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Article

Mitigating Microbial Artifacts in Laboratory Research on Underground Hydrogen Storage

by
Adnan Aftab
1,*,
Silvia J. Salgar-Chaparro
2,
Quan Xie
1,
Ali Saeedi
1 and
Mohammad Sarmadivaleh
1,*
1
Discipline of Energy Engineering, WA School of Mines: Minerals, Energy and Chemical Engineering, Curtin University, 26 Dick Perry Avenue, Kensington, WA 6151, Australia
2
Curtin Corrosion Centre, WA School of Mines: Minerals, Energy and Chemical Engineering, Curtin University, Bentley, WA 6151, Australia
*
Authors to whom correspondence should be addressed.
Fuels 2025, 6(3), 52; https://doi.org/10.3390/fuels6030052
Submission received: 28 March 2025 / Revised: 30 May 2025 / Accepted: 10 June 2025 / Published: 1 July 2025
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Abstract

The global energy sector is aiming to substantially reduce CO2 emissions to meet the UN climate goals. Among the proposed strategies, underground storage solutions such as radioactive disposal, CO2, NH3, and underground H2 storage (UHS) have emerged as promising options for mitigating anthropogenic emissions. These approaches require rigorous research and development (R&D), often involving laboratory-scale experiments to establish their feasibility before being scaled up to pilot plant operations. Microorganisms, which are ubiquitous in laboratory environments, can significantly influence geochemical reactions under variable experimental conditions of porous media and a salt cavern. We have selected a consortium composed of Bacillus sp., Enterobacter sp., and Cronobacter sp. bacteria, which are typically present in the laboratory environment. These microorganisms can contaminate the rock sample and develop experimental artifacts in UHS experiments. Hence, it is pivotal to sterilize the rock prior to conduct experimental research related to effects of microorganisms in the porous media and the salt cavern for the investigation of UHS. This study investigated the efficacy of various disinfection and sterilization methods, including ultraviolet irradiation, autoclaving, oven heating, ethanol treatments, and gamma irradiation, in removing the microorganisms from silica sand. Additionally, the consideration of their effects on mineral properties are reviewed. A total of 567 vials, each filled with 9 mL of acid-producing bacteria (APB) media were used to test killing efficacy of the cleaning methods. We conducted serial dilutions up to 10−8 and repeated them three times to determine whether any deviation occurred. Our findings revealed that gamma irradiation and autoclaving were the most effective techniques for eradicating microbial contaminants, achieving sterilization without significantly altering the mineral characteristics. These findings underscore the necessity of robust cleaning protocols in hydrogeochemical research to ensure reliable, reproducible data, particularly in future studies where microbial contamination could induce artifacts in laboratory research.

