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Insights into Nitrate-Reducing Fe(II) Oxidation Mechanisms through Analysis of Cell-Mineral Associations, Cell Encrustation, and Mineralogy in the Chemolithoautotrophic Enrichment Culture KS.

ABSTRACT: Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic and depend on organic cosubstrates for growth. Encrustation of cells in Fe(III) minerals has been observed for mixotrophic NRFeOB but not for autotrophic phototrophic and microaerophilic Fe(II) oxidizers. So far, little is known about cell-mineral associations in the few existing autotrophic NRFeOB. Here, we investigate whether the designated autotrophic Fe(II)-oxidizing strain (closely related to Gallionella and Sideroxydans) or the heterotrophic nitrate reducers that are present in the autotrophic nitrate-reducing Fe(II)-oxidizing enrichment culture KS form mineral crusts during Fe(II) oxidation under autotrophic and mixotrophic conditions. In the mixed culture, we found no significant encrustation of any of the cells both during autotrophic oxidation of 8 to 10 mM Fe(II) coupled to nitrate reduction and during cultivation under mixotrophic conditions with 8 to 10 mM Fe(II), 5 mM acetate, and 4 mM nitrate, where higher numbers of heterotrophic nitrate reducers were present. Two pure cultures of heterotrophic nitrate reducers (Nocardioides and Rhodanobacter) isolated from culture KS were analyzed under mixotrophic growth conditions. We found green rust formation, no cell encrustation, and only a few mineral particles on some cell surfaces with 5 mM Fe(II) and some encrustation with 10 mM Fe(II). Our findings suggest that enzymatic, autotrophic Fe(II) oxidation coupled to nitrate reduction forms poorly crystalline Fe(III) oxyhydroxides and proceeds without cellular encrustation while indirect Fe(II) oxidation via heterotrophic nitrate-reduction-derived nitrite can lead to green rust as an intermediate mineral and significant cell encrustation. The extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen species depends on Fe(II) concentrations and is probably negligible under environmental conditions in most habitats.IMPORTANCE Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic (their growth depends on organic cosubstrates) and can become encrusted in Fe(III) minerals. Encrustation is expected to be harmful and poses a threat to cells if it also occurs under environmentally relevant conditions. Nitrite produced during heterotrophic denitrification reacts with Fe(II) abiotically and is probably the reason for encrustation in mixotrophic NRFeOB. Little is known about cell-mineral associations in autotrophic NRFeOB such as the enrichment culture KS. Here, we show that no encrustation occurs in culture KS under autotrophic and mixotrophic conditions while heterotrophic nitrate-reducing isolates from culture KS become encrusted. These findings support the hypothesis that encrustation in mixotrophic cultures is caused by the abiotic reaction of Fe(II) with nitrite and provide evidence that Fe(II) oxidation in culture KS is enzymatic. Furthermore, we show that the extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen species depends on Fe(II) concentrations and is probably negligible in most environmental habitats.

SUBMITTER: Nordhoff M

PROVIDER: S-EPMC5478975 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Most isolated nitrate-reducing Fe(II)-oxidizing microorganisms are mixotrophic, meaning that Fe(II) is chemically oxidized by nitrite that forms during heterotrophic denitrification, and it is debated to which extent Fe(II) is enzymatically oxidized. One exception is the chemolithoautotrophic enrichment culture KS, a consortium consisting of a dominant Fe(II) oxidizer, Gallionellaceae sp., and less abundant heterotrophic strains (e.g., Bradyrhizobium sp., Nocardioides sp.). Currently, this is the only nitrate-reducing Fe(II)-oxidizing culture for which autotrophic growth has been demonstrated convincingly for many transfers over more than 2 decades. We used 16S rRNA gene amplicon sequencing and physiological growth experiments to analyze the community composition and dynamics of culture KS with various electron donors and acceptors. Under autotrophic conditions, an operational taxonomic unit (OTU) related to known microaerophilic Fe(II) oxidizers within the family Gallionellaceae dominated culture KS. With acetate as an electron donor, most 16S rRNA gene sequences were affiliated with Bradyrhizobium sp. Gallionellaceae sp. not only was able to oxidize Fe(II) under autotrophic and mixotrophic conditions but also survived over several transfers of the culture on only acetate, although it then lost the ability to oxidize Fe(II). Bradyrhizobium spp. became and remained dominant when culture KS was cultivated for only one transfer under heterotrophic conditions, even when conditions were reverted back to autotrophic in the next transfer. This study showed a dynamic microbial community in culture KS that responded to changing substrate conditions, opening up questions regarding carbon cross-feeding, metabolic flexibility of the individual strains in KS, and the mechanism of Fe(II) oxidation by a microaerophile in the absence of O2IMPORTANCE Nitrate-reducing Fe(II)-oxidizing microorganisms are present in aquifers, soils, and marine and freshwater sediments. Most nitrate-reducing Fe(II) oxidizers known are mixotrophic, meaning that they need organic carbon to continuously oxidize Fe(II) and grow. In these microbes, Fe(II) was suggested to be chemically oxidized by nitrite that forms during heterotrophic denitrification, and it remains unclear whether or to what extent Fe(II) is enzymatically oxidized. In contrast, the enrichment culture KS was shown to oxidize Fe(II) autotrophically coupled to nitrate reduction. This culture contains the designated Fe(II) oxidizer Gallionellaceae sp. and several heterotrophic strains (e.g., Bradyrhizobium sp.). We showed that culture KS is able to metabolize Fe(II) and a variety of organic substrates and is able to adapt to dynamic environmental conditions. When the community composition changed and Bradyrhizobium became the dominant community member, Fe(II) was still oxidized by Gallionellaceae sp., even when culture KS was cultivated with acetate/nitrate [Fe(II) free] before being switched back to Fe(II)/nitrate.

