Abstract
Reduction of crystalline Fe(III) oxides is one of the most important electron sinks for organic compound oxidation in natural environments. Yet the limited number of isolates makes it difficult to understand the physiology and ecological impact of the microorganisms involved. Here, two-stage cultivation was implemented to selectively enrich and isolate crystalline iron(III) oxide reducing microorganisms in soils and sediments. Firstly, iron reducers were enriched and other untargeted eutrophs were depleted by 2-years successive culture on a crystalline ferric iron oxide (i.e., goethite, lepidocrocite, hematite, or magnetite) as electron acceptor. Fifty-eight out of 136 incubation conditions allowed the continued existence of microorganisms as confirmed by PCR amplification. High-throughput Illumina sequencing and clone library analysis based on 16S rRNA genes revealed that the enrichment cultures on each of the ferric iron oxides contained bacteria belonging to the Deltaproteobacteria (mainly Geobacteraceae), followed by Firmicutes and Chloroflexi, which also comprised most of the operational taxonomic units (OTUs) identified. Venn diagrams indicated that the core OTUs enriched with all of the iron oxides were dominant in the Geobacteraceae while each type of iron oxides supplemented selectively enriched specific OTUs in the other phylogenetic groups. Secondly, 38 enrichment cultures including novel microorganisms were transferred to soluble-iron(III) containing media in order to stimulate the proliferation of the enriched iron reducers. Through extinction dilution-culture and single colony isolation, six strains within the Deltaproteobacteria were finally obtained; five strains belonged to the genus Geobacter and one strain to Pelobacter. The 16S rRNA genes of these isolates were 94.8–98.1% identical in sequence to cultured relatives. All the isolates were able to grow on acetate and ferric iron but their physiological characteristics differed considerably in terms of growth rate. Thus, the novel strategy allowed to enrich and isolate novel iron(III) reducers that were able to thrive by reducing crystalline ferric iron oxides.
Highlights
Iron (Fe) is the fourth most common element in the Earth’s crust, and the vast majority is distributed as iron minerals in natural environments such as soils and sediments (Stumm and Sulzberger, 1992; Davison, 1993; Weber et al, 2006)
At the end of the longterm incubation, the continued existence of microorganisms was confirmed by PCR amplification with genomic DNAs extracted from the enrichment cultures
Concerning the iron oxides employed, 14, 16, 10, and 18 enrichment cultures came from incubations with goethite, lepidocrocite, hematite, and magnetite, respectively
Summary
Iron (Fe) is the fourth most common element in the Earth’s crust, and the vast majority is distributed as iron minerals in natural environments such as soils and sediments (Stumm and Sulzberger, 1992; Davison, 1993; Weber et al, 2006). Microbial Fe(III) reduction is one of the most significant electron sinks for the oxidation of organic compounds under anoxic conditions prevailing in natural ecosystems (Yao et al, 1999; Lovley et al, 2004; Hori et al, 2010; Braunschweig et al, 2013). Thereby it exerts strong influences on global climate change as a competitor to methanogenesis that is the dominant terminal reduction process to produce the greenhouse effect gas methane (Conrad, 1996; Yvon-Durocher et al, 2014). A limited number of isolates makes it difficult to understand the physiology and ecological impact of the microorganisms. Lentini et al (2012) have enriched iron(III)reducing bacterial communities with iron-oxide minerals but not attempted to isolate the predominant microorganisms as pure cultures
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