1. Introduction

The introduction of geo-solutions is crucial in the energy transition phase to achieve carbon neutrality targets via compressed air, anthropogenic CO2, helium, and UHS [1,2,3]. Microorganisms, which are widely distributed in both surfaces and subsurface environments, can introduce uncertainties and risks to long-term implementations of UHS [4]. To assess the potential performance of reservoir rocks for UHS, various reservoir engineering experiments have been conducted, including measurement of porosity [5], permeability [5], capillary pressure [2], interfacial tension [2], H2-brine core flooding, wettability [6], and in situ loss of H2 and H2S generation [7,8]. However, microbial contamination from laboratory surfaces, such as benches and equipment surfaces or airborne bacteria, can compromise these experiments, leading to artifacts and unreliable data [9,10].
The effective sterilization of rock samples is crucial to mitigate microbial contamination without compromising the integrity of geological material. Importantly, little information is available regarding the microbial populations residing within the underground reservoir rock samples. Sterilized samples must be stored in controlled environments, such as isolation rooms or microbial-free zones, to prevent recontamination during further testing. There have been a variety of studies published which might not adopt the right cleaning technique for the rock for UHS research. Ali et al. conducted multiple studies and examined the wettability of rock. Nevertheless, the rock was cleaned with deionized water and surface residues were removed using the ultrapure nitrogen [11,12]. Thus, inadequate cleaning of the rock may fail to sterilize it and can introduce artifacts during wettability measurements. The presence of microorganisms could alter the wettability and other reservoir properties, such as permeability and porosity [13,14]. Therefore, the proper sterilization of the rock prior to the wettability, interfacial tension, and coreflooding tests is important to understand the UHS system.
Studies of Martian UV flux provide insight into sterilization challenges. On Mars, perchlorates exhibit bactericidal properties when exposed to UV radiation, including UVC (<280 nm) and UVB (280 to 315 nm) [15]. Furthermore, two components of the Red Planet, including iron oxides and hydrogen peroxide, induce a synergetic effect with irradiated perchlorates, causing a 10.8-fold increase in cell death for Bacillus subtilis compared to cells only exposed to UV rays for 60 s [16]. Despite these findings, the complete sterilization of rock samples using UV radiation remains a challenge as the irregular shapes and rough surfaces of rocks hinder UV penetration, leaving microorganisms within cracks and crevices unaffected [17].
Chemical disinfectants capable of penetrating microcapillaries may be required. However, chemical treatments, such as 70% and 96% ethanol, have shown limited effectiveness in creating aseptic conditions within deep cracks in limestone [18]. Furthermore, interactions between environmental microbes and rock surfaces can induce calcite dissolution and precipitation, potentially altering the rock’s mineralogy. In an overlooked phenomenon, it was illustrated that the entombment of microorganisms in a Si-rich precipitate in a nutrient-depleted environment indicates Si mobilization at ambient conditions [18]. These interactions underscore the need for sterilization methods that preserve the mineralogical integrity of geological samples.
Previous studies have employed autoclaving (~121 °C, 15 psi, and 30 min) as a sterilization method for geological samples [14,19,20]. However, it is reported that microorganisms can penetrate Berea sandstone rock quicker when sterilized by autoclaving. Additionally, the autoclaved sample showed an increased chloride content compared to dry heating, leading to aggregation and an uneven morphology in clay minerals [21]. For Mars analog rocks and minerals, gamma irradiation has been used as an alternative sterilization method. While high doses of gamma rays did not significantly affect the structural integrity of the rocks, some minerals exhibited darkening due to gamma radiation exposure. Despite this, gamma irradiation is considered a viable option for sterilizing the Mars-returned rock samples [22].
There is a need to develop a scientific approach for the sterilization of rocks intended for UHS research applications. Overall, oven heating, autoclaving, ethanol treatment, and gamma irradiation are promising candidates for microbial-focused laboratory environments. Their application can minimize microbial artifacts and ensure the reliability of experimental results in H2 and CO2 geological storage studies. In this study, we primarily focused on the effects of rock sterilization for UHS investigations. Nevertheless, given the wide range of existing literature on rock sterilization techniques for astrobiology applications, we have drawn upon these data to provide a comparative analysis of different sterilization techniques, including their merits and demerits (Table 1).

2. Materials and Methods

Figure 1 illustrates the research methodology used in this study to investigate the effects of different sterilization techniques on pre-inoculated silica sand. Silica sand (super fine) was purchased from Cook Industrial Minerals, WA, Australia. Table 2 lists the techniques and factors used to sterilize the silica sand mineral.

2.1. Sand Sample Preparation

To simulate core rock samples, 10 mL sterile glass vials were filled with 15.7 g of sand. Sand was aseptically transferred into each vial using sterile spatulas. A defined bacterial consortium composed of Bacillus sp., Enterobacter sp., and Cronobacter sp. was used in this study to recreate microbial contamination. Each strain was cultivated independently in separate vials under optimal growth conditions to obtain active cultures. From each culture, 1 mL was transferred into a 50 mL falcon tube containing PBS solution, resulting in a final bacterial concentration of ~107 cells/mL. 3 mL of this cell suspension were then used to inoculate the sand filled vials. Table S1 presents chemical composition of PBS.