Project description:Autotrophic nitrate-reducing Fe(II)-oxidizing (NRFeOx) microorganisms fix CO2 and oxidize Fe(II) coupled to denitrification, influencing carbon, iron, and nitrogen cycles in pH-neutral, anoxic environments. However, the distribution of electrons from Fe(II) oxidation to either biomass production (CO2 fixation) or energy generation (nitrate reduction) in autotrophic NRFeOx microorganisms has not been quantified. We therefore cultivated the autotrophic NRFeOx culture KS at different initial Fe/N ratios, followed geochemical parameters, identified minerals, analyzed N isotopes, and applied numerical modeling. We found that at all initial Fe/N ratios, the ratios of Fe(II)oxidized to nitratereduced were slightly higher (5.11 to 5.94 at Fe/N ratios of 10:1 and 10:0.5) or lower (4.27 to 4.59 at Fe/N ratios of 10:4, 10:2, 5:2, and 5:1) than the theoretical ratio for 100% Fe(II) oxidation being coupled to nitrate reduction (5:1). The main N denitrification product was N2O (71.88 to 96.29% at Fe/15N ratios of 10:4 and 5:1; 43.13 to 66.26% at an Fe/15N ratio of 10:1), implying that denitrification during NRFeOx was incomplete in culture KS. Based on the reaction model, on average 12% of electrons from Fe(II) oxidation were used for CO2 fixation while 88% of electrons were used for reduction of NO3- to N2O at Fe/N ratios of 10:4, 10:2, 5:2, and 5:1. With 10 mM Fe(II) (and 4, 2, 1, or 0.5 mM nitrate), most cells were closely associated with and partially encrusted by the Fe(III) (oxyhydr)oxide minerals, whereas at 5 mM Fe(II), most cells were free of cell surface mineral precipitates. The genus Gallionella (>80%) dominated culture KS regardless of the initial Fe/N ratios. Our results showed that Fe/N ratios play a key role in regulating N2O emissions, for distributing electrons between nitrate reduction and CO2 fixation, and for the degree of cell-mineral interactions in the autotrophic NRFeOx culture KS. IMPORTANCE 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. However, the question is how many electrons go into biomass production versus energy generation during autotrophic growth. Here, we demonstrated that in the autotrophic NRFeOx culture KS cultivated at Fe/N ratios of 10:4, 10:2, 5:2, and 5:1, ca. 12% of electrons went into biomass formation, while 88% of electrons were used for reduction of NO3- to N2O. Isotope analysis also showed that denitrification during NRFeOx was incomplete in culture KS and the main N denitrification product was N2O. Therefore, most electrons stemming from Fe(II) oxidation seemed to be used for N2O formation in culture KS. This is environmentally important for the greenhouse gas budget.

