Fe(II)-oxidizing Bacteria Research Articles

Microbe interactions drive the formation of floating iron films in circumneutral wetlands

Floating iron (Fe) films are widely found in wetlands that can form oxic-anoxic boundaries under circumneutral conditions. These films play a crucial role in the redox transformations and bioavailability of nutrients and trace metals. Current studies mainly focus on chemical oxidation during Fe film formation under circumneutral conditions. The functional microorganisms and associated microbial processes involved in Fe film formation have yet to be investigated in detail. Here, we investigated the microbial communities and involved microbial processes for the formation of floating Fe films in wetlands. Ferrihydrite was the dominant Fe(III) phase in films, accompanied by moderate levels of carbon and silicon. The Fe species and microbial analysis indicated that Fe films contain mixed-valent Fe and can form biotically. Microbial community analysis showed that the dominant genera in these Fe films were Fe-oxidizing and reducing bacteria and methanotrophs, including Leptothrix, Ferriphasclus, Gallionella, Geobacter and Methylococcales. Leptothrix, Ferriphasclus and Gallionella, as classical Fe(II)-oxidizing bacteria (FeOB), can oxidize Fe(II) with limited oxygen and form special structures that are consistent with Fe film morphology. Geobacter can provide a source of Fe(II) for FeOB growth, and Methylococcales can perform methane oxidation to provide energy for Fe cycling. The high ratios of Gallionella- and Geobacter-related microorganisms and carbon fixation genes proved the contribution of potential of Fe cycling and autotrophic microbial communities to the formation of Fe films. The diversity of microbial community suggested that Fe(II) oxidation could trigger carbon fixation, while Fe(III) reduction accelerated Fe and carbon cycling through anaerobic respiration and autotrophic chemosynthesis. These results highlight the contribution of these multiple microbial processes to Fe and carbon cycling during the formation of floating Fe films in wetlands. However, further studies are required to fully elucidate the interaction of functional microorganisms involved in floating film formation and their biogeochemical role in wetlands.

Metagenomic analysis of Fe(II)-oxidizing bacteria for Fe(III) mineral formation and carbon assimilation under microoxic conditions in paddy soil

Microbially mediated Fe(II) oxidation is prevalent and thought to be central to many biogeochemical processes in paddy soils. However, we have limited insights into the Fe(II) oxidation process in paddy fields, considered the world's largest engineered wetland, where microoxic conditions are ubiquitous. In this study, microaerophilic Fe(II) oxidizing bacteria (FeOB) from paddy soil were enriched in gradient tubes with FeS, FeCO3, and Fe3(PO4)2 as iron sources to investigate their capacity for Fe(II) oxidation and carbon assimilation. Results showed that the highest rate of Fe(II) oxidation (k = 0.836 mM d−1) was obtained in the FeCO3 tubes, and cells grown in the Fe3(PO4)2 tubes yielded maximum assimilation amounts of 13C-NaHCO3 of 1.74% on Day 15. Amorphous Fe(III) oxides were found in all the cell bands with iron substrates as a result of microbial Fe(II) oxidation. Metagenomics analysis of the enriched microbes targeted genes encoding iron oxidase Cyc2, oxygen-reducing terminal oxidase, and ribulose-bisphosphate carboxylase, with results indicated that the potential Fe(II) oxidizers include nitrate-reducing FeOB (Dechloromonas and Thiobacillus), Curvibacter, and Magnetospirillum. By combining cultivation-dependent and metagenomic approaches, our results found a number of FeOB from paddy soil under microoxic conditions, which provide insight into the complex biogeochemical interactions of iron and carbon within paddy fields. The contribution of the FeOB to the element cycling in rice-growing regions deserves further investigation.

‘Candidatus ferrigenium straubiae’ sp. nov., ‘Candidatus ferrigenium bremense’ sp. nov., ‘Candidatus ferrigenium altingense’ sp. nov., are autotrophic Fe(II)-oxidizing bacteria of the family Gallionellaceae

