.5 ± 0.6</td><td>6.0 ± 0.6</td><td>1.8 ± 0.03</td><td>6.4 ± 0.04</td></tr><tr><td>Basalt</td><td>6.5 ± 1.0</td><td>3.1 ± 0.5</td><td>1.4 ± 0.4</td><td>5.2 ± 0.9</td><td>2.5 ± 0.5</td><td>4.5 ± 0.9</td></tr><tr><td>Gypsum</td><td>4.3 ± 0.3</td><td>1.5 ± 0.2</td><td>1.5 ± 0.1</td><td>3.6 ± 0.3</td><td>1.5 ± 0.2</td><td>3.5 ± 0.3</td></tr></tbody></table>

The remaining amount of H₂ not consumed by these metabolisms [X] is then expressed as:

$$[X] = 100 - (400 \times (CH_4(g) + C_4H_4O_2(aq) + H_2S_T)/[H_2])$$

$$\text{with } H_2S_T = H_2S(g) + \sum (H_2S_{(aq)}, HS^-, S^{2-}) \text{ (in moles)}$$

Control incubations at 34 and 40 °C show that 70–80% of the consumed H₂ is converted into CH₄ while the production of H₂S and acetate remains minor, respectively 5–6% and 1–4%. At 25 °C, however, competition for hydrogen between methanogenesis and homoacetogenesis is slightly stronger. The share of CH₄ production falls to 57–62% and 14–16% of the H₂ is converted to acetate. Previous studies showed that homoacetogens may outcompete methanogens for H₂ in laboratory experiments at low temperature (6–17 °C; e.g., Ref. [59]), which could explain the increasing proportion of homoacetogenesis in incubations at 25 °C. It may be due to higher growth rates of homoacetogens at 25 °C (e.g., Ref. [60]). Indeed, reports of methanogenic archaea, which are not only tolerant but are especially adapted to life at low temperatures are not very common, indicating that methanogenic archaea potentially adapt better to high than to low temperatures. Moreover, methanogenesis is thermodynamically favored by high temperature, with a

mesophilic optimum around 30 °C and 50–70 °C for thermophilic species [61].

With gypsum powder, no acetate production was observed while bacterial sulfate reduction exceeds methanogenesis, accounting for 81 ± 5%, 68 ± 3%, and 41 ± 6% of the H₂ consumption at 25, 34 and 40 °C, respectively. Sulfate reducers are known to outcompete homoacetogens and methanogens when sulfate is not limiting, which is the case in incubations with gypsum [62,63]. This is explained by their kinetic properties (Ks, μmax) and given the favorable thermodynamic conditions [64,65]. Nevertheless, we can notice that sulfate reduction is decreasingly dominant with respect to methanogenesis with increasing temperature.

The presence of calcite, basalt and sandstone powder does not significantly alter the proportions of H₂ converted to CH₄, H₂S and acetate at 34 and 40 °C. A slight increase in the contribution of homoacetogenesis to 6–7% is nevertheless observed at 34 °C (against 1–4% in the control incubation). Meanwhile, at 25 °C, the H₂ conversion into acetate rises compared to the control in average to 46 ± 11% (n = 3), 66 ± 3% (n = 3) and 83 ± 5% (n = 3) respectively with sandstone, calcite and basalt powder. Several factors may explain these variations: differences in surface availability to form biofilms and the availability of bio-essential elements such as Mg, Ca or trace elements in the rock powders. Several studies consistently demonstrated the influence of surface area available for microbial colonization on stimulating microbial activity (e.g., Ref. [36,66,67]). Notably, Khajooie et al. (2024) recently observed a significant increase in methanogenic activity when utilizing rock fragments with varying specific surface areas, resulting in activity levels 8 to 10 times higher than in corresponding bulk solution, a trend correlated with pore space. Moreover, previous studies focused on biogas production in bioreactors showed that the chemical composition of the support materials can also impact the microorganisms activity. For instance, the utilization of zeolites modified with metals such as cobalt, nickel and magnesium is favorable to the growth of methanogens and hydrolytic bacteria [68].

### 3.3. Influence of the mineral substrate

To explore the link between mineralogy and acetate production by homoacetogenesis, and the effect of microbial activity on precipitation

<caption>**Table 3**</caption>

Gas and fluid analyses at time T0 and TF (19 days) of abiotic control experiments with powders (basalt, calcite, sandstone and gypsum) at 25, 34 and 40 °C.

