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Passive treatment based on iron biological oxidation is a promising strategy for Arsenic (As)-rich acid mine drainage (AMD) remediation. In the present study, we characterized by 16S rRNA metabarcoding the bacterial diversity in a field-pilot bioreactor treating extremely As-rich AMD in situ , over a 6 months monitoring period. Inside the bioreactor, the bacterial communities responsible for iron and arsenic removal formed a biofilm ("biogenic precipitate") whose composition varied in time and space. These communities evolved from a structure at first similar to the one of the feed water used as an inoculum to a structure quite similar to the natural biofilm developing in situ in the AMD. Over the monitoring period, iron-oxidizing bacteria always largely dominated the biogenic precipitate, with distinct populations ( Gallionella, Ferrovum, Leptospirillum, Acidithiobacillus, Ferritrophicum ), whose relative proportions extensively varied among time and space. A spatial structuring was observed inside the trays (arranged in series) composing the bioreactor. This spatial dynamic could be linked to the variation of the physico-chemistry of the AMD water between the raw water entering and the treated water exiting the pilot. According to redundancy analysis (RDA), the following parameters exerted a control on the bacterial communities potentially involved in the water treatment process: dissolved oxygen, temperature, pH, dissolved sulfates, arsenic and Fe(II) concentrations and redox potential. Appreciable arsenite oxidation occurring in the bioreactor could be linked to the stable presence of two distinct monophylogenetic groups of Thiomonas related bacteria. The ubiquity and the physiological diversity of the bacteria identified, as well as the presence of bacteria of biotechnological relevance, suggested that this treatment system could be applied to the treatment of other AMD.

According to the literature, pyrite (FeS 2 ) oxidation has been previously determined to involve thiosulfate as the first aqueous intermediate sulfur product, which is further oxidized to sulfate. In the present study, pyrite oxidation by Acidithiobacillus ferrooxidans was studied using electrochemical and metabolic approaches in an effort to extend existing knowledge on the oxidation mechanism. Due to the small surface area, the reaction rate of a compact pyrite electrode in the form of polycrystalline pyrite aggregate in A. ferrooxidans suspension was very slow at a spontaneously formed high redox potential. The slow rate made it possible to investigate the oxidation process in detail over a term of 100 days. Using electrochemical parameters from polarization curves and levels of released iron, the number of exchanged electrons per pyrite molecule was estimated. The values close to 14 and 2 electrons were determined for the oxidation with and without bacteria, respectively. These results indicated that sulfate was the dominant first aqueous sulfur species formed in the presence of bacteria and elemental sulfur was predominantly formed without bacteria. The stoichiometric calculations are consistent with high iron-oxidizing activities of bacteria that continually keep the released iron in the ferric form, resulting in a high redox potential. The sulfur entity of pyrite was oxidized to sulfate by Fe 3+ without intermediate thiosulfate under these conditions. Cell attachment on the corroded pyrite electrode surface was documented although pyrite surface corrosion by Fe 3+ was evident without bacterial participation. Attached cells may be important in initiating the oxidation of the pyrite surface to release iron from the mineral. During the active phase of oxidation of a pyrite concentrate sample, the ATP levels in attached and planktonic bacteria were consistent with previously established ATP content of iron-oxidizing cells. No significant upregulation of three essential genes involved in energy metabolism of sulfur compounds was observed in the planktonic cells, which represented the dominant biomass in the pyrite culture. The study demonstrated the formation of sulfate as the first dissolved sulfur species with iron-oxidizing bacteria under high redox potential conditions. Minor aqueous sulfur intermediates may be formed but as a result of side reactions.

Arsenic removal consecutive to biological iron oxidation and precipitation is an effective process for treating As-rich acid mine drainage (AMD). We studied the effect of hydraulic retention time (HRT)-from 74 to 456 min-in a bench-scale bioreactor exploiting such process. The treatment efficiency was monitored during 19 days, and the final mineralogy and bacterial communities of the biogenic precipitates were characterized by X-ray absorption spectroscopy and high-throughput 16S rRNA gene sequencing. The percentage of Fe(II) oxidation (10-47%) and As removal (19-37%) increased with increasing HRT. Arsenic was trapped in the biogenic precipitates as As(III)-bearing schwertmannite and amorphous ferric arsenate, with a decrease of As/Fe ratio with increasing HRT. The bacterial community in the biogenic precipitate was dominated by Fe-oxidizing bacteria whatever the HRT. The proportion of Gallionella and Ferrovum genera shifted from respectively 65 and 12% at low HRT to 23 and 51% at high HRT, in relation with physicochemical changes in the treated water. aioA genes and Thiomonas genus were detected at all HRT although As(III) oxidation was not evidenced. To our knowledge, this is the first evidence of the role of HRT as a driver of bacterial community structure in bioreactors exploiting microbial Fe(II) oxidation for AMD treatment.