Iron(II) [Fe(II)] oxidation coupled to denitrification is recognized as an environmentally important process in many ecosystems. However, the Fe(II)-oxidizing bacteria (FeOB) dominating autotrophic nitrate-reducing Fe(II)-oxidizing enrichment cultures, affiliated with the family Gallionellaceae, remain poorly taxonomically defined due to lack of representative isolates. We describe the taxonomic classification of three novel FeOB based on metagenome-assembled genomes (MAGs) acquired from the autotrophic nitrate-reducing enrichment cultures KS, BP and AG. Phylogenetic analysis of nearly full-length 16S rRNA gene sequences demonstrated that these three FeOB were most closely affiliated to the genera Ferrigenium, Sideroxydans and Gallionella, with up to 96.5%, 95.4% and 96.2% 16S rRNA gene sequence identities to representative isolates of these genera, respectively. In addition, average amino acid identities (AAI) of the genomes compared to the most closely related genera revealed highest AAI with Ferrigenium kumadai An22 (76.35–76.74%), suggesting that the three FeOB are members of this genus. Phylogenetic analysis of conserved functional genes further supported that these FeOB represent three novel species of the genus Ferrigenium. Moreover, the three novel FeOB likely have characteristic features, performing partial denitrification coupled to Fe(II) oxidation and carbon fixation. Scanning electron microscopy of the enrichment cultures showed slightly curved rod-shaped cells, ranging from 0.2-0.7 μm in width and 0.5–2.3 μm in length. Based on the phylogenetic, genomic and physiological characteristics, we propose that these FeOB represent three novel species, ‘Candidatus Ferrigenium straubiae’ sp. nov., ‘Candidatus Ferrigenium bremense’ sp. nov. and ‘Candidatus Ferrigenium altingense’ sp. nov. that might have unique metabolic features among the genus Ferrigenium.

Unraveling Fe(II)-Oxidizing Mechanisms in a Facultative Fe(II) Oxidizer, Sideroxydans lithotrophicus Strain ES-1, via Culturing, Transcriptomics, and Reverse Transcription-Quantitative PCR.

ABSTRACTSideroxydans lithotrophicus ES-1 grows autotrophically either by Fe(II) oxidation or by thiosulfate oxidation, in contrast to most other isolates of neutrophilic Fe(II)-oxidizing bacteria (FeOB). This provides a unique opportunity to explore the physiology of a facultative FeOB and constrain the genes specific to Fe(II) oxidation. We compared the growth of S. lithotrophicus ES-1 on Fe(II), thiosulfate, and both substrates together. While initial growth rates were similar, thiosulfate-grown cultures had higher yield with or without Fe(II) present, which may give ES-1 an advantage over obligate FeOB. To investigate the Fe(II) and S oxidation pathways, we conducted transcriptomics experiments, validated with reverse transcription-quantitative PCR (RT-qPCR). We explored the long-term gene expression response at different growth phases (over days to a week) and expression changes during a short-term switch from thiosulfate to Fe(II) (90 min). The dsr and sox sulfur oxidation genes were upregulated in thiosulfate cultures. The Fe(II) oxidase gene cyc2 was among the top expressed genes during both Fe(II) and thiosulfate oxidation, and addition of Fe(II) to thiosulfate-grown cells caused an increase in cyc2 expression. These results support the role of Cyc2 as the Fe(II) oxidase and suggest that ES-1 maintains readiness to oxidize Fe(II), even in the absence of Fe(II). We used gene expression profiles to further constrain the ES-1 Fe(II) oxidation pathway. Notably, among the most highly upregulated genes during Fe(II) oxidation were genes for alternative complex III, reverse electron transport, and carbon fixation. This implies a direct connection between Fe(II) oxidation and carbon fixation, suggesting that CO2 is an important electron sink for Fe(II) oxidation.IMPORTANCE Neutrophilic FeOB are increasingly observed in various environments, but knowledge of their ecophysiology and Fe(II) oxidation mechanisms is still relatively limited. Sideroxydans isolates are widely observed in aquifers, wetlands, and sediments, and genome analysis suggests metabolic flexibility contributes to their success. The type strain ES-1 is unusual among neutrophilic FeOB isolates, as it can grow on either Fe(II) or a non-Fe(II) substrate, thiosulfate. Almost all our knowledge of neutrophilic Fe(II) oxidation pathways comes from genome analyses, with some work on metatranscriptomes. This study used culture-based experiments to test the genes specific to Fe(II) oxidation in a facultative FeOB and refine our model of the Fe(II) oxidation pathway. We gained insight into how facultative FeOB like ES-1 connect Fe, S, and C biogeochemical cycling in the environment and suggest a multigene indicator would improve understanding of Fe(II) oxidation activity in environments with facultative FeOB.

