Nitrate removal in oligotrophic environments is often limited by the availability of suitable organic electron donors. Chemolithoautotrophic bacteria may play a key role in denitrification in aquifers depleted in organic carbon. Under anoxic and circumneutral pH conditions, iron(II) was hypothesized to serve as an electron donor for microbially mediated nitrate reduction by Fe(II)-oxidizing (NRFeOx) microorganisms. However, lithoautotrophic NRFeOx cultures have never been enriched from any aquifer and as such there are no model cultures available to study the physiology and geochemistry of this potentially environmentally relevant process. Using iron(II) as an electron donor, we enriched a lithoautotrophic NRFeOx culture from nitrate-containing groundwater of a pyrite-rich limestone aquifer. In the enriched NRFeOx culture that does not require additional organic co-substrates for growth, within 7-11 days 0.3-0.5 mM of nitrate was reduced and 1.3-2 mM of iron(II) was oxidized leading to a stoichiometric NO
3
-
/Fe(II) ratio of 0.2, with N
2
and N
2
O identified as the main nitrate reduction products. Short-range ordered Fe(III) (oxyhydr)oxides were the product of iron(II) oxidation. Microorganisms were observed to be closely associated with formed minerals but only few cells were encrusted, suggesting that most of the bacteria were able to avoid mineral precipitation at their surface. Analysis of the microbial community by long-read 16S rRNA gene sequencing revealed that the culture is dominated by members of the
Gallionellaceae
family that are known as autotrophic, neutrophilic, microaerophilic iron(II)-oxidizers. In summary, our study suggests that NRFeOx mediated by lithoautotrophic bacteria can lead to nitrate removal in anthropogenically impacted aquifers.
Importance
Removal of nitrate by microbial denitrification in groundwater is often limited by low concentrations of organic carbon. In these carbon-poor ecosystems, nitrate-reducing bacteria that can use inorganic compounds such as Fe(II) (NRFeOx) as electron donors could play a major role in nitrate removal. However, no lithoautotrophic NRFeOx culture has been successfully isolated or enriched from this type of environment and as such there are no model cultures available to study the rate-limiting factors of this potentially important process. Here we present the physiology and microbial community composition of a novel lithoautotrophic NRFeOx culture enriched from a fractured aquifer in southern Germany. The culture is dominated by a putative Fe(II)-oxidizer affiliated with the
Gallionellaceae
family and performs nitrate reduction coupled to Fe(II) oxidation leading to N
2
O and N
2
formation without the addition of organic substrates. Our analyses demonstrate that lithoautotrophic NRFeOx can potentially lead to nitrate removal in nitrate-contaminated aquifers.
Neutrophilic microbial
pyrite (FeS2) oxidation coupled
to denitrification is thought to be an important natural nitrate attenuation
pathway in nitrate-contaminated aquifers. However, the poor solubility
of pyrite raises questions about its bioavailability and the mechanisms
underlying its oxidation. Here, we investigated direct microbial pyrite
oxidation by a neutrophilic chemolithoautotrophic nitrate-reducing
Fe(II)-oxidizing culture enriched from a pyrite-rich aquifer. We used
pyrite with natural abundance (NA) of Fe isotopes (NAFe-pyrite)
and 57Fe-labeled siderite to evaluate whether the oxidation
of the more soluble Fe(II)-carbonate (FeCO3) can indirectly
drive abiotic pyrite oxidation. Our results showed that in setups
where only pyrite was incubated with bacteria, direct microbial pyrite
oxidation contributed ca. 26% to overall nitrate reduction. The rest
was attributed to the oxidation of elemental sulfur (S0), present as a residue from pyrite synthesis. Pyrite oxidation was
evidenced in the NAFe-pyrite/57Fe-siderite setups
by maps of 56FeO and 32S obtained using a combination
of SEM with nanoscale secondary ion MS (NanoSIMS), which showed the presence of 56Fe(III) (oxyhydr)oxides
that could solely originate from 56FeS2. Based
on the fit of a reaction model to the geochemical data and the Fe-isotope
distributions from NanoSIMS, we conclude that anaerobic oxidation
of pyrite by our neutrophilic enrichment culture was mainly driven
by direct enzymatic activity of the cells. The contribution of abiotic
pyrite oxidation by Fe3+ appeared to be negligible in our
experimental setup.
Autotrophic NRFeOx microorganisms that oxidize Fe(II), reduce nitrate, and produce biomass play a key role in carbon, iron, and nitrogen cycles in pH-neutral, anoxic environments. Electrons from Fe(II) oxidation are used for the reduction of both carbon dioxide and nitrate.
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