Indigenous iron-oxidizing bacteria were isolated on modified selective 9KFe 2+ medium from Baiyin copper mine stope, China. Three distinct acidophilic bacteria were isolated and identified by analyzing the sequences of 16S rRNA gene. Based on published sequences of 16S rRNA gene in the GenBank, a phylogenetic tree was constructed. The sequence of isolate WG101 showed 99% homology with Acidithiobacillus ferrooxidans strain AS2. Isolate WG102 exhibited 98% similarity with Leptospirillum ferriphilum strain YSK. Similarly, isolate WG103 showed 98% similarity with Leptospirillum ferrooxidans strain L15. Furthermore, the biotechnological potential of these isolates in consortia form was evaluated to recover copper and zinc from their ore. Under optimized conditions, 77.68 ± 3.55% of copper and 70.58 ± 3.77% of zinc were dissolved. During the bioleaching process, analytical study of pH and oxidation-reduction potential fluctuations were monitored that reflected efficient activity of the bacterial consortia. The FTIR analysis confirmed the variation in bands after treatment with consortia. The impact of consortia on iron speciation within bioleached ore was analyzed using Mössbauer spectroscopy and clear changes in iron speciation was reported. The use of indigenous bacterial consortia is more efficient compared to pure inoculum. This study provided the basic essential conditions for further upscaling bioleaching application for metal extraction.

Lithotrophic iron-oxidizing bacteria depend on reduced iron, Fe(II), as their primary energy source, making them natural candidates for growing in association with steel infrastructure and potentially contributing to microbially influenced corrosion (MIC). This review summarizes recent work on the role of iron-oxidizing bacteria (FeOB) in MIC. By virtue of producing complex 3-dimensional biofilms that result from the accumulation of iron-oxides, FeOB may aid in the colonization of steel surfaces by other microbes involved in MIC. Evidence points to a successional pattern occurring whereby FeOB are early colonizers of mild steel (MS), followed by sulfate-reducing bacteria and other microbes, although studies of aged corrosion products indicate that FeOB do establish a long-term presence. There is evidence that only specific clades of FeOB, with unique adaptations for growing on steel surfaces are part of the MIC community. These are discussed in the context of the larger MIC microbiome.

An iron-oxidizing bacterium, designated strain An22 T , which was isolated from a paddy field soil in Anjo, Japan, was described taxonomically. Strain An22 T was motile by a single polar flagellum, curved-rod, Gram-negative bacterium that was able to grow at 12-37 °C (optimally at 25-30 °C) and at pH 5.2-6.8 (pH 5.9-6.1). The strain grew microaerobically and autotrophically by oxidizing ferrous iron, but did not form stalks, a unique structure of iron oxides. The major cellular fatty acids were C16 : 0 and C16 : 1ω7c/C16 : 1ω6c. The major respiratory quinones were UQ-10 and UQ-8. The strain possessed ribulose-1,5-bisphosphate carboxylase/oxygenase indicating an autotrophic nature via the Calvin-Benson-Bassham cycle. The total DNA G+C content was 61.4 mol%. 16S rRNA gene sequence analysis revealed that strain An22 T was affiliated with the class Betaproteobacteria and clustered with iron-oxidizing bacteria, Gallionella ferrugineaJohan (94.8 % similarity) and Ferriphaselus amnicola OYT1 T (94.4 %) in the family Gallionellaceae. Based on the low 16S rRNA gene sequence similarity to the phylogenetically closest genera and the combination of unique morphological, physiological and biochemical characteristics, strain An22 T represents a novel genus and species within the family Gallionellaceae, for which the name Ferrigenium kumadai gen. nov., sp. nov. is proposed. The type strain is An22 T (=JCM 30584 T =NBRC 112974 T =ATCC TSD-51 T ).

