Isolation of microorganisms involved in reduction of crystalline iron(III) oxides in natural environments.
Isolation of microorganisms involved in reduction of crystalline iron(III) oxides in natural environments.
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DOI：
10.3389/fmicb.2015.00386
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发表时间：
2015
影响因子：
5.2
通讯作者：
Kamagata Y
中科院分区：
文献类型：
--
作者：
Hori T;Aoyagi T;Itoh H;Narihiro T;Oikawa A;Suzuki K;Ogata A;Friedrich MW;Conrad R;Kamagata Y
晶体Fe（III）氧化物的还原是自然环境中有机化合物氧化的最重要的电子汇之一。然而，由于分离株的数量有限，很难了解所涉及的微生物的生理和生态影响。在这里，两个阶段的培养实施，以选择性地丰富和隔离结晶氧化铁（III）还原微生物在土壤和沉积物。首先，通过在结晶氧化铁上连续培养2年来富集铁还原剂并耗尽其他非靶向的富养生物（即，针铁矿、纤铁矿、赤铁矿或磁铁矿）作为电子受体。PCR扩增证实，136种孵育条件中有58种允许微生物继续存在。基于16 S rRNA基因的高通量Illumina测序和克隆文库分析显示，在每种三价铁氧化物上的富集培养物含有属于三角形变形菌门（主要是地杆菌科）的细菌，其次是厚壁菌门和绿球藻门，其也包含所鉴定的大多数操作分类单位（OTU）。维恩图表明，核心OTU富含所有的铁氧化物是占主导地位的土杆菌科，而每种类型的铁氧化物补充选择性地富集特定的OTU在其他系统发育的群体。其次，将包括新微生物的38个富集培养物转移到含有可溶性铁（III）的培养基中，以刺激富集的铁还原剂的增殖。通过稀释培养和单菌落分离，最终得到6株三角形变形菌，其中5株为Geetrium属，1株为Peletrium属。这些分离物的16 S rRNA基因序列与近缘种的同源性为94.8-98.1%。所有的菌株都能够生长在醋酸盐和三价铁，但他们的生理特性差异很大，在生长速度方面。因此，新策略允许富集和分离能够通过还原结晶三价铁氧化物而茁壮成长的新型铁（III）还原剂。
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.