2.2. Sterilization Methods

UV irradiation: Three vials of sandstone–bacteria–PBS inoculum were placed inside the biosafety cabinet. The UV light was turned on, and the cabinet windows were covered to prevent external light exposure. UV irradiation was conducted for 30 min, each vial was rotated ~180° clockwise every 15 min. The rubber seal were removed during this process to allow maximum UV penetration.
Ethanol washing: ethanol was used at two different concentrations to sterilize the rock sample. The 95% and 75% ethanol solutions were prepared using ethanol pure 99.9%. A total of 95 mL of ethanol and 5 mL of water were added to a 200 mL regent bottle to make a solution of Ethanol 95 wt%. Similarly, 75 wt% ethanol was prepared using 75 mL of pure ethanol and 35 mL of water. Three sandstone–bacteria–PBS inoculum vials were washed three times with 95% ethanol, and another 3 vials were washed with 75% ethanol. The ethanol was not completely removed from the vials, so that the sandstone may remain soaked for at least 15 min.
Oven heating: A Venticell 111-Eco line oven was used to sterilize the sandstone–bacteria–PBS inoculum vials. The oven was switched on and preheated until the set temperature reached 200 °C. Three sandstone–bacteria–PBS inoculum vials were placed in the oven for approximately 2 h at a constant 200 °C.
Autoclave: the benchtop autoclave model 3870EL-D was used. Three sandstone–bacteria–PBS inoculum vials were autoclaved using the liquid cycle at 121 °C, 15 psia pressure for 30 min.
Gamma irradiation: three vials of sand–bacteria–PBS inoculum samples were provided to ChemCenter Government of Western Australia at Curtin University Campus for the irradiation of the gamma ray. The samples were irradiated for at least 32 h. The center is equipped with a gamma irradiation unit named Gammacell 220.

2.3. Microbial Quantification

The most probable number (MPN) method was employed to evaluate the efficacy of various sterilization techniques in eliminating microorganisms from rock samples. This method involves a series of dilutions and inoculation into a suitable culture medium to estimate the concentration of viable microorganisms in the sample. The culture medium used for microbial recovery was red phenol broth which supports the growth of a broad spectrum of microorganisms. The media was prepared according to standard protocols, sterilized, and poured into sterile vials (Figure 2A–C). Table S2 provides the chemical composition of culture media APB.
An amount of 2 g of the sterilized samples were inoculated into falcon tubes containing 20 mL of PBS solution. Tubes were sonicated in cycles of 15 s on and 15 s off to detach cells from the silica sand. Tubes were vortexed and 1 mL aliquots were used to inoculate vials containing the red phenol medium. Samples were processed in triplicate with a dilution factor of 10−8. After inoculation, the vials were incubated at 40 °C for 3 weeks and microbial growth was recorded using colorimetric change in the media.

3. Results and Discussions

Figure 3A illustrates the concentration of surviving cells in the silica sand after sterilization process. The control (not subjected to sterilization) indicated that the concentration of cells in the test bottles was in the order of average 107 cell/g before sterilization treatment. Most probable number (MPN) results indicate that autoclave, oven heating, and gamma irradiation were able to eliminate all cells in the sand. These findings were well matched with those of previous studies [35,36]. However, oven heating, autoclave, and gamma rays could induce mineralogical changes. For instance, oven heating is reported for microcracking in quartz mineralogy of sand [32]. Gamma rays caused the darkening of quartz mineral (Figure 4) in silica sand, which is in line with previous findings [22].
Figure 4 illustrates that the glass of serum vials and sand changed to a blackish color after treating with gamma irradiation. These changes could affect the in-situ reservoir properties, in particular porosity, permeability, interfacial tension, capillary pressure, and wettability at the micron scale, resulting in adverse effects on the laboratory research for geological H2 storage.
Ethanol and UV radiation were not able to inactivate all the cells and a concentration between 105 to 107 cells/g surviving bacteria was still detected. Our results provide a clear comparison of the performance of cell sterilization with that of the different techniques. UV sterilization shows no effect on the sterilization of sand. We found that ethanol concentrations, including 75 wt% and 95 wt%, illustrate the lowest killing efficiency; thus, more living cells were detected compared to the other sterilization methods evaluated. (Figure 3B). This figure shows the killing efficiency of each sterilization method. UV irradiation was an inefficient technique, with a killing efficiency of 0%. This finding may be attributed to less penetration of UV from a glass of the vial into the rock. Notably, 75% ethanol achieved a killing efficiency of 96.3%, whereas 95% ethanol reached a killing efficiency of 99.2% (Figure 3B). Although killing efficiency values are high, it is important to consider that the surviving population of cells was also high, in the order of 106 and 105, respectively. This survival percentage could restore microbial activity in long-time core flooding and salt cavern bioreactor experimental setups under the influence of anaerobic conditions. Additionally, the effects of precipitation, changes in minerology, and brittle behavior of the formation were reported in calcite, clay, and sand, respectively, after the use of ethanol as a sterilizing substance. Figure 5A,B illustrates the total number of vials incubated after inoculation with cell suspension. Table S3 provides data of different sterilization methods, MPN, and dilution series.
Overall, physical methods, such as UV irradiation, oven heating, and autoclaving, are widely used for different kinds of sterilization. UV irradiation revealed a not considerable level of sterilization. UV can be useful for the sterilization of surface and air. Nevertheless, the limited penetration depth restricts its effectiveness for bulk sterilization. Gamma irradiation was very effective and a very high sterilization. Gamma irradiation damages cellular components, such as DNA, which results in microbial killing. This is a very effective technique with strong penetration capability. Similarly, autoclave revealed very strong sterilization. Autoclave shows a variety of merits, such as faster cycle time, in-house control and accessibility, lower cost, and less impact on the properties of materials in comparison to gamma irradiation. Autoclave disrupts bacterial proteins and cellular structure, leading to better and rapid sterilization.

