Functional Gene Analysis of Freshwater Iron-Rich Flocs at Circumneutral pH and Isolation of a Stalk-Forming Microaerophilic Iron-Oxidizing Bacterium

Abstract

bIron-rich flocs often occur where anoxic water containing ferrous iron encounters oxygenated environments. Culture-independent molecular analyses have revealed the presence of 16S rRNA gene sequences related to diverse bacteria, including autotrophic iron oxidizers and methanotrophs in iron-rich flocs; however, the metabolic functions of the microbial communities remain poorly characterized, particularly regarding carbon cycling. In the present study, we cultivated iron-oxidizing bacteria (FeOB) and performed clone library analyses of functional genes related to carbon fixation and methane oxidization (cbbM and pmoA, respectively), in addition to bacterial and archaeal 16S rRNA genes, in freshwater iron-rich flocs at groundwater discharge points. The analyses of 16S rRNA, cbbM, and pmoA genes strongly suggested the coexistence of autotrophic iron oxidizers and methanotrophs in the flocs. Furthermore, a novel stalk-forming microaerophilic FeOB, strain OYT1, was isolated and characterized phylogenetically and physiologically. The 16S rRNA and cbbM gene sequences of OYT1 are related to those of other microaerophilic FeOB in the family Gallionellaceae, of the Betaproteobacteria, isolated from freshwater environments at circumneutral pH. The physiological characteristics of OYT1 will help elucidate the ecophysiology of microaerophilic FeOB. Overall, this study demonstrates functional roles of microorganisms in iron flocs, suggesting several possible linkages between Fe and C cycling.

Ferriphaselus amnicola gen. nov., sp. nov., a neutrophilic, stalk-forming, iron-oxidizing bacterium isolated from an iron-rich groundwater seep

“…As reported previously, cells of strain OYT1 T produced stalks in an HM-agarose-stabilized medium at pH 7.1-7.3, but not at pH 6.5 or below (Kato et al, 2013). Instead, they produced extracellular particulate oxides under lower pH (Fig.…”

“…The stalks contain carboxyl-rich polysaccharides and may prevent cell encrustation with iron oxides (Chan et al, 2009(Chan et al, , 2011. We isolated another stalk-forming, iron-oxidizing betaproteobacterium, strain OYT1 T , from an iron-rich groundwater seep and briefly determined its physiological and phylogenetic characteristics (Kato et al, 2013). Here, we describe the detailed physiological and phylogenetic characteristics of OYT1 T and propose that this isolate represents a novel species in a new genus.…”

“…The stalks consisted of thin filaments (20-200 nm wide), determined by transmission electron microscopy ( Fig. 1; Kato et al, 2013), which resemble those produced by G. ferruginea (Vatter & Wolfe, 1956), strain R-1 (Krepski et al, 2012) and M. ferrooxydans (Chan et al, 2011). All stalks represented a left-handed (S-type) helical structure.…”

“…OYT1 T was affiliated with the Gallionella-related cluster within the class Betaproteobacteria, as shown by phylogenetic analysis using the 16S rRNA gene sequence of OYT1 T (Kato et al, 2013). In addition, we checked the 16S rRNA gene sequence by direct sequencing of PCR products amplified from the extracted DNA.…”

“…Strain OYT1 T was isolated from an iron-rich floc sample collected from a groundwater seep in Ohyato Park, Tokyo, Japan, by a serial dilution-to-extinction method using 96-well plates (Kato et al, 2013). OYT1 T was maintained using an agarose-stabilized gradient culture technique as described previously (Emerson & Merrill Floyd, 2005 The temperature and pH ranges and optima for growth, requirement for energy sources and growth rate have been reported previously (Kato et al, 2013). In brief, OYT1 T grew at 8-30 u C and pH 5.6-7.3.…”