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Nitrate Removal by a Novel Lithoautotrophic Nitrate-Reducing, Iron(II)-Oxidizing Culture Enriched from a Pyrite-Rich Limestone Aquifer.

ABSTRACT: 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 cosubstrates for growth, within 7 to 11 days, 0.3 to 0.5 mM nitrate was reduced and 1.3 to 2 mM iron(II) was oxidized, leading to a stoichiometric NO₃^-/Fe(II) ratio of 0.2, with N₂ and N₂O 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, and microaerophilic iron(II) oxidizers. In summary, our study suggests that NRFeOx mediated by lithoautotrophic bacteria can lead to nitrate removal in anthropogenically affected 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 N₂O and N₂ formation without the addition of organic substrates. Our analyses demonstrate that lithoautotrophic NRFeOx can potentially lead to nitrate removal in nitrate-contaminated aquifers.

SUBMITTER: Jakus N

PROVIDER: S-EPMC8373248 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UNLABELLED:This study demonstrates the prevalence, phylogenetic diversity, and physiology of nitrate-reducing microorganisms capable of utilizing reduced humic acids (HA) as electron donors in agricultural soils. Most probable number (MPN) enumeration of agricultural soils revealed large populations (10(4) to 10(6) cells g(-1) soil) of microorganisms capable of reducing nitrate while oxidizing the reduced HA analog 2,6-anthrahydroquinone disulfonate (AH(2)DS) to its corresponding quinone. Nitrate-dependent HA-oxidizing organisms isolated from agricultural soils were phylogenetically diverse and included members of the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Advective up-flow columns inoculated with corn plot soil and amended with reduced HA and nitrate supported both HA oxidation and enhanced nitrate reduction relative to no-donor or oxidized HA controls. The additional electron donating capacity of reduced HA could reasonably be attributed to the oxidation of reduced functional groups. Subsequent 16S rRNA gene-based high-density oligonucleotide microarray (PhyloChip) indicated that reduced HA columns supported the development of a bacterial community enriched with members of the Acidobacteria, Firmicutes, and Betaproteobacteria relative to the no-donor control and initial inoculum. This study identifies a previously unrecognized role for HA in stimulating denitrification processes in saturated soil systems. Furthermore, this study indicates that reduced humic acids impact soil geochemistry and the indigenous bacterial community composition. IMPORTANCE:This study identifies a new metabolic capacity in soil microbial communities that may be responsible for the mediation of significant nitrogen losses from soil systems. Nitrate-dependent humic acid (HA)-oxidizing organisms isolated from agricultural soils were phylogenetically diverse and included members of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Advective up-flow columns inoculated with corn plot soil and amended with reduced HA and nitrate supported both HA oxidation and enhanced nitrate reduction relative to no-donor or oxidized HA controls. The additional electron donating capacity of reduced HA could reasonably be attributed to the oxidation of reduced functional groups.

Project description:Nitrate decreases sulfide release in wastewater treatment plants (WWTP), but little is known on how it affects the microzonation and kinetics of related microbial processes within the biofilm. The effect of nitrate addition on these properties for sulfate reduction, sulfide oxidation, and oxygen respiration were studied with the use of microelectrodes in microaerophilic wastewater biofilms. Mass balance calaculations and community composition analysis were also performed. At basal WWTP conditions, the biofilm presented a double-layer system. The upper microaerophilic layer (~300 ?m) showed low sulfide production (0.31 ?mol cm-3 h-1) and oxygen consumption rates (0.01 ?mol cm-3 h-1). The anoxic lower layer showed high sulfide production (2.7 ?mol cm-3 h-1). Nitrate addition decreased net sulfide production rates, caused by an increase in sulfide oxidation rates (SOR) in the upper layer, rather than an inhibition of sulfate reducing bacteria (SRB). This suggests that the indigenous nitrate reducing-sulfide oxidizing bacteria (NR-SOB) were immediately activated by nitrate. The functional vertical structure of the biofilm changed to a triple-layer system, where the previously upper sulfide-producing layer in the absence of nitrate split into two new layers: 1) an upper sulfide-consuming layer, whose thickness is probably determined by the nitrate penetration depth within the biofilm, and 2) a middle layer producing sulfide at an even higher rate than in the absence of nitrate in some cases. Below these layers, the lower net sulfide-producing layer remained unaffected. Net SOR varied from 0.05 to 0.72 ?mol cm-3 h-1 depending on nitrate and sulfate availability. Addition of low nitrate concentrations likely increased sulfate availability within the biofilm and resulted in an increase of both net sulfate reduction and net sulfide oxidation by overcoming sulfate diffusional limitation from the water phase and the strong coupling between SRB and NR-SOB syntrophic relationship.

Project description:Oil reservoir souring and associated material integrity challenges are of great concern to the petroleum industry. The bioengineering strategy of nitrate injection has proven successful for controlling souring in some cases, but recent reports indicate increased corrosion in nitrate-treated produced water reinjection facilities. Sulfide-oxidizing, nitrate-reducing bacteria (soNRB) have been suggested to be the cause of such corrosion. Using the model soNRB Sulfurimonas sp. strain CVO obtained from an oil field, we conducted a detailed analysis of soNRB-induced corrosion at initial nitrate-to-sulfide (N/S) ratios relevant to oil field operations. The activity of strain CVO caused severe corrosion rates of up to 0.27?millimeters per year (mm y-1) and up to 60-?m-deep pitting within only 9 days. The highest corrosion during the growth of strain CVO was associated with the production of zero-valent sulfur during sulfide oxidation and the accumulation of nitrite, when initial N/S ratios were high. Abiotic corrosion tests with individual metabolites confirmed biogenic zero-valent sulfur and nitrite as the main causes of corrosion under the experimental conditions. Mackinawite (FeS) deposited on carbon steel surfaces accelerated abiotic reduction of both sulfur and nitrite, exacerbating corrosion. Based on these results, a conceptual model for nitrate-mediated corrosion by soNRB is proposed.IMPORTANCE Ambiguous reports of corrosion problems associated with the injection of nitrate for souring control necessitate a deeper understanding of this frequently applied bioengineering strategy. Sulfide-oxidizing, nitrate-reducing bacteria have been proposed as key culprits, despite the underlying microbial corrosion mechanisms remaining insufficiently understood. This study provides a comprehensive characterization of how individual metabolic intermediates of the microbial nitrogen and sulfur cycles can impact the integrity of carbon steel infrastructure. The results help explain the dramatic increases seen at times in corrosion rates observed during nitrate injection in field and laboratory trials and point to strategies for reducing adverse integrity-related side effects of nitrate-based souring mitigation.

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Nitrate Removal by a Novel Lithoautotrophic Nitrate-Reducing, Iron(II)-Oxidizing Culture Enriched from a Pyrite-Rich Limestone Aquifer.

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