Project description:Nitrate reduction coupled to Fe(II) oxidation (NRFO) has been recognized as an environmentally important microbial process in many freshwater ecosystems. However, well-characterized examples of autotrophic nitrate-reducing Fe(II)-oxidizing bacteria are rare, and their pathway of electron transfer as well as their interaction with flanking community members remain largely unknown. Here, we applied meta-omics (i.e., metagenomics, metatranscriptomics, and metaproteomics) to the nitrate-reducing Fe(II)-oxidizing enrichment culture KS growing under autotrophic or heterotrophic conditions and originating from freshwater sediment. We constructed four metagenome-assembled genomes with an estimated completeness of ≥95%, including the key players of NRFO in culture KS, identified as Gallionellaceae sp. and Rhodanobacter sp. The Gallionellaceae sp. and Rhodanobacter sp. transcripts and proteins likely involved in Fe(II) oxidation (e.g., mtoAB, cyc2, and mofA), denitrification (e.g., napGHI), and oxidative phosphorylation (e.g., respiratory chain complexes I to V) along with Gallionellaceae sp. transcripts and proteins for carbon fixation (e.g., rbcL) were detected. Overall, our results indicate that in culture KS, the Gallionellaceae sp. and Rhodanobacter sp. are interdependent: while Gallionellaceae sp. fixes CO2 and provides organic compounds for Rhodanobacter sp., Rhodanobacter sp. likely detoxifies NO through NO reduction and completes denitrification, which cannot be performed by Gallionellaceae sp. alone. Additionally, the transcripts and partial proteins of cbb3- and aa3-type cytochrome c suggest the possibility for a microaerophilic lifestyle of the Gallionellaceae sp., yet culture KS grows under anoxic conditions. Our findings demonstrate that autotrophic NRFO is performed through cooperation among denitrifying and Fe(II)-oxidizing bacteria, which might resemble microbial interactions in freshwater environments. IMPORTANCE Nitrate-reducing Fe(II)-oxidizing bacteria are widespread in the environment, contribute to nitrate removal, and influence the fate of the greenhouse gases nitrous oxide and carbon dioxide. The autotrophic growth of nitrate-reducing Fe(II)-oxidizing bacteria is rarely investigated and not fully understood. The most prominent model system for this type of study is the enrichment culture KS. To gain insights into the metabolism of nitrate reduction coupled to Fe(II) oxidation in the absence of organic carbon and oxygen, we performed metagenomic, metatranscriptomic, and metaproteomic analyses of culture KS and identified Gallionellaceae sp. and Rhodanobacter sp. as interdependent key Fe(II) oxidizers in culture KS. Our work demonstrates that autotrophic nitrate reduction coupled to Fe(II) oxidation is not performed by an individual strain but is a cooperation of at least two members of the bacterial community in culture KS. These findings serve as a foundation for our understanding of nitrate-reducing Fe(II)-oxidizing bacteria in the environment.

Project description:The enrichment culture KS is one of the few existing autotrophic, nitrate-reducing, Fe(II)-oxidizing cultures that can be continuously transferred without an organic carbon source. We used a combination of catalyzed amplification reporter deposition fluorescence in situ hybridization (CARD-FISH) and nanoscale secondary ion mass spectrometry (NanoSIMS) to analyze community dynamics, single-cell activities, and interactions among the two most abundant microbial community members (i.e., Gallionellaceae sp. and Bradyrhizobium spp.) under autotrophic and heterotrophic growth conditions. CARD-FISH cell counts showed the dominance of the Fe(II) oxidizer Gallionellaceae sp. under autotrophic conditions as well as of Bradyrhizobium spp. under heterotrophic conditions. We used NanoSIMS to monitor the fate of 13C-labeled bicarbonate and acetate as well as 15N-labeled ammonium at the single-cell level for both taxa. Under autotrophic conditions, only the Gallionellaceae sp. was actively incorporating 13C-labeled bicarbonate and 15N-labeled ammonium. Interestingly, both Bradyrhizobium spp. and Gallionellaceae sp. became enriched in [13C]acetate and [15N]ammonium under heterotrophic conditions. Our experiments demonstrated that Gallionellaceae sp. was capable of assimilating [13C]acetate while Bradyrhizobium spp. were not able to fix CO2, although a metagenomics survey of culture KS recently revealed that Gallionellaceae sp. lacks genes for acetate uptake and that the Bradyrhizobium sp. carries the genetic potential to fix CO2 The study furthermore extends our understanding of the microbial reactions that interlink the nitrogen and Fe cycles in the environment.IMPORTANCE Microbial mechanisms by which Fe(II) is oxidized with nitrate as the terminal electron acceptor are generally referred to as "nitrate-dependent Fe(II) oxidation" (NDFO). NDFO has been demonstrated in laboratory cultures (such as the one studied in this work) and in a variety of marine and freshwater sediments. Recently, the importance of NDFO for the transport of sediment-derived Fe in aquatic ecosystems has been emphasized in a series of studies discussing the impact of NDFO for sedimentary nutrient cycling and redox dynamics in marine and freshwater environments. In this article, we report results from an isotope labeling study performed with the autotrophic, nitrate-reducing, Fe(II)-oxidizing enrichment culture KS, which was first described by Straub et al. (1) about 20 years ago. Our current study builds on the recently published metagenome of culture KS (2).

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Insights into Nitrate-Reducing Fe(II) Oxidation Mechanisms through Analysis of Cell-Mineral Associations, Cell Encrustation, and Mineralogy in the Chemolithoautotrophic Enrichment Culture KS.

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