Geographical Distribution of Iron Redox Cycling Bacterial Community in Peatlands: Distinct Assemble Mechanism Across Environmental Gradient.

Microbial-mediated iron (Fe) oxidation and reduction greatly contribute to the biogeochemistry and mineralogy of ecosystems. However, knowledge regarding the composition and distribution patterns of iron redox cycling bacteria in peatlands remains limited. Here, using high-throughput sequencing, we compared biogeographic patterns and assemblies of the iron redox cycling bacterial community between soil and water samples obtained from different types of peatland across four regions in Northeast China. A total of 48 phylotypes were identified as potential iron redox bacteria, which had greater than 97% similarity with Fe(II)-oxidizing bacteria (FeOB) and Fe(III)-reducing bacteria (FeRB). Among them, Rhodoferax, Clostridium, Geothrix, Sideroxydans, Geobacter, Desulfovibrio, and Leptothrix could be used as bioindicators in peatlands for characterizing different hydrological conditions and nutrient demands. Across all samples, bacterial communities associated with iron redox cycling were mainly affected by pH, dissolved organic carbon (DOC), and Fe2+. Distance–decay relationship (DDR) analysis indicated that iron redox cycling bacterial communities in soil, but not in water, were highly correlated with geographic distance. Additionally, null model analysis revealed that stochastic processes substituted deterministic processes from minerotrophic fens to ombrotrophic bogs in soils, whereas deterministic processes were dominant in water. Overall, these observations suggest that bacteria involved in iron redox cycling are widespread in diverse habitats and exhibit distinct patterns of distribution and community assembly mechanisms between soil and water in peatlands.

The Weathering Microbiome of an Outcropping Granodiorite.

Microorganisms have long been recognized for their capacity to catalyze the weathering of silicate minerals. While the vast majority of studies on microbially mediated silicate weathering focus on organotrophic metabolism linked to nutrient acquisition, it has been recently demonstrated that chemolithotrophic ferrous iron [Fe(II)] oxidizing bacteria (FeOB) are capable of coupling the oxidation of silicate mineral Fe(II) to metabolic energy generation and cellular growth. In natural systems, complex microbial consortia with diverse metabolic capabilities can exist and interact to influence the biogeochemical cycling of essential elements, including iron. Here we combine microbiological and metagenomic analyses to investigate the potential interactions among metabolically diverse microorganisms in the near surface weathering of an outcrop of the Rio Blanco Quartz Diorite (DIO) in the Luquillo Mountains of Puerto Rico. Laboratory based incubations utilizing ground DIO as metabolic energy source for chemolithotrophic FeOB confirmed the ability of FeOB to grow via the oxidation of silicate-bound Fe(II). Dramatically accelerated rates of Fe(II)-oxidation were associated with an enrichment in microorganisms with the genetic capacity for iron oxidizing extracellular electron transfer (EET) pathways. Microbially oxidized DIO displayed an enhanced susceptibility to the weathering activity of organotrophic microorganisms compared to unoxidized mineral suspensions. Our results suggest that chemolithotrophic and organotrophic microorganisms are likely to coexist and contribute synergistically to the overall weathering of the in situ bedrock outcrop.

Changes in the microbial community during microbial microaerophilic Fe(II) oxidation at circumneutral pH enriched from paddy soil.

Fe(II)-oxidizing bacteria (FeOB) are important catalysts for iron cycling in iron-rich marine, groundwater, and freshwater environments. However, few studies have reported the distribution and diversity of these bacteria in flooded paddy soils. This study investigates the microbial structure and diversity of microaerophilic Fe(II)-oxidizing bacteria (mFeOB) and their possible role in Fe(II) oxidation in iron-rich paddy soils. Using enrichment experiments that employed serial transfers, the changes in microaerophilic microbial community were examined via 16S rRNA gene high-throughput sequencing. During enrichments, the Fe(II) oxidation rate decreased as transfers increased, and the maximum rate of Fe(II) oxidation was observed in the first transfer (0.197mMday-1). Results from X-ray diffraction of minerals and scanning electron microscopy of the cell-mineral aggregates revealed that cell surfaces in all transfers were partly covered with amorphous iron oxide formed by FeOB. After four transfers, the phyla of Proteobacteria had a dominant presence that reached up to 95%. Compared with the original soil, the relative abundances of Cupriavidus, Massilia, Pseudomonas, Ralstonia, Sphingomonas, and Variovorax increased in FeS gradient tubes and became dominant genera after transfers. Cupriavidus, Pseudomonas, and Ralstonia have been identified as FeOB previously. Furthermore, the structure of the microbial community tended to be stable as transfers increased, indicating that other bacterial species might perform important roles in Fe(II) oxidation. These results suggest the potential involvement of mFeOB and these other microorganisms in the Fe(II)-oxidizing process of soils. It will be helpful for future studies to consider their role in related biogeochemical processes, such as transformation of organic matters and heavy metals.