<table><thead><tr><th rowspan="2">T (°C)</th><th rowspan="2">Mineralogy</th><th rowspan="2">Time (day)</th><th colspan="4">Gas Composition (µmol)</th><th colspan="5">Fluid composition</th></tr><tr><th>H2</th><th>CH4</th><th>CO2</th><th>H2S</th><th>pH</th><th>Sulfide (mM)</th><th>Sulfate (mM)</th><th>Acetate (±0.1 g/l)</th><th>Formate (±0.1 g/l)</th></tr></thead><tbody><tr><td rowspan="2">25</td><td rowspan="2">Sandstone</td><td>0</td><td>9111</td><td>1</td><td>2559</td><td>0</td><td>6.9</td><td>0.1</td><td>3.5</td><td>1.0</td><td>2.0</td></tr><tr><td>19</td><td>8729</td><td>1</td><td>2365</td><td>0</td><td>6.7</td><td>0.2</td><td>3.4</td><td>1.0</td><td>1.9</td></tr><tr><td rowspan="2">25</td><td rowspan="2">Calcite</td><td>0</td><td>9074</td><td>1</td><td>2595</td><td>0</td><td>6.9</td><td>0.1</td><td>3.1</td><td>1.1</td><td>2.0</td></tr><tr><td>19</td><td>8726</td><td>1</td><td>2417</td><td>0</td><td>6.8</td><td>0.3</td><td>3.3</td><td>1.0</td><td>1.9</td></tr><tr><td rowspan="2">25</td><td rowspan="2">Basalt</td><td>0</td><td>9048</td><td>1</td><td>2588</td><td>0</td><td>6.9</td><td>0.1</td><td>3.1</td><td>1.1</td><td>2.0</td></tr><tr><td>19</td><td>8644</td><td>1</td><td>2369</td><td>8</td><td>6.8</td><td>0.6</td><td>2.3</td><td>1.0</td><td>1.8</td></tr><tr><td rowspan="2">25</td><td rowspan="2">Gypsum</td><td>0</td><td>9041</td><td>2</td><td>2588</td><td>0</td><td>6.9</td><td>0.1</td><td>22.8</td><td>1.1</td><td>2.0</td></tr><tr><td>19</td><td>8640</td><td>1</td><td>2379</td><td>0</td><td>6.7</td><td>0.1</td><td>32.1</td><td>1.0</td><td>1.9</td></tr><tr><td rowspan="2">34</td><td rowspan="2">Sandstone</td><td>0</td><td>9289</td><td>1</td><td>2610</td><td>0</td><td>6.9</td><td>0.1</td><td>3.5</td><td>1.0</td><td>2.0</td></tr><tr><td>19</td><td>8836</td><td>1</td><td>2372</td><td>0</td><td>6.7</td><td>0.3</td><td>3.4</td><td>1.0</td><td>1.9</td></tr><tr><td rowspan="2">34</td><td rowspan="2">Calcite</td><td>0</td><td>9212</td><td>1</td><td>2634</td><td>0</td><td>6.9</td><td>0.1</td><td>3.1</td><td>1.1</td><td>2.0</td></tr><tr><td>19</td><td>8761</td><td>1</td><td>2339</td><td>0</td><td>6.8</td><td>0.2</td><td>3.3</td><td>1.0</td><td>1.9</td></tr><tr><td rowspan="2">34</td><td rowspan="2">Basalt</td><td>0</td><td>9137</td><td>1</td><td>2614</td><td>0</td><td>6.9</td><td>0.1</td><td>3.1</td><td>1.1</td><td>2.0</td></tr><tr><td>19</td><td>8679</td><td>1</td><td>2312</td><td>0</td><td>6.7</td><td>0.2</td><td>3.4</td><td>1.0</td><td>1.9</td></tr><tr><td rowspan="2">34</td><td rowspan="2">Gypsum</td><td>0</td><td>9098</td><td>2</td><td>2604</td><td>0</td><td>6.9</td><td>0.1</td><td>22.8</td><td>1.1</td><td>2.0</td></tr><tr><td>19</td><td>8582</td><td>1</td><td>2407</td><td>0</td><td>6.7</td><td>0.2</td><td>28.8</td><td>1.0</td><td>1.9</td></tr><tr><td rowspan="2">40</td><td rowspan="2">Sandstone</td><td>0</td><td>9280</td><td>1</td><td>2607</td><td>0</td><td>6.9</td><td>0.1</td><td>3.5</td><td>1.0</td><td>2.0</td></tr><tr><td>19</td><td>8601</td><td>1</td><td>2274</td><td>0</td><td>6.7</td><td>0.1</td><td>3.3</td><td>1.0</td><td>1.9</td></tr><tr><td rowspan="2">40</td><td rowspan="2">Calcite</td><td>0</td><td>8527</td><td>1</td><td>2439</td><td>0</td><td>6.9</td><td>0.1</td><td>3.1</td><td>1.1</td><td>2.0</td></tr><tr><td>19</td><td>8079</td><td>1</td><td>2271</td><td>0</td><td>6.7</td><td>0.1</td><td>3.0</td><td>1.0</td><td>1.9</td></tr><tr><td rowspan="2">40</td><td rowspan="2">Basalt</td><td>0</td><td>9168</td><td>1</td><td>2623</td><td>0</td><td>6.9</td><td>0.1</td><td>3.1</td><td>1.1</td><td>2.0</td></tr><tr><td>19</td><td>8610</td><td>1</td><td>2288</td><td>0</td><td>6.7</td><td>0.2</td><td>3.3</td><td>1.0</td><td>1.9</td></tr><tr><td rowspan="2">40</td><td rowspan="2">Gypsum</td><td>0</td><td>9165</td><td>2</td><td>2623</td><td>0</td><td>6.9</td><td>0.1</td><td>22.8</td><td>1.1</td><td>2.0</td></tr><tr><td>19</td><td>8610</td><td>2</td><td>2397</td><td>0</td><td>6.7</td><td>0.2</td><td>32.2</td><td>1.0</td><td>1.9</td></tr></tbody></table>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Cumulative production of CH₄ (A–C), H₂S (solid lines) and HS⁻ (dashed lines; D–F) evidencing methanogenesis and sulfate reduction activity in incubations of an anaerobic microbial consortium at 25, 34 and 40 °C with different rock powders (sandstone, calcite, basalt, and gypsum; in colors) and in a powder-free negative control (in black). The data points and lines represent the mean concentrations in µmol obtained from triplicate experiments, and the reproducibility is indicated by shaded envelopes. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Acetate production (g/L) by homoacetogenesis in incubations of an anaerobic microbial consortium at 25, 34 and 40 °C with different rock powders (sandstone, calcite, basalt, and gypsum) and in a powder-free negative control. Concentrations are the mean of triplicate incubations (with 1σ error bars). They were measured after 90% H₂ consumption (i.e. day 16 at 25 °C and day 7 at 34 and 40 °C) and corrected for the initial concentration in the culture medium.</figcaption></figure>