4. Conclusions

Currently, underground hydrogen storage (UHS) experiments are being carried out, often impacting the integrity of rock samples by introducing a variety of anthropogenic microorganisms. In response, we have developed a laboratory protocol and procedure for rock sterilization. Our experiments demonstrated that the eradication of microorganisms can be achieved in UHS experiments when differentiating between biological activity and abiotic processes is critical. This research provides valuable information to safeguard the accuracy and reliability of future hydrogen geological laboratory-scale experiments. UV irradiation was not an efficient technique, with a killing efficiency of 0%. This finding may be attributed to lower penetration of UV into the rock. Notably, 75% ethanol achieved a killing efficiency of 96.3%, whereas 95% ethanol reached a killing efficiency of 99.2%. We conclude that autoclave process, oven heating, and gamma irradiation methods achieved 100% killing efficiency and absolutely eliminate microbial life from the rock. However, gamma irradiation caused the discoloration of sand, and oven heating may induce micro-cracks, potentially compromising the integrity of the sand. Therefore, we recommend the autoclave process as the most suitable sterilization method due to its relatively low operating temperature and pressure, which are well suited for sand sterilization. Further research is needed to assess the impact of these sterilization methods on the mineralogy of rocks, including petrophysical properties and surface behavior for large-scale geological hydrogen storage experiments.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fuels6030052/s1, Table S1: Chemical composition of PBS 1000 mL; Table S2: Chemical composition of culture media APB 1000 mL; Table S3: provides data of different sterilization methods, MPN, and dilution series.

Author Contributions

Conceptualization, A.A. and M.S.; methodology, S.J.S.-C.; software, A.A.; validation, S.J.S.-C.; formal analysis, S.J.S.-C. and M.S.; investigation, A.A.; resources, M.S. and A.S.; data curation, A.A.; writing—original draft preparation, A.A.; writing—review and editing, A.A. and S.J.S.-C.; visualization, A.A. and M.S.; supervision, M.S., S.J.S.-C. and Q.X.; project administration, M.S. and A.S.; funding acquisition, M.S. All authors have read and agreed to the published version of the manuscript.

Funding

The authors would like to thank Curtin University, Australia and Government of Australia for funding the first author’s PhD scholarship through the Research Training Program.

Data Availability Statement

All the data included in this study is presented in the manuscript.