Involvement of microaerophilic iron-oxidizing bacteria in the iron-oxidizing process at the surface layer of flooded paddy field soil

To reveal whether microaerophilic Fe(II)-oxidizing bacteria (FeOB) participate in the Fe(II) oxidation at the oxic-anoxic interface in flooded paddy field soil, distribution of microaerophilic FeOB belonging to Gallionellaceae (Gallionella-related FeOB) in the surface layer of a flooded paddy soil microcosm and O2 conditions for the Fe(II) oxidation by a microaerophilic Fe(II) oxidizer, Ferrigenium kumadai An22, were investigated. Flooded paddy soil microcosms were incubated for 30 days. Five soil layers were sampled at 2-mm intervals from the soil surface after the incubation. The community structure of Gallionella-related FeOB was analyzed with qPCR and DGGE methods. In the culture experiment, O2 and Fe(II) profiles in F. kumadai An22-inoculated and non-inoculated gel-stabilized gradient tubes were analyzed, in which an opposing gradient of O2 and Fe(II) was formed. A thin oxic layer was formed at the soil surface after the incubation. The copy number of 16S rRNA genes of Gallionella-related FeOB was highest at the top 0–2 mm layer of the soil. DGGE analysis showed that several bands derived from Gallionella-related FeOB newly appeared at the top soil layer with high intensity. In the culture experiment, F. kumadai A22 grew at the oxic side of the oxic-anoxic interface with the formation of Fe oxides. The present study indicated that Gallionella-related microaerophilic FeOB proliferated in the surface layer of flooded paddy field soil, presumably by oxidizing Fe(II) in the oxic-anoxic interface.

Micro- and nano-scale mineralogical characterization of Fe(II)-oxidizing bacterial stalks.

Neutrophilic, microaerobic Fe(II)-oxidizing bacteria (FeOB) from marine and freshwater environments are known to generate twisted ribbon-like organo-mineral stalks. These structures, which are extracellularly precipitated, are susceptible to chemical influences in the environment once synthesized. In this paper, we characterize the minerals associated with freshwater FeOB stalks in order to evaluate key organo-mineral mechanisms involved in biomineral formation. Micro-Raman spectroscopy and Field Emission Scanning Electron Microscopy revealed that FeOB isolated from drinking water wells in Sweden produced stalks with ferrihydrite, lepidocrocite and goethite as main mineral components. Based on our observations made by micro-Raman Spectroscopy, field emission scanning electron microscopy and scanning transmission electron microscope combined with electron energy-loss spectroscopy, we propose a model that describes the crystal-growth mechanism, the Fe-oxidation state, and the mineralogical state of the stalks, as well as the biogenic contribution to these features. Our study suggests that the main crystal-growth mechanism in stalks includes nanoparticle aggregation and dissolution/re-precipitation reactions, which are dominant near the organic exopolymeric material produced by the microorganism and in the peripheral region of the stalk, respectively.

Microbial communities and biogenic Mn-oxides in an on-site biofiltration system for cold Fe-(II)- and Mn(II)-rich groundwater treatment