and dissolution of rock powders, we investigated variations in the major elements' composition of incubation solutions and abiotic controls.

In the incubations at 34 and 40 °C, the concentration of calcium

(Ca²⁺) is higher in the presence of calcite (CaCO₃) compared to basalt, sandstone, and the control without added powder (Fig. S3, Table S2). This trend is consistent in abiotic experiments, indicating a partial dissolution of calcite due to thermodynamic equilibrium with the culture medium (Fig. S3). However, at 25 °C, calcite dissolution is notably more pronounced when the consortium is present, resulting in a significant increase in [Ca²⁺] from 1.3 to 12.5 mM compared to 1.7 mM in abiotic conditions. This suggests that the biological activity of the consortium may be responsible for the increase in calcite dissolution. In contrast, the Ca released by gypsum dissolution is equivalent in biotic and abiotic experiments suggesting no biological effect; gypsum dissolution is only controlled by thermodynamic equilibrium with the culture medium.

High levels of acetate production are also observed with basalt and sandstone, which contain little to no calcium. With basalt, variations in magnesium (Mg²⁺) concentrations are observed only at 25 °C in the presence of the consortium, potentially indicating alteration of the mineral substrate due to microbial activity (Fig. S3). In the case of sandstone (SiO₂), there is a substantial decrease in Mg²⁺ concentration compared to the powder-free control incubations, declining from 4.6 ± 0.4 mM to 1.3–3.6 mM. Overall, these chemical variations at 25 °C suggest interactions between the consortium and the mineral substrates, which may serve as surface to form biofilms. Measurements of specific surface area using BET analysis also reveal differences related to the mineralogy. Basalt has the largest surface area (0.88 m²/g), followed by calcite (0.64 m²/g) and sandstone (<0.08 m²/g; Table S3), which is consistent with the variations in acetate production observed with these rock powders.

In summary, low temperatures favor the development of homoacetogenic bacteria over methanogenic archaea. However, variations in acetate and methane production with mineralogy also strongly suggest that the presence of a mineral substrate to form biofilms influences the competition for H₂, which is consistent with the recent results of Dohrmann and Krüger [63] and Khajooie et al. [36]. Furthermore, the results indicate that the microbial activity of the consortium can alter
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Proportions of H₂ (in %) converted into methane, acetate, sulfuric acid and unknown products respectively by methanogenesis, homoacetogenesis, sulfate reduction, and other metabolisms at 25, 34 and 40 °C with different rock powders (sandstone, calcite, basalt, and gypsum) and in a powder-free negative control. These proportions were calculated by mass balance based on the metabolic reactions of H₂ consumption (see text for details).</figcaption></figure>

the thermodynamic equilibria between the fluid and rock powders, and, in our case, promote mineral alteration (calcite and basalt). Further experiments with varying particle sizes and chemical compositions may help us to better understand and predict these effects.