Acknowledgments

We would like to extend our sincere thanks to Adeeba Hussain for her invaluable assistance with the experimental work conducted in the B601 MIC Laboratory at the Curtin Corrosion Centre, Australia.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fuels 06 00052 g001
Figure 1. Research methodology: 1. preparation of pre-inoculated silica sand mineral and exposure to different sterilization methods; 2. sampling of treated silica sand; 3. resuspension of silica sand in PBS solution; 4–5. cell detachment by ultrasonication; 6. rapid mixing of samples using the vortex machine; 7–8. aliquot for the most probable number quantification; and finally, 9. incubation of the culture media to grow surviving microorganisms.
Figure 1. Research methodology: 1. preparation of pre-inoculated silica sand mineral and exposure to different sterilization methods; 2. sampling of treated silica sand; 3. resuspension of silica sand in PBS solution; 4–5. cell detachment by ultrasonication; 6. rapid mixing of samples using the vortex machine; 7–8. aliquot for the most probable number quantification; and finally, 9. incubation of the culture media to grow surviving microorganisms.
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Figure 2. (A) Schematic of the preparation of anaerobic culture medium. (B) Images show deoxygenation of 10 mL vials and (C) APB stock solution deoxygenating with N2 gas.
Figure 2. (A) Schematic of the preparation of anaerobic culture medium. (B) Images show deoxygenation of 10 mL vials and (C) APB stock solution deoxygenating with N2 gas.
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Figure 3. Effect of different techniques on the sterilization of the sand: (A) bacteria concentration in cells/g, and (B) bacteria-killing efficiency in percentage.
Figure 3. Effect of different techniques on the sterilization of the sand: (A) bacteria concentration in cells/g, and (B) bacteria-killing efficiency in percentage.
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Figure 4. Illustrated difference between color of control vials (replicates A,B,C) (left), and gamma rays treated vials (replicates A,B,C) (right).
Figure 4. Illustrated difference between color of control vials (replicates A,B,C) (left), and gamma rays treated vials (replicates A,B,C) (right).
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Fuels 06 00052 g005aFuels 06 00052 g005b
Figure 5. (A) Control from left ran parallel with 95 wt% ethanol sterilization. (A,B) Another control ran for 75 wt% ethanol, oven heating experiments, UV, and autoclave sterilization techniques. (B) The second last control was run parallel to the Gamma ray. There are 567 vials, each 10 mL, as illustrated in the figure. We conducted serial dilutions up to 10−7, and repeated it three times to determine if any deviation occurred in the values. Note: Red color vials show no growth. Yellow color vials illustrate positive growth of microorganisms in the APB media solution.
Figure 5. (A) Control from left ran parallel with 95 wt% ethanol sterilization. (A,B) Another control ran for 75 wt% ethanol, oven heating experiments, UV, and autoclave sterilization techniques. (B) The second last control was run parallel to the Gamma ray. There are 567 vials, each 10 mL, as illustrated in the figure. We conducted serial dilutions up to 10−7, and repeated it three times to determine if any deviation occurred in the values. Note: Red color vials show no growth. Yellow color vials illustrate positive growth of microorganisms in the APB media solution.
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Table 1. Effects of different sterilization techniques on the minerology of rocks related to geoscience and astrobiology applications.
Table 1. Effects of different sterilization techniques on the minerology of rocks related to geoscience and astrobiology applications.
Sterilization TechniqueOverall Effect on MineralogyGeoscience ApplicationsAstrobiology ApplicationsStudy
UV
  • Unpenetrated to core of the rock
  • Unpenetrated UV radiation could not produce the expected results due to the irregular shape of the rock and rough surface.
  • It was observed that only one side of the rock could be irradiated at a time and required a change in position of the sample during exposure to UV.
  • A photochemical system consisting of four circular UV lamps allows adequate sterilization of rough surfaces of geological material while killing the microorganism possibly living in the core sample.
  • Martian UV flux made surface of perchlorates bactericidal.
  • The surface of the planet is exposed to both UVC radiation of <280 nm and UVB of 280 to 315 nm when compared to surface of the Earth.