Leptothrix ochracea is known for producing large volumes of iron oxyhydroxide sheaths that alter wetland biogeochemistry. For over a century, these delicate structures have fascinated microbiologists and geoscientists. Because L. ochracea still resists long-term in vitro culture, the debate regarding its metabolic classification dates back to 1885. We developed a novel culturing technique for L. ochracea using in situ natural waters and coupled this with single-cell genomics and nanoscale secondary-ion mass spectrophotometry (nanoSIMS) to probe L. ochracea 's physiology. In microslide cultures L. ochracea doubled every 5.7 h and had an absolute growth requirement for ferrous iron, the genomic capacity for iron oxidation, and a branched electron transport chain with cytochromes putatively involved in lithotrophic iron oxidation. Additionally, its genome encoded several electron transport chain proteins, including a molybdopterin alternative complex III (ACIII), a cytochrome bd oxidase reductase, and several terminal oxidase genes. L. ochracea contained two key autotrophic proteins in the Calvin-Benson-Bassham cycle, a form II ribulose bisphosphate carboxylase, and a phosphoribulose kinase. L. ochracea also assimilated bicarbonate, although calculations suggest that bicarbonate assimilation is a small fraction of its total carbon assimilation. Finally, L. ochracea 's fundamental physiology is a hybrid of those of the chemolithotrophic Gallionella- type iron-oxidizing bacteria and the sheathed, heterotrophic filamentous metal-oxidizing bacteria of the Leptothrix-Sphaerotilus genera. This allows L. ochracea to inhabit a unique niche within the neutrophilic iron seeps. IMPORTANCE Leptothrix ochracea was one of three groups of organisms that Sergei Winogradsky used in the 1880s to develop his hypothesis on chemolithotrophy. L. ochracea continues to resist cultivation and appears to have an absolute requirement for organic-rich waters, suggesting that its true physiology remains unknown. Further, L. ochracea is an ecological engineer; a few L. ochracea cells can generate prodigious volumes of iron oxyhydroxides, changing the ecosystem's geochemistry and ecology. Therefore, to determine L. ochracea 's basic physiology, we employed new single-cell techniques to demonstrate that L. ochracea oxidizes iron to generate energy and, despite having predicted genes for autotrophic growth, assimilates a fraction of the total CO 2 that autotrophs do. Although not a true chemolithoautotroph, L. ochracea 's physiological strategy allows it to be flexible and to extensively colonize iron-rich wetlands., (Copyright © 2018 American Society for Microbiology.)

Contamination by heavy metals and metalloids is a serious environmental and health concern. Acidic wastewaters are often associated with toxic metals which may enter and spread into agricultural soils. Several biological assays have been developed to detect toxic metals; however, most of them can only detect toxic metals in a neutral pH, not in an acidic environment. In this study, an acidophilic iron-oxidizing bacterium (IOB) Strain Y10 was isolated, characterized, and used to detect toxic metals toxicity in acidic water at pH 2.5. The colorimetric acidophilic IOB biosensor was based on the inhibition of the iron oxidizing ability of Strain Y10, an acidophilic iron-oxidizing bacterium, by metals toxicity. Our results showed that Strain Y10 is acidophilic iron-oxidizing bacterium. Thiobacillus caldus medium (TCM) (pH 2.5) supplied with both S 4 O 6 2- and glucose was the optimum growth medium for Strain Y10. The optimum temperature and pH for the growth of Strain Y10 was 45 °C and pH 2.5, respectively. Our study demonstrates that the color-based acidophilic IOB biosensor can be semi-quantitatively observed by eye or quantitatively measured by spectrometer to detect toxicity from multiple toxic metals at pH 2.5 within 45 min. Our study shows that monitoring toxic metals in acidic water is possible by using the acidophilic IOB biosensor. Our study thus provides a novel approach for rapid and cost-effective detection of toxic metals in acidic conditions that can otherwise compromise current methods of chemical analysis. This method also allows for increased efficiency when screening large numbers of environmental samples., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In recent years, the role of microorganisms inhabiting rice rhizosphere in promoting arsenic contamination has emerged. However, little is known concerning the species and metabolic properties involved in this phenomenon. In this study, the influence of water management on the rhizosphere microbiota in relation to arsenic dissolution in soil solution was tested. Rice plants were cultivated in macrocosms under different water regimes: continuous flooding, continuous flooding with a 2-week period drainage before flowering, and dry soil watered every 10 days. The active bacterial communities in rhizosphere soil and in rhizoplane were characterized by 16S rRNA pyrosequencing. An in-depth analysis of microbial taxa with direct or indirect effects on arsenic speciation was performed and related contribution was evaluated. Continuous flooding promoted high diversity in the rhizosphere, with the plant strongly determining species richness and evenness. On the contrary, under watering the communities were uniform, with little differences between rhizosphere soil and rhizoplane. Arsenic-releasing and arsenite-methylating bacteria were selected by continuous flooding, where they represented 8% of the total. On the contrary, bacteria decreasing arsenic solubility were more abundant under watering, with relative abundance of 10%. These values reflected arsenic concentrations in soil solution: 135 μg L -1 and negligible in continuous flooding and under watering, respectively. When short-term drainage was applied before flowering, intermediate conditions were achieved. This evidence strongly indicates an active role of the rhizosphere microbiota in driving arsenic biogeochemistry in rice paddies, influenced by water management, explaining amounts and speciation of arsenic often found in rice grains.