### 3.4. DNA-based microbial community analyses

To obtain a general qualitative view of microbial community dynamic under different conditions and identify key microorganisms potentially involved in H₂ consumption, high-throughput 16S rRNA sequencing was performed on 24 samples before 60% and after 90% H₂ consumption. For archaea between 25816 and 97017 sequences and for bacteria between 22016 and 83291 sequences were identified and respectively assembled into 1 and 10 OTUs. Relative abundances of bacterial genera are presented in Figs. 5 and 6. Preculture samples collected just before the beginning of the incubations were considered as

the initial inoculum. Bacterial OTU tables are presented in Supplementary Material (Tables S5–S7).

In all samples, a single methanogenic archaeon belonging to *Methanobacterium* was detected with a relative abundance of 100%. Regarding bacteria, sulfate reducers detected in the inoculum mainly belong to *Desulfovibrio* genus with relative abundances of 5% at 25 °C, 15% at 34 °C and 22% at 40 °C. In all incubations where the sulfate concentration was limited (i.e. without gypsum), *Desulfovibrio* relative abundances remained constant or decreased along the experiments. In opposition, *Desulfovibrio* became predominant with gypsum powder at 25 °C and 34 °C with a relative abundance respectively up to 48% and 40%. Meanwhile, at 40 °C, only 14% of the OTUs were assigned to *Desulfovibrio* whereas *Clostridium* relative abundance increased up to 51% (compared to 38% in the inoculum). The *Clostridium* OTU is 100% identical to *Clostridium thiosulfatireducens* (OR342400).

Despite the inability to reduce sulfate, mesophilic *Clostridium* species
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Relative abundances of the main bacterial genera generated by 16S rRNA gene sequencing in incubations at 34 °C and 40 °C with different rock powders (sandstone, calcite, basalt, and gypsum) and in a powder-free negative control. Relative abundances obtained at H₂ consumption rates <60% and >90% are compared with the inoculum population (T₀).</figcaption></figure>

are known to produce sulfides from thiosulfate and elemental sulfur in the presence of peptone, yeast extract and glucose [69]. Thus, our results tend to suggest that at 40 °C, *Clostridium thiosulfatireducens* may be involved in the high H₂S production with those of *Desulfovibrio* genus. It can also be noted that at 34 °C, relative abundances of *Clostridium* temporarily increased during the experiments with basalt, sandstone and calcite and the control (<60% H₂ consumption) probably until the complete consumption of oxidized sulfur species.

Homoacetogenetic bacteria mainly belonging to *Acetobacterium* were not detected in the inoculum but increased along the experiments at 25 °C and 34 °C. Their relative abundance remained limited at 34 °C to 12%, 7%, and 9% respectively with sandstone, calcite, and basalt. At 25 °C, however, up to 47%, 59% and 64% of the sequences respectively with sandstone, calcite and basalt were assigned to *Acetobacterium*. Moreover, *Acetobacterium* abundance is highly variable between trilicate flasks with sandstone, calcite and basalt powders, which is consistent with the variable acetate production. Finally, *Acetobacterium* was

not detected with gypsum. It confirms the previous results of [70] which report that the growth of this genus is faster at low temperature, while being inhibited by high sulfate concentrations.

### 3.5. Relevance of the results for future UHS

Hydrogen injected into a porous reservoir is known to change the chemical equilibrium between the formation pore water, dissolved gases and the rock matrix, as it notably induces reduced Eh conditions. Resulting geochemical reactions could lead to: (i) significant loss of hydrogen; (ii) the production of other gases (e.g., CH₄, H₂S); (iii) mineral dissolution/precipitation leading to enhanced or reduced injectivity, opening of migration pathways through the caprock, and modification of the mechanical properties of the reservoir and the caprock [10]. Microbial activity is also known to have an impact on these geochemical equilibria and may therefore play a very important role in the evolution of H₂ geological storage [6,17,19]. In this study, we
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Relative abundances of the main bacterial genera generated by 16S rRNA gene sequencing in incubations at 25 °C with different rock powders (sandstone, calcite, basalt, and gypsum) and in a powder-free negative control (-). Relative abundances obtained before 60% (A) and after 90% (B) of H₂ consumption are compared with the inoculum population (T₀). Samples were clustered by mineralogy and experimental triplicates (R1, R2 and R3) are shown for sandstone, calcite and basalt to illustrate variability in abundance between bioreactors.</figcaption></figure>

suggested that the presence of a mineral substrate may influence competition for H₂ consumption, especially at low temperatures. This is consistent with previous studies showing the influence of surface area available for microbial colonization on stimulation of microbial activity (e.g., Ref. [36,66,67]). We also showed that in our experiments, the dissolution of calcite and basalt is promoted by the presence of the microbial consortium at low temperature, adding Ca and Mg to the fluids. It confirms that microorganisms can potentially influence water-rock equilibria and therefore promote the dissolution of the reservoir minerals or, in other cases, the precipitation of secondary minerals. This has also been suggested by Ranchou-Peyruse et al. [38], where a pH increase induced by lithoautotrophic microbial metabolisms appeared to promote the mineralization of CO₂ dissolved in carbonates.

