Periodic Reporting for period 1 - MinOx (Geomicrobiology of Fe(II)-bearing Minerals and Nitrate-Reducing Iron-Oxidizing microbial cultures)
Reporting period: 2024-05-01 to 2026-04-30
Summary of the context and overall objectives of the project
Nitrate pollution is one of the most widespread threats to groundwater quality in Europe and worldwide, largely driven by intensive agriculture and insufficient natural attenuation. In many aquifers, nitrate concentrations exceed safe limits, posing risks to drinking water supplies and ecosystem health. At the same time, subsurface environments often lack organic carbon, which limits the efficiency of conventional microbial nitrate removal processes.
In this context, alternative pathways for nitrate attenuation are increasingly important. One such pathway involves microorganisms that can use iron minerals as an energy source to remove nitrate from water. These microorganisms, known as nitrate-reducing Fe(II)-oxidizing (NRFeOx) microbes, play a key role in linking the iron, nitrogen and carbon cycles in anoxic environments. However, despite their environmental relevance, the mechanisms that allow these microbial communities to function and remain active under natural conditions are still poorly understood.
The MinOx project addresses this knowledge gap by investigating how NRFeOx microbial communities interact with iron-bearing minerals and how these interactions control mineral transformation and nutrient cycling in the subsurface. The project combines laboratory experiments, advanced microscopy and molecular biology techniques to identify which microorganisms are involved, how they grow on mineral surfaces, and how they influence the chemical reactivity of iron. Another key objective is to understand how these microorganisms compete with abiotic (non-biological) reactions and with other microbial processes, particularly under conditions where multiple energy sources are available. By clarifying these mechanisms, the project aims to provide a more complete picture of how iron and nitrate are transformed in natural environments.
The expected impact of the project is to improve our understanding of microbially driven processes that contribute to nitrate removal in groundwater systems, especially under low-organic-carbon conditions. This knowledge can support the development of more accurate biogeochemical models and inform future strategies for sustainable groundwater management and pollution mitigation. In the longer term, the project contributes to broader environmental goals related to water quality, climate-relevant biogeochemical cycles, and the sustainable use of subsurface resources.
In this context, alternative pathways for nitrate attenuation are increasingly important. One such pathway involves microorganisms that can use iron minerals as an energy source to remove nitrate from water. These microorganisms, known as nitrate-reducing Fe(II)-oxidizing (NRFeOx) microbes, play a key role in linking the iron, nitrogen and carbon cycles in anoxic environments. However, despite their environmental relevance, the mechanisms that allow these microbial communities to function and remain active under natural conditions are still poorly understood.
The MinOx project addresses this knowledge gap by investigating how NRFeOx microbial communities interact with iron-bearing minerals and how these interactions control mineral transformation and nutrient cycling in the subsurface. The project combines laboratory experiments, advanced microscopy and molecular biology techniques to identify which microorganisms are involved, how they grow on mineral surfaces, and how they influence the chemical reactivity of iron. Another key objective is to understand how these microorganisms compete with abiotic (non-biological) reactions and with other microbial processes, particularly under conditions where multiple energy sources are available. By clarifying these mechanisms, the project aims to provide a more complete picture of how iron and nitrate are transformed in natural environments.
The expected impact of the project is to improve our understanding of microbially driven processes that contribute to nitrate removal in groundwater systems, especially under low-organic-carbon conditions. This knowledge can support the development of more accurate biogeochemical models and inform future strategies for sustainable groundwater management and pollution mitigation. In the longer term, the project contributes to broader environmental goals related to water quality, climate-relevant biogeochemical cycles, and the sustainable use of subsurface resources.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
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.
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.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
This project has generated new knowledge on the role of nitrate-reducing Fe(II)-oxidizing (NRFeOx) microbial communities in the transformation of Fe(II)-bearing minerals under anoxic conditions. The main results can be summarized as follows:
First, we demonstrated that NRFeOx enrichment cultures are able to grow using Fe(II)-bearing minerals, including naturally occurring crystalline pyrite and magnetite, as electron donors under strictly anoxic conditions. These results provide experimental support for mineral-based NRFeOx as a biologically relevant process.
