The MinOx project has focused on understanding the mechanisms underlying nitrate-reducing Fe(II) oxidation (NRFeOx) and the interaction between microorganisms and Fe(II)-bearing minerals under anoxic conditions.
First, stable autotrophic NRFeOx enrichment cultures (KS, BP and AG) were maintained and further characterized under controlled laboratory conditions. These cultures, which represent three of the four systems where true NRFeOx has been demonstrated, were used as model systems to investigate microbial activity, community composition and functional roles when using Fe(II)-bearing minerals as energy sources. Particular attention was given to the dominant Fe(II)-oxidizing microorganisms affiliated with the genus Ca. Ferrigenium and their interaction with flanking community members.
A major part of the work addressed the capacity of these enrichment cultures to use Fe(II)-bearing minerals as electron donors. Batch incubation experiments were performed with different minerals, including pyrite, magnetite and mackinawite. These experiments demonstrated that both KS and BP cultures are able to grow using solid-phase Fe(II) minerals, with clear differences depending on the mineral type and the microbial flanking community. In particular, pyrite was shown to be actively solubilized and used as an energy source, coupled to nitrate reduction and microbial growth. Quantitative analyses of Fe(II)/Fe(III), nitrate consumption and cell abundance confirmed that mineral-based metabolism can sustain autotrophic growth under anoxic conditions.
Comparative experiments revealed distinct metabolic behaviors between enrichment cultures. For example, BP generally showed higher activity than KS when grown on minerals, and different Fe(II)-to-nitrate stoichiometries were observed, indicating variability in metabolic pathways and efficiencies. These results highlight the importance of community composition in controlling NRFeOx activity and suggest that flanking microorganisms play a key role in enabling or enhancing this metabolism.
In parallel, the role of microbial interactions was investigated using available genomic and community data. The results support a model in which Ca. Ferrigenium spp. depend on "flanking" microorganisms to complete denitrification steps and/or to mediate electron transfer processes. Evidence from enrichment cultures indicates that these interactions are essential to sustain Fe(II) oxidation coupled to nitrate reduction, reinforcing the concept of a community-driven metabolism rather than a purely organism-specific process.
Another key achievement of the project was the development and optimisation of a correlative microscopy workflow combining fluorescence microscopy (FISH), Raman spectroscopy and scanning electron microscopy (SEM). This workflow enables the direct linkage between microbial identity, mineralogy and spatial organisation at the microscale. Methodological challenges such as sample preparation, fluorescence preservation and compatibility between techniques were systematically addressed, resulting in a robust protocol for analysing microbe–mineral interactions in complex samples. Using this approach, it was possible to observe microbial attachment to mineral surfaces and to assess how different microorganisms interact with and potentially modify Fe(II)-bearing minerals. These observations provide direct evidence for spatial organisation and physical interactions that are critical for understanding electron transfer processes and mineral transformation mechanisms. In addition, mineral products resulting from microbial Fe(II) oxidation were characterised using advanced microscopy techniques, including high-resolution electron microscopy (e.g. STEM), allowing the identification of structural and morphological changes associated with microbial activity.
Overall, the work performed demonstrates that (i) NRFeOx microorganisms can use Fe(II)-bearing minerals as energy sources under anoxic conditions, (ii) microbial community composition strongly controls metabolic activity and efficiency, and (iii) microbe–mineral interactions occur at the microscale and can be partially resolved using correlative analytical approaches. These findings provide important insights into the coupling of the nitrogen, carbon and iron cycles in subsurface environments and establish a methodological framework to study these processes in detail.