In addition, recent studies indicated that microbial growth and metabolic activities are controlled by several parameters such as temperature, pH, salinity, pressure and nutrient supply [21]. This study is the first to highlight the control of reservoir mineralogy on microbial competition for H₂ consumption. However, these batch experiments

were performed at near atmospheric pressure, which is not representative of H₂ storage conditions. Further investigations into how microbial consortia interact with reservoir rocks under higher pressure and high H₂ concentration, through experiments in porous media, will be necessary to understand the behavior of microorganisms and accurately assess the magnitude of H₂ consumption in reservoir conditions.

## 4. Conclusions

As the first study of its kind, these experiments are intended to assess the control of the mineralogy and temperature on the competition for hydrogen consumption of hydrogenotrophic communities. Interestingly, it was shown that both temperature and mineralogy affect H₂ consumption kinetics, as well as the metabolic activity of the microbial consortium towards H₂ substrate. The main observations are.

* The presence of gypsum favors sulfate reduction over methanogenesis and homoacetogenesis, especially at low temperature.
* Conversely, methanogenesis is dominant at 34 and 40 °C with all sulfate-free mineralogies (basalt, calcite, sandstone), with only minor H₂S and acetate production (<7%).

* At 25 °C, the development of homoacetogenic bacteria is favored over methanogens.

* Acetate production increases in the presence of mineral substrate and varies with mineralogy. This suggests interactions of the microbial community with the rock powders, which may serve as a surface to form biofilms.

* Radical changes in microbial populations are in line with these metabolic shifts. Members, which consume H₂, like sulfate reducers of the genus *Desulfovibrio*, homoacetogens of the genus *Acetobacterium* and methanogens of the genus *Methanobacterium*, as well as other members not directly involved in H₂ consumption like thiosulfate reducers of the genus *Clostridium* vary depending on temperature and mineralogy.

* Microbial activity promotes the dissolution of calcite and basalt in experiments at 25 °C. It confirms that microorganisms may influence water-rock equilibria and promote mineral precipitation or dissolution.

This study reveals the importance of considering mineralogy as well as temperature in assessing potential underground storage reservoirs, as they may significantly impact indigenous microbial populations and H₂ consumption dynamics. Further quantitative investigation into these factors is warranted for comprehensive site evaluations.

## Ethics approval and consent to participate

Not applicable.

## Consent for publication

All the authors mutually consent for publication this manuscript and state that has not been previously published in another journal.

## CRediT authorship contribution statement

**Elodie Muller:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.
**Julia Guélard:** Writing – review & editing, Writing – original draft, Validation, Methodology, Funding acquisition, Formal analysis, Conceptualization.
**Olivier Sissmann:** Writing – review & editing, Writing – original draft, Validation, Methodology, Funding acquisition, Formal analysis, Conceptualization.
**Ambre Tafit:** Writing – review & editing, Writing – original draft, Investigation.
**Simon Poirier:** Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgement

We thank Céline Liorzou for performing ICP-AES analyses at Pôle Spectrométrie Océan (Laboratoire Géo-Océan, Brest, France). We also thank the anonymous reviewers for their constructive reviews. This study was funded by IFPEN.

# Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijhydene.2024.08.024.

## References

[1] Clark WW, Rifkin J. A green hydrogen economy. Energy Pol 2006;34(17):2630-9.

[2] Reitenbach V, Ganzer L, Albrecht D, Hagemann B. Influence of added hydrogen on underground gas storage: a review of key issues. Environ Earth Sci 2015;7(11):6927-37.

[3] Haddad PG, Ranchou-Peyruse M, Guignard M, Mura J, Casteran F, Ronjon-Magand L, Senechal P, Isaire M-P, Moonen P, Hoareau G, Dequidt D, Chiquet P, Caumette G, Cezac P, Ranchou-Peyruse A. Geological storage of hydrogen in deep aquifers – an experimental multidisciplinary study. Energy Environ Sci 2022;15(8):3400-15.

[4] Zivar D, Kumar S, Foroozesh J. Underground hydrogen storage: a comprehensive review. Int J Hydrogen Energy 2021;46(45):23436-62.