  • Iron oxides and hydrogen peroxide induce a synergetic effect with irradiated perchlorates causing a 10.8-fold increase in cell death of Bacillus subtilis compared to cells exposed by UV rays for 60 s
  • However, the absolute killing of bacteria from the rock remains a challenge through UV sterilization.
[15,16,17]
75% ethanol and
95% ethanol
  • Calcite can precipitate
  • Clay minerology can change
  • Brittle film formed on sandstone
  • High-Mg calcite can precipitate at ambient temperatures by partially replacing water with 75% ethanol; additionally, it bypasses the hydration barrier.
  • Ethanol concentration could change the polymorph of calcite, vaterite, or aragonite in Mg-free environment.
  • The specific polarizability decreases as ethanol concentration increased from 0, 10, and 20% v/v. Ethanol concentration on clay-driven polarization is relative to changes in clay minerology.
  • Ethanol does not develop alkoxysilane–sandstone compatibility. Ethanol formed a brittle film.
  • Ethanol was used instead of water to prevent interaction of clay–water cation exchange reactions prior to starting experiment.
Nil[23,24,25,26,27,28]
SolarizationSoil
  • Solarization was conducted through covering the soil with sterile transparent plastic film to trap solar radiation for heating which could rise temperature above 70 °C and considered as an adequate range to kill a variety of plant pathogens.
Nil[29]
Oven heating
  • Micro cracking in quartz sandstone at very high temperature
  • Micro-cracking in quartz sandstone at the grain boundaries revealed increase in the porosity at 600 °C and within the grains and mineralogical changes at 750 °C.
  • The structure of clay mineral collapse at 600 °C and chlorite above 600 °C. The high temperature might affect the physical properties of mineral structure [30,31].
Nil[32]
Autoclave
  • Could increase bacteria penetration in porous rock
  • Steam treatment provides a better solution to clean pests in soil.
Nil[33]
Gamma Irradiation
  • Very low, discoloration of the rock
Physico-chemical properties of natural sediments:
  • Major part of clay mineralogy unchanged
  • pH slightly changes after irradiation
  • Irradiation samples reduced the cation exchange capacity
  • Irradiated samples reduced iron oxide
  • Effects on organic and inorganic fractions were observed
Mars-returned rock samples sterilization:
  • Gamma photons from 60Co (1.17 and 1.33 MeV)
  • Doses as high as 3 × 107 rads
  • No effect detected on basalt rock and quartz mineral
  • No change in concentration of elements before and after irradiation
  • Crystal structure of mineral not affected
  • No change in grain density except a small effect on halite
  • Specific surface area of rock was not affected
  • Dose induces no radioactivity in the rock
  • No change in their isotopic composition
  • No change in chemical composition
  • No change in crystallographic structure
  • Effects on the visible and near-infrared spectral region
  • Discoloration of quartz
  • Darkening of quartz and halite
  • Increases thermoluminescence of quartz and plagioclase
[22,34]
Table 2. Sterilization methods and parameters used in this study.
Table 2. Sterilization methods and parameters used in this study.
TechniquesTemperaturePressureIntensityExposure TimeConcentration
UV irradiationAmbientAmbient280 to 100 nm30 min both sidesNA
Autoclave121 °C15 psiNA30 minNA
Oven heating200 °CAmbientNA2 hNA
Ethanol washingAmbientAmbientNA15 min × 3 times75 wt%
95 wt%
Gamma irradiationNANANA32 hNA
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Aftab, A.; Salgar-Chaparro, S.J.; Xie, Q.; Saeedi, A.; Sarmadivaleh, M. Mitigating Microbial Artifacts in Laboratory Research on Underground Hydrogen Storage. Fuels 2025, 6, 52. https://doi.org/10.3390/fuels6030052

AMA Style

Aftab A, Salgar-Chaparro SJ, Xie Q, Saeedi A, Sarmadivaleh M. Mitigating Microbial Artifacts in Laboratory Research on Underground Hydrogen Storage. Fuels. 2025; 6(3):52. https://doi.org/10.3390/fuels6030052

Chicago/Turabian Style

Aftab, Adnan, Silvia J. Salgar-Chaparro, Quan Xie, Ali Saeedi, and Mohammad Sarmadivaleh. 2025. "Mitigating Microbial Artifacts in Laboratory Research on Underground Hydrogen Storage" Fuels 6, no. 3: 52. https://doi.org/10.3390/fuels6030052

APA Style

Aftab, A., Salgar-Chaparro, S. J., Xie, Q., Saeedi, A., & Sarmadivaleh, M. (2025). Mitigating Microbial Artifacts in Laboratory Research on Underground Hydrogen Storage. Fuels, 6(3), 52. https://doi.org/10.3390/fuels6030052

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