# Real time monitoring of hydrogenotrophic methanogenesis under deep saline aquifers conditions

## TL;DR

Researchers developed a high-pressure reactor to study how a methanogenic microbe responds to CO2 and H2 under deep underground conditions, finding that methane production increases with pressure and stirring affects growth.

## Contribution

A new optically transparent high-pressure reactor was developed to study microbial growth under deep underground conditions without decompression biases.

## Key findings

- Methane production increased with higher CO2 and H2 partial pressures up to 15 bar for unstirred and 30 bar for stirred conditions.
- Biofilm formation under unstirred conditions favored methane production and extended the range of H2/CO2 partial pressures for growth.
- The reactor allows studying deep microbial strains under natural pressure conditions, eliminating decompression biases.

## Abstract

To investigate the microbial response to deep underground gas injection, specifically CO2 and H2, a new optically transparent high-pressure reactor was developed to monitor autotrophic microbial growth via in situ and ex situ characterization techniques. The main advantages rely on avoiding any decompression phases during the entire process, thanks to direct optical access. Here, we monitored the growth of the model methanogenic strain Methanothermococcus thermolithotrophicus by applying different H2/CO2 partial pressures at a total pressure of 100 bar, which is representative of the deep underground storage environment. These reactors can be used to investigate deep microbial strains under pressure conditions close to their natural environments, eliminating decompression biases. Additionally, we measured the methane production of the strain at the end of the incubation, with or without stirring. The results showed an increase in methane production with increasing CO2 and H2 partial pressures, up to p(H2/CO2, 80/20 mol%) = 15 bar for unstirred conditions and p(H2/CO2, 80/20 mol%) = 30 bar for stirred conditions, respectively. This difference in behavior depending on stirring conditions was clearly observed through biofilm formation in the absence of stirring, favoring methane production and extending the range of H2 / CO2 partial pressures under which the strain can grow.

The online version contains supplementary material available at 10.1186/s13036-025-00584-y.