Chemolithotrophic iron-oxidizing bacteria (FeOB) could theoretically inhabit any environment where Fe(II) and O 2 (or nitrate) coexist. Until recently, marine Fe-oxidizing Zetaproteobacteria had primarily been observed in benthic and subsurface settings, but not redox-stratified water columns. This may be due to the challenges that a pelagic lifestyle would pose for Zetaproteobacteria, given low Fe(II) concentrations in modern marine waters and the possibility that Fe oxyhydroxide biominerals could cause cells to sink. However, we recently cultivated Zetaproteobacteria from the Chesapeake Bay oxic-anoxic transition zone, suggesting that they can survive and contribute to biogeochemical cycling in a stratified estuary. Here we describe the isolation, characterization, and genomes of two new species, Mariprofundus aestuarium CP-5 and Mariprofundus ferrinatatus CP-8, which are the first Zetaproteobacteria isolates from a pelagic environment. We looked for adaptations enabling strains CP-5 and CP-8 to overcome the challenges of living in a low Fe redoxcline with frequent O 2 fluctuations due to tidal mixing. We found that the CP strains produce distinctive dreadlock-like Fe oxyhydroxide structures that are easily shed, which would help cells maintain suspension in the water column. These oxides are by-products of Fe(II) oxidation, likely catalyzed by the putative Fe(II) oxidase encoded by the cyc2 gene, present in both CP-5 and CP-8 genomes; the consistent presence of cyc2 in all microaerophilic FeOB and other FeOB genomes supports its putative role in Fe(II) oxidation. The CP strains also have two gene clusters associated with biofilm formation (Wsp system and the Widespread Colonization Island) that are absent or rare in other Zetaproteobacteria. We propose that biofilm formation enables the CP strains to attach to FeS particles and form flocs, an advantageous strategy for scavenging Fe(II) and developing low [O 2 ] microenvironments within more oxygenated waters. However, the CP strains appear to be adapted to somewhat higher concentrations of O 2 , as indicated by the presence of genes encoding aa 3 -type cytochrome c oxidases, but not the cbb 3 -type found in all other Zetaproteobacteria isolate genomes. Overall, our results reveal adaptations for life in a physically dynamic, low Fe(II) water column, suggesting that niche-specific strategies can enable Zetaproteobacteria to live in any environment with Fe(II).

A novel iron-oxidizing chemolithoautotrophic bacterium, strain ET2 T , was isolated from a deep-sea sediment in a hydrothermal field of the Bayonnaise knoll of the Izu-Ogasawara arc. Cells were bean-shaped, curved short rods. Growth was observed at a temperature range of 15-30 °C (optimum 25 °C, doubling time 24 h) and a pH range of 5.8-7.0 (optimum pH 6.4) in the presence of NaCl at a range of 1.0-4.0 % (optimum 2.75 %). The isolate was a microaerophilic, strict chemolithoautotroph capable of growing using ferrous iron and molecular oxygen (O 2 ) as the sole electron donor and acceptor, respectively; carbon dioxide as the sole carbon source; and either ammonium or nitrate as the sole nitrogen source. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the new isolate was related to the only previously isolated Mariprofundus species, M. ferrooxydans. Although relatively high 16S rRNA gene similarity (95 %) was found between the new isolate and M. ferrooxydans, the isolate was distinct in terms of cellular fatty acid composition, genomic DNA G+C content and cell morphology. Furthermore, genomic comparison between ET2 T and M. ferrooxydans PV-1 indicated that the genomic dissimilarity of these strains met the standard for species-level differentiation. On the basis of its physiological and molecular characteristics, strain ET2 T (= KCTC 15556 T  = JCM 30585 T ) represents a novel species of Mariprofundus, for which the name Mariprofundus micogutta is proposed. We also propose the subordinate taxa Mariprofundales ord. nov. and Zetaproteobacteria classis nov. in the phylum Proteobacteria.