This study investigated relationships between microbial communities, groundwater chemistry, and geochemical and mineralogical characteristics in field-aged biofilter media from a two-stage, pilot-scale, flow-through biofiltration unit designed to remove Fe(II) and Mn(II) from cold groundwater (8 to 15 °C). High-throughput 16S rRNA gene amplicon sequencing of influent groundwater and biofilter samples (solids, effluents, and backwash water) revealed significant differences in the groundwater, Fe filter, and Mn filter communities. These community differences reflect conditions in each filter that select for populations that biologically oxidize Fe(II) and Mn(II) in the two filters, respectively. Genera identified in both filters included relatives of known Fe(II)-oxidizing bacteria (FeOB), Mn(II)-oxidizing bacteria (MnOB), and ammonia-oxidizing bacteria (AOB). Relatives of AOB and nitrite-oxidizing bacteria were abundant in sequencing reads from both filters. Relatives of FeOB in class Betaproteobacteria dominated the Fe filter. Taxa related to Mn-oxidizing organisms were minor members of the Mn-filter communities; intriguingly, while Alphaproteobacteria dominated (40 ± 10% of sequencing reads) the Mn filter community, these Alphaproteobacteria did not classify as known MnOB. Isolates from Fe and Mn filter backwash enrichment studies provide insight on the identity of MnOB in this system. Novel putative MnOB isolates included Azospirillum sp. CDMB, Solimonas soli CDMK, and Paenibacillus sp. CDME. The isolate Hydrogenophaga strain CDMN can oxidize Mn(II) at 8 °C; this known FeOB is likely capable of Mn(II) oxidation in this system. Synchrotron-based X-ray near-edge spectroscopy (XANES) coupled with electron paramagnetic resonance (EPR) revealed the dominant Mn-oxide that formed was biogenic birnessite. Co-existence of amorphous and crystallized Mn-oxide surface morphologies on the Mn-filter media suggest occurrence of both biological and autocatalytic Mn(II) oxidation in the biofilter. This study provides evidence that biofiltration is a viable approach to remove iron, manganese, and ammonia in cold groundwater systems, and that mineralogical and microbiological approaches can be used to monitor biofiltration system efficacy and function.

Community structure of microaerophilic iron-oxidizing bacteria in Japanese paddy field soils

ABSTRACT Redox cycle of iron (Fe) is the central process in the biogeochemistry of paddy field soil. Although Fe(II)-oxidizing process is mediated by both abiotic and biotic reactions, microorganisms involved in the process have not been well studied in paddy field soil. The present study investigated the community structure of microaerophilic Fe(II)-oxidizing bacteria (FeOB) in the family Gallionellaceae in the plow layer of paddy fields located in the central (Anjo) and northeastern (Omagari) Japan since the members in the family are the typical FeOB in circumneutral freshwater environments and possibly have the significant role for Fe(II) oxidation in paddy field soils. A primer set targeting 16S rRNA gene of Gallionella-related FeOB was newly designed for the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR (qPCR) analyses. DGGE analysis showed significant differences in the band patterns between the field sites. Besides, minor differences were observed in the patterns between the soil depths (0–1 cm and below 1 cm) in the Anjo field, while the patterns were relatively stable in the Omagari field during the annual rice cultivation practices. In total 54 bands were sequenced and clustered into 20 operational taxonomic units (OTUs) on the basis of the 97% similarity. Eighteen out of twenty OTUs (50 of 54 bands) were affiliated within the FeOB cluster of Gallionellaceae, some of which were clustered with known microaerophilic FeOB, Ferrigenium kumadai, Ferriphaselus amnicola, ‘Sideroxydans lithotrophicus’ and ‘S. paludicola’. The number of the 16S rRNA gene copies was 105–107 and 106–108 copies g−1 dried soil in the two paddy fields and negatively correlated to the contents of acetate-extractable Fe(II) in the soils during the rice cultivation period. These results suggested inhabitance of considerable number of diverse Gallionella-related FeOB and their potential involvement in the Fe(II)-oxidizing process of soil, especially during the rice cultivation period in the paddy field soils.

Effects of Mn(II) on the Oxidation of Fe in Soils and the Uptake of Cadmium by Rice (Oryza sativa)

Soil properties affect the bioavailability of cadmium (Cd). In general, soils in southern China are rich in iron (Fe) and contain manganese (Mn), which form ferromanganese nodules through biological or chemical reactions. In this study, it is hypothesized that the formation of Fe/Mn oxides can reduce the bioavailability of Cd and play an important role in reducing the uptake of Cd by rice. In this study, the effects of Fe and Mn on the contents of Fe (II)-oxidizing bacteria (FeOB) communities, Fe oxides, and Cd speciation in Cd-contaminated paddy soils were studied in a pot experiment. Results showed that, compared with the CK treatment, the addition of Mn (II) increased the abundance of FeOB (Thiobacillus sp.) in the soil, thereby increasing the content of amorphous Fe oxides in the rhizosphere. Amorphous Fe oxides effectively immobilized Cd in the soil, that is, with an increasing content of Cd bound to amorphous Fe/Mn oxides in the rhizosphere and non-rhizosphere soils, the mobility and bioavailability of Cd were reduced. After the addition of Mn (II) and Fe (II), the Fe content in the root Fe plaque of rice increased significantly, while the Cd content decreased significantly. The increase of Fe in the root Fe plaque inhibited rice uptake of soil Cd; thus, the Cd content in rice shoots was significantly lower than the Cd content in the CK treatment. Compared with the addition of Fe (II), the addition of Mn (II) effectively reduced the Cd content in rice shoots. These findings suggested that the addition of Mn (II) can promote the formation of Fe oxides in soils and reduce the Cd content in rice. These research results provide support for using Mn-based soil remediation materials to remedy Cd-contaminated soils.