[5] van Gessel SF, Hajibeygi H, Edlmann Katriona, Dopffel Nicole, Xie Quan, Ed Hough Remco Groenenberg, Yallup Christine, Greco Gianluca, Réveillère Arnaud, Schultz Richard, Fernández Cristina Yuste, Fournier Cyriane, Draganov Deyan, Machado de Sousa Diana, Slob Evert, Juez-Larré Joaquim, van Winden Julia, Bisdom Kevin, Ramesh Kumar Kishan, Truche Laurent, Zeng Lingping, Noort Reinier van, Pérez Ricardo Ariel, Murúa Sebastián, Jansen Stefan, Hangx Suzanne, Schacht Ulrike. Hydrogen TCP-Task42 technology monitor 2023. 2023.

[6] Dopffel N, Jansen S, Gerritse J. Microbial side effects of underground hydrogen storage – knowledge gaps, risks and opportunities for successful implementation. Int J Hydrogen Energy 2021;46(12):8594-606.

[7] Lankof L, Tarkowski R. Assessment of the potential for underground hydrogen storage in bedded salt formation. Int J Hydrogen Energy 2020;45(38):19479-92.

[8] Michalski J, Bünger U, Crotogino F, Donadei S, Schneider G-S, Pregger T, Cao K-K, Heide D. Hydrogen generation by electrolysis and storage in salt caverns: potentials, economics and systems aspects with regard to the German energy transition. Int J Hydrogen Energy 2017;42(19):13427-43.

[9] Tarkowski R. Underground hydrogen storage: characteristics and prospects. Renew Sustain Energy Rev 2019;105:86-94.

[10] Heinemann N, Alcalde J, Miocic JM, Hangx SJT, Kallmeyer J, Ostertag-Henning C, Hassanpouryouzband A, Thaysen EM, Strobel GJ, Schmidt-Hattenberger C, Edlmann K, Wilkinson M, Bentham M, Stuart Haszeldine R, Carbonell R, Rudloff A. Enabling large-scale hydrogen storage in porous media – the scientific challenges. Energy Environ Sci 2021;14(2):853-64.

[11] Flesch S, Pudlo D, Albrecht D, Jacob A, Ennzmann F. Hydrogen underground storage—petrographic and petrophysical variations in reservoir sandstones from laboratory experiments under simulated reservoir conditions. Int J Hydrogen Energy 2018;43(45):20822-35.

[12] Hemme C, van Berk W. Potential risk of H2S generation and release in salt cavern gas storage. J Nat Gas Sci Eng 2017;47:114-23.

[13] Crotogino F, Schneider G-S, Evans DJ. Renewable energy storage in geological formations. Proc Inst Mech Eng A J Power Energy 2018;232(1):100-14.

[14] Bardelli F, Mondelli C, Didier M, Vitillo JG, Cavicchia DR, Robinet J-C, Leone L, Charlet L. Hydrogen uptake and diffusion in Callovo-Oxfordian clay rock for nuclear waste disposal technology. Appl Geochem 2014;49:168-77.

[15] Foh, et al. Underground hydrogen storage: final report. 1979. Upton NY (USA).

[16] Yekta AE, Manceau J-C, Gaboreau S, Pichavant M, Audigane P. Determination of hydrogen-water relative permeability and capillary pressure in sandstone: application to underground hydrogen injection in sedimentary formations. Transport Porous Media 2018;122(2):333-56.

[17] Ebigbo A, Golfer F, Quintard M. A coupled, pore-scale model for methanogenic microbial activity in underground hydrogen storage. Adv Water Resour 2013;61:74-85.

[18] Hemme C, van Berk W. Hydrogeochemical modeling to identify potential risks of underground hydrogen storage in depleted gas fields. Appl Sci 2018;8(11):2282.

[19] Ivanova AE, Borzenkov IA, Tarasov AL, Milekhina EI, Belyaev SS. A microbiological study of an underground gas storage in the process of gas injection. Microbiology 2007;76(4):453-60.

[20] Panfilov M. Underground storage of hydrogen: in situ self-organisation and methane generation. Transport Porous Media 2010;85(3):841-65.

[21] Thaysen EM, McMahon S, Strobel GJ, Butler IB, Ngwenya BT, Heinemann N, Wilkinson M, Hassanpouryouzband A, McDermott CI, Edlmann K. Estimating microbial growth and hydrogen consumption in hydrogen storage in porous media. Renew Sustain Energy Rev 2021;151:111481.

[22] Tremosa J, Jakobsen R, Le Gallo Y. Assessing and modeling hydrogen reactivity in underground hydrogen storage: a review and models simulating the Lobodice town gas storage. Front Energy Res 2023;11.

[23] Aüllo T, Ranchou-Peyruse A, Ollivier B, Magot M. Desulfotomaculum spp. and related gram-positive sulfate-reducing bacteria in deep subsurface environments. Front Microbiol 2013;4:362.

[24] Berlendis S, Lascourreges J-F, Schraauwers B, Sivadon P, Magot M. Anaerobic biodegradation of BTEX by original bacterial communities from an underground gas storage aquifer. Environmental science & technology 2010;44(9):3621-8.

