title: Isolation of microorganisms involved in reduction of crystalline iron(III) oxides in natural environments.
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.
authors.affiliation: Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology Tsukuba, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Hori
authors.affiliation: Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology Tsukuba, Japan.
authors.forename: Tomo
authors.initials: T
authors.lastname: Aoyagi
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology Sapporo, Japan.
authors.forename: Hideomi
authors.initials: H
authors.lastname: Itoh
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology Tsukuba, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Narihiro
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology Sapporo, Japan.
authors.forename: Azusa
authors.initials: A
authors.lastname: Oikawa
authors.affiliation: Methane Hydrate Research & Development Division, Japan Oil, Gas and Metals National Corporation Chiba, Japan.
authors.forename: Kiyofumi
authors.initials: K
authors.lastname: Suzuki
authors.affiliation: Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology Tsukuba, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ogata
authors.affiliation: Microbial Ecophysiology Group, Faculty of Biology/Chemistry and MARUM, University of Bremen Bremen, Germany.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Friedrich
authors.affiliation: Max Planck Institute for Terrestrial Microbiology Marburg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Conrad
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology Tsukuba, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kamagata
keywords: Geobacter; crystalline iron(III) oxide; high-throughput sequencing; iron-reducing bacteria; isolation