Second, the project showed that oxidation of Fe(II)-bearing minerals coupled to nitrate reduction is strongly dependent on microbial community composition. Different enrichment cultures displayed distinct Fe(II) oxidation and nitrate reduction dynamics, indicating that this metabolism is controlled by community interactions rather than being an intrinsic property of a single organism.
Finally, we established and optimized a correlative microscopy workflow combining fluorescence microscopy (FISH), Raman spectroscopy and scanning electron microscopy. This approach allows the direct spatial correlation between microbial identity and mineral transformation, providing mechanistic insights into microbe–mineral interactions at the microscale.
These findings have several potential impacts. At the environmental level, they improve our understanding of nitrate attenuation in anoxic, carbon-limited subsurface environments, where Fe(II)-bearing minerals are abundant. This is particularly relevant for groundwater systems affected by nitrate pollution. At the scientific level, the project advances the field of geomicrobiology by providing new mechanistic insights into microbially driven mineral transformations and by introducing methodological approaches that can be applied to other complex environmental systems.
The developed correlative microscopy workflow also has broader applicability for the study of biominerals and biosignatures, including potential applications in environmental monitoring and astrobiology.
To ensure further uptake and maximise the impact of these results, several key needs have been identified:
- Further research is required to elucidate the exact metabolic pathways and electron transfer mechanisms involved in mineral-based NRFeOx, particularly the role of individual community members and their interactions.
- Determining the presence, distribution, abundance and the composition of NRFeOx communities in natural environments is needed to assess the relevance of these processes under field conditions and their contribution to nitrate removal in natural aquifers.
- Integration into predictive models will be necessary to incorporate mineral-based NRFeOx into reactive transport and biogeochemical models used for environmental management.
Overall, the project contributes to bridging the gap between laboratory-based studies and environmental processes, opening new avenues for the understanding and potential application of microbially driven mineral transformations in natural and engineered systems.
First, we demonstrated that NRFeOx enrichment cultures are able to grow using Fe(II)-bearing minerals, including naturally occurring crystalline pyrite and magnetite, as electron donors under strictly anoxic conditions. These results provide experimental support for mineral-based NRFeOx as a biologically relevant process.
Second, the project showed that oxidation of Fe(II)-bearing minerals coupled to nitrate reduction is strongly dependent on microbial community composition. Different enrichment cultures displayed distinct Fe(II) oxidation and nitrate reduction dynamics, indicating that this metabolism is controlled by community interactions rather than being an intrinsic property of a single organism.
Finally, we established and optimized a correlative microscopy workflow combining fluorescence microscopy (FISH), Raman spectroscopy and scanning electron microscopy. This approach allows the direct spatial correlation between microbial identity and mineral transformation, providing mechanistic insights into microbe–mineral interactions at the microscale.
These findings have several potential impacts. At the environmental level, they improve our understanding of nitrate attenuation in anoxic, carbon-limited subsurface environments, where Fe(II)-bearing minerals are abundant. This is particularly relevant for groundwater systems affected by nitrate pollution. At the scientific level, the project advances the field of geomicrobiology by providing new mechanistic insights into microbially driven mineral transformations and by introducing methodological approaches that can be applied to other complex environmental systems.
The developed correlative microscopy workflow also has broader applicability for the study of biominerals and biosignatures, including potential applications in environmental monitoring and astrobiology.
To ensure further uptake and maximise the impact of these results, several key needs have been identified:
- Further research is required to elucidate the exact metabolic pathways and electron transfer mechanisms involved in mineral-based NRFeOx, particularly the role of individual community members and their interactions.
- Determining the presence, distribution, abundance and the composition of NRFeOx communities in natural environments is needed to assess the relevance of these processes under field conditions and their contribution to nitrate removal in natural aquifers.
- Integration into predictive models will be necessary to incorporate mineral-based NRFeOx into reactive transport and biogeochemical models used for environmental management.
Overall, the project contributes to bridging the gap between laboratory-based studies and environmental processes, opening new avenues for the understanding and potential application of microbially driven mineral transformations in natural and engineered systems.