Growth and Population Dynamics of the Anaerobic Fe(II)-Oxidizing and Nitrate-Reducing Enrichment Culture KS.

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.

Insights into Carbon Metabolism Provided by Fluorescence In Situ Hybridization-Secondary Ion Mass Spectrometry Imaging of an Autotrophic, Nitrate-Reducing, Fe(II)-Oxidizing Enrichment Culture.

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).

Metagenomic Analyses of the Autotrophic Fe(II)-Oxidizing, Nitrate-Reducing Enrichment Culture KS.

Nitrate-dependent ferrous iron [Fe(II)] oxidation (NDFO) is a well-recognized chemolithotrophic pathway in anoxic sediments. The neutrophilic chemolithoautotrophic enrichment culture KS originally obtained from a freshwater sediment (K. L. Straub, M. Benz, B. Schink, and F. Widdel, Appl Environ Microbiol 62:1458-1460, 1996) has been used as a model system to study NDFO. However, the primary Fe(II) oxidizer in this culture has not been isolated, despite extensive efforts to do so. Here, we present a metagenomic analysis of this enrichment culture in order to gain insight into electron transfer pathways and the roles of different bacteria in the culture. We obtained a near-complete genome of the primary Fe(II) oxidizer, a species in the family Gallionellaceae, and draft genomes from its flanking community members. A search of the putative extracellular electron transfer pathways in these genomes led to the identification of a homolog of the MtoAB complex [a porin-multiheme cytochromec system identified in neutrophilic microaerobic Fe(II)-oxidizing Sideroxydans lithotrophicus ES-1] in a Gallionellaceae sp., and findings of other putative genes involving cytochromecand multicopper oxidases, such as Cyc2 and OmpB. Genome-enabled metabolic reconstruction revealed that this Gallionellaceae sp. lacks nitric oxide and nitrous oxide reductase genes and may partner with flanking populations capable of complete denitrification to avoid toxic metabolite accumulation, which may explain its resistance to growth in pure culture. This and other revealed interspecies interactions and metabolic interdependencies in nitrogen and carbon metabolisms may allow these organisms to cooperate effectively to achieve robust chemolithoautotrophic NDFO. Overall, the results significantly expand our knowledge of NDFO and suggest a range of genetic targets for further exploration.