[25] Gregory SP, Barnett MJ, Field LP, Milodowski AE. Subsurface microbial hydrogen cycling: natural occurrence and implications for industry. Microorganisms 2019;7(2).
[26] Ranchou-Peyruse M, Gasc C, Guignard M, Aullo T, Dequidt D, Peyret P, Ranchou-Peyruse A. The sequence capture by hybridization: a new approach for revealing the potential of mono-aromatic hydrocarbons bioattenuation in a deep oligotrophic aquifer. Microb Biotechnol 2017;10(2):469–79.

[27] Ranchou-Peyruse M, August J-C, Mazière C, Restrepo-Ortiz CX, Guignard M, Dequidt D, Chiquet P, Cézac P, Ranchou-Peyruse A. Geological gas-storage shapes deep life. Environ Microbiol 2019;21(10):3953–64.

[28] Basso O, Lascourreges J-F, Le Borgne F, Le Goff C, Magot M. Characterization by culture and molecular analysis of the microbial diversity of a deep subsurface gas storage aquifer. Res Microbiol 2009;160(2):107–16.

[29] Panfilov M. Underground and pipeline hydrogen storage. Compendium of Hydrogen Energy; 2016. p. 91–115.

[30] Weijma J, Gubbels F, Hulshoff Pol LW, Stams A, Lens P, Lettinga G. Competition for H2 between sulfate reducers, methanogens and homoacetogens in a gas-lift reactor. Water Sci Technol 2002;45(10):75–80.

[31] RAG. Underground Sun Storage (USS): final report public. 2020.

[32] Amigán P, Greksak M, Kozánková J, Buzek F, Onderka V, Wolf I. Methanogenic bacteria as a key factor involved in changes of town gas stored in an underground reservoir. FEMS 1990;6(3).

[33] Gniese C, Bombach P, Rakoczy J, Hoth N, Schlömann M, Richnow H-H, Krüger M. Relevance of deep-subsurface microbiology for underground gas storage and geothermal energy production. Advances in biochemical engineering/biotechnology 2014;142:95–121.

[34] Buzek F, Onderka V, Vancura P, Wolf I. Carbon isotope study of methane production in a town gas storage reservoir. Fuel 1994;73(5):747–52.

[35] Yin W, Wang Y, Liu L, He J. Biofilms: the microbial "protective clothing" in extreme environments. Int J Mol Sci 2019;20(14):3423.

[36] Khajooie S, Gaus G, Dohrmann AB, Krüger M, Littke R. Methanogenic conversion of hydrogen to methane in reservoir rocks: an experimental study of microbial activity in water-filled pore space. Int J Hydrogen Energy 2024;50:272–90.

[37] Mura J, Ranchou-Peyruse M, Guignard M, Haddad PG, Ducoussou M, Casteran F, Sénéchal P, Larregieu M, Isaire M-P, Moonen P, Le Hécho I, Hoareau G, Baldy A, Lafont A, Petit A, Chiquet P, Caumette G, Cézac P, Ranchou-Peyruse A. Comparative study of three H2 geological storages in deep aquifers simulated in high-pressure reactors, vol. 63; 2024. p. 330–45. 0360-3199.

[38] Ranchou-Peyruse M, Guignard M, Chiquet P, Caumette G, Cézac P, Ranchou-Peyruse A. Assessment of the in situ biomethanation potential of a deep aquifer used for natural gas storage. FEMS (Fed Eur Microbiol Soc) Microbiol Ecol 2024; 100(6).

[39] Martinez F, Hey R. Mantle melting, lithospheric strength and transform fault stability: insights from the North Atlantic. Earth Planet Sci Lett 2022;579:117351.

[40] Aloisi G, Guibourdenche L, Natalicchio M, Caruso A, Haffert L, El Kilany A, Dela Pierre F. The geochemical riddle of “low-salinity gypsum” deposits. Geochem Cosmochim Acta 2022;327:247–75.

[41] Borgomano JVM, Pimienta L, Fortin J, Guéguen Y. Dispersion and attenuation measurements of the elastic moduli of a dual-porosity limestone. J Geophys Res Solid Earth 2017;122(4):2690–711.

[42] Clapeyron B-PE. Mémoire sur la puissance motrice de la chaleur. Journal de L'Ecole Royale Polytechnique 1834;0:153–90.

[43] Cord-Ruwisch R. A quick method for the determination of dissolved and precipitated sulfides in cultures of sulfate-reducing bacteria. 1985.

[44] Brunauer S, Emmett PH, Teller E. Adsorption of gases in multimolecular layers. J Am Chem Soc 1938;60(2):309–19.