Microaerobic Fe(II) oxidation coupled to carbon assimilation processes driven by microbes from paddy soil

Microaerobic Fe(II) oxidation process at neutral pH, driven by microbes can couple to carbon assimilation process in iron-rich freshwater and marine environments; however, few studies report such coupled processes in paddy soil of the critical zone in South China. In this study, rhizosphere soil from flooded paddy field was used as the inoculum to enrich the microaerophilic Fe(II)-oxidizing bacteria (FeOB) in gradient tubes with different Fe(II) substrates (FeS and FeCO3) and 13C-biocarbonate as inorganic carbon source to track the carbon assimilation. Kinetics of Fe(II) oxidation and biomineralization were analyzed, and the composition and abundance of the microbial community were profiled using 16S rRNA gene-based high-throughput sequencing. Results showed that microbial cell bands were formed 0.5–1.0 cm below the medium surface in the inoculated tubes with Fe(II) substances, while no cell band was found in the non-inocula controls. The protein concentrations in the cell bands reached the highest values at 18.7–22.9 mg mL-1 on 6 d in the inocula tubes with Fe(II) substrates. A plateau of the yields of 13C-biocarbonate incorporation was observed during 6–15 d at 0.44–0.54% and 1.61–1.98% in the inocula tubes with FeS and FeCO3, respectively. The inocula tube with FeS showed a higher Fe(II) oxidation rate of 0.156 mmol L-1 d-1 than that with FeCO3 (0.106 mmol L-1 d-1). Analyses of X-ray diffraction and scanning electron microscopy with energy-dispersive X-ray spectroscopy revealed that amorphous iron oxide was formed on the surface of rod-shaped bacteria after Fe(II) oxidation. Relative to the agar only control, the abundances of Clostridium and Pseudogulbenkiania increased in the inocula tube with FeS, while those of Vogesella, Magnetospirillum, Solitalea, and Oxalicibacterium increased in the inocula tube with FeCO3, all of which might be the potential microaerophilic FeOB in paddy soil. The findings in this study suggest that microbes that couple microaerobic Fe(II) oxidation to carbon assimilation existed in the paddy soil, which provides an insight into the iron-carbon coupling transformation under microaerobic conditions in the critical zone of the iron-rich red soil.

High diversity of potential nitrate-reducing Fe(II)-oxidizing bacteria enriched from activated sludge.

Nitrate-dependent Fe(II) oxidation (NDFO) has been discovered in various environments including activated sludge and can potentially be used to remove nitrate from wastewater. In this study, NDFO sludge was successfully enriched from activated sludge under high Fe(II) concentrations over 100days and the denitrification rate achieved 1.37mmolN/(gVSSday). High-throughput sequencing of the bacterial 16S rRNA gene was used to investigate the microbial community structure dynamics during the enrichment process. The results showed that the microbial community changed significantly and high diversity of potential Fe(II)-oxidizing bacteria (FeOB) was observed in the enriched sludge. Thermomonas and Gallionella were the dominant bacterial genera in the enriched sludge and their relative abundances accounted for 9.49 and 4.08%, respectively. Furthermore, it was found that potential FeOB were also abundantly present in activated sludge samples of common municipal wastewater treatment plants. Collectively, this study demonstrated that NDFO could be successfully performed by enriched activated sludge and high diversity of bacteria is involved in this process, and the results also provide baseline information for future research and engineering application of NDFO process.

Comparative Genomic Analysis of Neutrophilic Iron(II) Oxidizer Genomes for Candidate Genes in Extracellular Electron Transfer.