Previous studies of microbial communities in deep-sea hydrothermal ferric deposits have demonstrated that members of Zetaproteobacteria play significant ecological roles in biogeochemical iron-cycling. However, the ecophysiological characteristics and interaction between other microbial members in the habitat still remain largely unknown. In this study, we investigated microbial communities in a core sample obtained from shallow hydrothermal iron-oxyhydroxide deposits at Nagahama Bay of Satsuma Iwo-Jima, Japan. Scanning electron microscopic observation showed numerous helical stalk structures, suggesting the occurrence of iron-oxidizing bacteria. Analysis of 16S rRNA gene sequences indicated the co-occurrence of iron-oxidizing Zetaproteobacteria and iron-reducing bacteria such as the genera Deferrisoma and Desulfobulbus with strong correlations on the sequence abundance. CARD-FISH indicated that the numbers of Zetaproteobacteria were not always consistent to the frequency of stalk structures. In the stalk-abundant layers with relatively small numbers of Zetaproteobacteria cells, accumulation of polyphosphate was observed inside Zetaproteobacteria cells, whereas no polyphosphate grains were observed in the topmost layers with fewer stalks and abundant Zetaproteobacteria cells. These results suggest that Zetaproteobacteria store intracellular polyphosphates during active iron oxidation that contributes to the mineralogical growth and biogeochemical iron cycling.

Neutrophilic microaerophilic iron-oxidizing bacteria (FeOB) are thought to play a significant role in cycling of carbon, iron and associated elements in both freshwater and marine iron-rich environments. However, the roles of the neutrophilic microaerophilic FeOB are still poorly understood due largely to the difficulty of cultivation and lack of functional gene markers. Here, we analyze the genomes of two freshwater neutrophilic microaerophilic stalk-forming FeOB, Ferriphaselus amnicola OYT1 and Ferriphaselus strain R-1. Phylogenetic analyses confirm that these are distinct species within Betaproteobacteria; we describe strain R-1 and propose the name F. globulitus. We compare the genomes to those of two freshwater Betaproteobacterial and three marine Zetaproteobacterial FeOB isolates in order to look for mechanisms common to all FeOB, or just stalk-forming FeOB. The OYT1 and R-1 genomes both contain homologs to cyc2, which encodes a protein that has been shown to oxidize Fe in the acidophilic FeOB, Acidithiobacillus ferrooxidans. This c-type cytochrome common to all seven microaerophilic FeOB isolates, strengthening the case for its common utility in the Fe oxidation pathway. In contrast, the OYT1 and R-1 genomes lack mto genes found in other freshwater FeOB. OYT1 and R-1 both have genes that suggest they can oxidize sulfur species. Both have the genes necessary to fix carbon by the Calvin-Benson-Basshom pathway, while only OYT1 has the genes necessary to fix nitrogen. The stalk-forming FeOB share xag genes that may help form the polysaccharide structure of stalks. Both OYT1 and R-1 make a novel biomineralization structure, short rod-shaped Fe oxyhydroxides much smaller than their stalks; these oxides are constantly shed, and may be a vector for C, P, and metal transport to downstream environments. Our results show that while different FeOB are adapted to particular niches, freshwater and marine FeOB likely share common mechanisms for Fe oxidation electron transport and biomineralization pathways.

This study was conducted to assess the abundance of iron-oxidizing bacteria and biological sulfur oxidation potential from soil impacted by coal and coal refuse from two coal-burning electric power facilities located at the U.S. Department of Energy's Savannah River Site (Aiken, S.C.) and the South Carolina Electric and Gas Site at Beech Island, S. C. Significantly higher MPN counts of iron-oxidizing bacteria were obtained from samples collected at the confluence of a coal storage runoff containment basin, a coal reject (refuse) pile, and an adjacent wetland at the Savannah River Site. Significant differences in pH, sulfate-S, ferrous- and ferric-iron were also obtained between sampling locations. No significant differences in ferric/ferrous ratioswere determined. These ratios however, exceeded a value of 2.0 when sample pH values were less than 4.5. Under optimal conditions, biological thiosulfate-S oxidation potentials (in vitro) showed a 4- to 7-day lag in the appearance of sulfate-S, and a final pH (after twenty-four days of perfusion) of 1.97 to 3.90. These results indicate that contamination of subsurface water by acidic leachate derived from thionic bacterial activity will occur if coal and coal refuse piles are not confined by an impermeable surface or containment facility. [ABSTRACT FROM AUTHOR]