[45] Tjørve KMC, Tjørve E. The use of Gompertz models in growth analyses, and new Gompertz-model approach: an addition to the Unified-Richards family. PLoS One 2017;12(6):e0178691.

[46] Herlemann DP, Labrenz M, Jürgens K, Bertilsson S, Waniek JJ, Andersson AF. Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea. The ISME journal 2011;5(10):1571–9.

[47] Quince C, Lanzen A, Davenport RJ, Turnbaugh PJ. Removing noise from pyrosequenced amplicons. BMC Bioinf 2011;12:38.

[48] Gantner S, Andersson AF, Alonso-Sáez L, Bertilsson S. Novel primers for 16S rRNA-based archaeal community analyses in environmental samples. J Microbiol Methods 2011;84(1):12–8.

[49] Apprill A, McNally S, Parsons R, Weber L. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat Microb Ecol 2015;75(2):129–37.

[50] Andrews S. FastQC: a quality control tool for high throughput sequence data. 2010.

[51] Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 2016;32(19): 3047–8.

[52] Escudié F, Auer L, Bernard M, Cauquil L, Maman S, Mariadassou M, Combes S, Hernandez Raquet G, Pascal G. FROGS training on Galaxy : metagenomics november 2017. 2017.

[53] Rognes T, Flouri T, Nichols B, Quince C, Mahé F. VSEARCH: a versatile open source tool for metagenomics. PeerJ 2016;4:e2584.

[54] Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal. 2011;17(1):10.

[55] Mahé F, Rognes T, Quince C, Vargas C de, Dunthorn M. Swarm: robust and fast clustering method for amplicon-based studies. PeerJ 2014;2:e593.

[56] Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI, Knight R, Mills DA, Caporaso JG. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat Methods 2013;10(1):57–9.

[57] Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. 2017.

[58] Dopffel N, Mayers K, Kedir A, Alagic E, An-Stepec BA, Djurhuus K, Boldt D, Beeder J, Hoth S. Microbial hydrogen consumption leads to a significant pH increase under high-saline-conditions- implications for hydrogen storage in salt caverns. 2023.

[59] Ye R, Jin Q, Bohannan B, Keller JK, Bridgham SD. Homoacetogenesis: a potentially underappreciated carbon pathway in peatlands. Soil Biol Biochem 2014;68: 385–91.

[60] Kotsyurbenko OR, Glagolev MV, Nozhevnikova AN, Conrad R. Competition between homoacetogenic bacteria and methanogenic archaea for hydrogen at low temperature. FEMS (Fed Eur Microbiol Soc) Microbiol Ecol 2001;38(2–3):153–9.

[61] Conrad R. Complexity of temperature dependence in methanogenic microbial environments. Front Microbiol 2023;14:1232946.

[62] Dar SA, Kleerebezem R, Stams AJM, Kuenen JG, Muyzer G. Competition and coexistence of sulfate-reducing bacteria, acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio. Appl Microbiol Biotechnol 2008;78(6):1045–55.

[63] Dohrmann AB, Krüger M. Microbial H2 consumption by a formation fluid from a natural gas field at high-pressure conditions relevant for underground H2 storage. 2023.

[64] Robinson JA, Tiedje JM. Competition between sulfate-reducing and methanogenic bacteria for H2 under resting and growing conditions. Arch Microbiol 1984;137(1): 26–32.

[65] Achtlich C, Bak F, Conrad R. Competition for electron donors among nitrate reducers, ferric iron reducers, sulfate reducers, and methanogens in anoxic paddy soil. Biol Fertil Soils 1995;19(1):65–72.

[66] Stark WH, Stadler J, McCoy E. Some factors affecting the bacterial population of freshwater lakes. 1938.

[67] Dang H, Lovell CR. Microbial surface colonization and biofilm development in marine environments. Microbiology and molecular biology reviews : MMBR (Microbiol Mol Biol Rev) 2016;80(1):91–138.

[68] Milán Z, Villa P, Sánchez E, Montalvo S, Borja R, Ilangovan K, Briones R. Effect of natural and modified zeolite addition on anaerobic digestion of piggery waste. Water Sci Technol 2003;48(6):263–9.

[69] Hernández-Eugenio G, Fardeau M-L, Cayol J-L, Patel BKC, Thomas P, Macarie H, García J-L, Ollivier B. Clostridium thiosulfatreducens sp. nov., a proteolytic, thiosulfate- and sulfur-reducing bacterium isolated from an upflow anaerobic sludge blanket (UASB) reactor. Int J Syst Evol Microbiol 2002;52(Pt 5):1461–8.

[70] Kotelnikova S, Pedersen K. Distribution and activity of methanogens and homoacetogens in deep granitic aquifers at Å.spÅ Hard Rock Laboratory, Sweden. FEMS (Fed Eur Microbiol Soc) Microbiol Ecol 1998;26(2):121–34.