Extracellular electron transfer (EET) is recognized as a key biochemical process in circumneutral pH Fe(II)-oxidizing bacteria (FeOB). In this study, we searched for candidate EET genes in 73 neutrophilic FeOB genomes, among which 43 genomes are complete or close-to-complete and the rest have estimated genome completeness ranging from 5 to 91%. These neutrophilic FeOB span members of the microaerophilic, anaerobic phototrophic, and anaerobic nitrate-reducing FeOB groups. We found that many microaerophilic and several anaerobic FeOB possess homologs of Cyc2, an outer membrane cytochrome c originally identified in Acidithiobacillus ferrooxidans. The “porin-cytochrome c complex” (PCC) gene clusters homologous to MtoAB/PioAB are present in eight FeOB, accounting for 19% of complete and close-to-complete genomes examined, whereas PCC genes homologous to OmbB-OmaB-OmcB in Geobacter sulfurreducens are absent. Further, we discovered gene clusters that may potentially encode two novel PCC types. First, a cluster (tentatively named “PCC3”) encodes a porin, an extracellular and a periplasmic cytochrome c with remarkably large numbers of heme-binding motifs. Second, a cluster (tentatively named “PCC4”) encodes a porin and three periplasmic multiheme cytochromes c. A conserved inner membrane protein (IMP) encoded in PCC3 and PCC4 gene clusters might be responsible for translocating electrons across the inner membrane. Other bacteria possessing PCC3 and PCC4 are mostly Proteobacteria isolated from environments with a potential niche for Fe(II) oxidation. In addition to cytochrome c, multicopper oxidase (MCO) genes potentially involved in Fe(II) oxidation were also identified. Notably, candidate EET genes were not found in some FeOB, especially the anaerobic ones, probably suggesting EET genes or Fe(II) oxidation mechanisms are different from the searched models. Overall, based on current EET models, the search extends our understanding of bacterial EET and provides candidate genes for future research.

Divalent metal cation adsorption onto Leptothrix cholodnii SP-6SL bacterial cells

Bacteriogenic iron oxide (BIOS) particles have a high affinity to adsorb inorganic nutrients such as Ca, Mg, Zn, and Ni, raising the question of how Fe(II) oxidizing bacteria (FeOB) compete for these same cations while the BIOS are in such close proximity to the cells. Answering this question requires a detailed understanding of metal binding properties of FeOB.In this study, the adsorption behaviors of aqueous Ni, Cu, Zn, Sr, Cd, and Pb onto the sheathless FeOB Leptothrix cholodnii SP-6SL were measured separately, and the adsorption behaviors were compared to that of Bacillus subtilis in order to determine if the iron oxidizing bacterial species exhibits similar binding properties to those of most previously studied bacterial species. The experiments for both species were performed aerobically; ionic strength was held constant with 0.1M NaClO4; the experiments were conducted as a function of pH over the range of 2 to 9; and biomass and metal concentrations were 10g (wet weight)/L and 2ppm, respectively.Our results show that the two studied bacterial species exhibit similar adsorption of the tested metals, and that although some iron oxides formed during L. cholodnii SP-6SL liquid culture growth, they are present in low enough concentrations not to significantly affect the extent of metal adsorption. We apply a linear free-energy approach to define relationships between the stability constants for site-specific metal-bacterial surface complexes and corresponding metal-acetate binding constants. We use these relationships to estimate binding constants for L. cholodnii for some metals that have not been studied, and we apply these results to calculate the ability of L. cholodnii to compete for nutrients with BIOS particles that form during Fe(II) oxidation by the bacteria.

Iron encrustations on filamentous algae colonized by <i>Gallionella</i>-related bacteria in a metal-polluted freshwater stream

Abstract. Filamentous macroscopic algae were observed in slightly acidic to circumneutral (pH 5.9–6.5), metal-rich stream water that leaked out from a former uranium mining district (Ronneburg, Germany). These algae differed in color and morphology and were encrusted with Fe-deposits. To elucidate their potential interaction with Fe(II)-oxidizing bacteria (FeOB), we collected algal samples at three time points during summer 2013 and studied the algae-bacteria-mineral compositions via confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectra, and a 16S and 18S rRNA gene-based bacterial and algae community analysis. Surprisingly, sequencing analysis of 18S rRNA gene regions of green and brown algae revealed high homologies with the freshwater algae Tribonema (99.9–100 %). CLSM imaging indicated a loss of active chloroplasts in the algae cells, which may be responsible for the change in color in

Microbial mineral colonization across a subsurface redox transition zone.

This study employed 16S rRNA gene amplicon pyrosequencing to examine the hypothesis that chemolithotrophic Fe(II)-oxidizing bacteria (FeOB) would preferentially colonize the Fe(II)-bearing mineral biotite compared to quartz sand when the minerals were incubated in situ within a subsurface redox transition zone (RTZ) at the Hanford 300 Area site in Richland, WA, USA. The work was motivated by the recently documented presence of neutral-pH chemolithotrophic FeOB capable of oxidizing structural Fe(II) in primary silicate and secondary phyllosilicate minerals in 300 Area sediments and groundwater (Benzine et al., 2013). Sterilized portions of sand+biotite or sand alone were incubated in situ for 5 months within a multilevel sampling (MLS) apparatus that spanned a ca. 2-m interval across the RTZ in two separate groundwater wells. Parallel MLS measurements of aqueous geochemical species were performed prior to deployment of the minerals. Contrary to expectations, the 16S rRNA gene libraries showed no significant difference in microbial communities that colonized the sand+biotite vs. sand-only deployments. Both mineral-associated and groundwater communities were dominated by heterotrophic taxa, with organisms from the Pseudomonadaceae accounting for up to 70% of all reads from the colonized minerals. These results are consistent with previous results indicating the capacity for heterotrophic metabolism (including anaerobic metabolism below the RTZ) as well as the predominance of heterotrophic taxa within 300 Area sediments and groundwater. Although heterotrophic organisms clearly dominated the colonized minerals, several putative lithotrophic (NH4+, H2, Fe(II), and HS- oxidizing) taxa were detected in significant abundance above and within the RTZ. Such organisms may play a role in the coupling of anaerobic microbial metabolism to oxidative pathways with attendant impacts on elemental cycling and redox-sensitive contaminant behavior in the vicinity of the RTZ.

Vertical distribution of Fe and Fe(III)-reducing bacteria in the sediments of Lake Donghu, China.

In lake sediments, iron (Fe) is the most versatile element, and the redox cycling of Fe has a wide influence on the biogeochemical cycling of organic and inorganic substances. The aim of the present study was to analyze the vertical distribution of Fe and Fe(III)-reducing bacteria (FeRB) in the surface sediment (30 cm) of Lake Donghu, China. At the 3 sites we surveyed, FeRB and Fe(II)-oxidizing bacteria (FeOB) coexisted in anoxic sediments. Geobacter-related FeRB accounted for 5%-31% of the total Bacteria, while Gallionella-related FeOB accounted for only 0.1%-1.3%. A significant correlation between the relative abundance of poorly crystalline Fe and Geobacter spp. suggested that poorly crystalline Fe favored microbial Fe(III) reduction. Poorly crystalline Fe and Geobacter spp. were significantly associated with solid-phase Fe(II) and total inorganic phosphorus levels. Pore water Fe(II) concentrations negatively correlated with NO3(-) at all sites. We concluded that Geobacter spp. were abundant in the sediments of Lake Donghu, and the redox of Fe might participate in the cycling of nitrogen and phosphorus in sediments. These observations provided insight into the roles of microbial Fe cycling in lake sediments.

Metagenomic insights into the dominant Fe(II) oxidizing Zetaproteobacteria from an iron mat at Lō´ihi, Hawai´l

Zetaproteobacteria are among the most prevalent Fe(II)-oxidizing bacteria (FeOB) at deep-sea hydrothermal vents; however, knowledge about their environmental significance is limited. We provide metagenomic insights into an iron mat at the Lō´ihi Seamount, Hawai´l, revealing novel genomic information of locally dominant Zetaproteobacteria lineages. These lineages were previously estimated to account for ~13% of all local Zetaproteobacteria based on 16S clone library data. Biogeochemically relevant genes include nitrite reductases, which were previously not identified in Zetaproteobacteria, sulfide:quinone oxidases, and ribulose-1,5-bisphosphate carboxylase (RuBisCo). Genes assumed to be involved in Fe(II) oxidation correlate in synteny and share 87% amino acid similarity with those previously identified in the related Zetaproteobacterium Mariprofundus ferrooxydans PV-1. Overall, Zetaproteobacteria genes appear to originate primarily from within the Proteobacteria and the Fe(II)-oxidizing Leptospirillum spp. and are predicted to facilitate adaptation to a deep-sea hydrothermal vent environment in addition to microaerophilic Fe(II) and H2S oxidation. This dataset represents the first metagenomic study of FeOB from an iron oxide mat at a deep-sea hydrothermal habitat.