Microbial Ferric Iron Reduction Research Articles

A one-million-year isotope record from siderites formed in modern ferruginous sediments

Abstract Ancient iron formations hold important records of environmental conditions during the Precambrian eons. Reconstructions of past oceanic systems require investigation of modern ferruginous analogs to disentangle water column and diagenetic signals recorded in iron-bearing minerals. We analyzed oxygen, iron, and carbon isotopes in siderite, a ferrous carbonate phase commonly used as an environmental proxy, from a 100-m-long record spanning a 1 Ma depositional history in ferruginous Lake Towuti, Indonesia. Combining bulk sediment and pore water geochemistry, we traced processes controlling siderite isotope signatures. We show that siderite oxygen isotope compositions (δ18O) reflect in-lake hydrological and depositional conditions. Low iron isotope values (δ56Fe) record water column oxygenation events over geological timescales, with minor diagenetic partitioning of Fe isotopes by microbial iron reduction after deposition. The carbon isotope compositions (δ13C) reflect the incorporation of biogenic HCO3–, which is consistent with sediment organic matter remineralization lasting over ca. 200 ka after burial. Positive δ13C excursions indicate an increased production of biogenic methane that escaped the sediment during low lake levels. Diffusion across the sediment–water interface during initial formation of siderites tends to align the isotope signatures of bottom waters to those of pore waters. As microbial reduction of ferric iron and oxidation of organic matter proceed and saturate pore water conditions with respect to siderite, overgrowth on nuclei partially mutes the environmental signal inherited from past bottom waters over ca. 1 Ma. Because high depositional fluxes of ferric iron and organic matter in early oceans would have promoted similar microbial processes in ferruginous deposits prior to lithification, the environmental record contained in siderite grains can successively integrate depositional and early diagenetic signals over short geological timescales.

Methane emission suppression in flooded soil from Amazonia

The coupling between ferrous iron and methane production has important global implications, with iron ions acting as electron acceptors for anaerobic oxidation of methane (AOM) and inhibitors of methanogenesis in different environments, including floodplain soils. In this sense, we analyzed the relationship between Fe(II) concentration and methane production in soil layers collected at 0–15 cm and 15–30 cm from flooded-forest and -agroforestry in Amazonian clear water floodplain incubated in anaerobic batch reactors using acetate, formate and glucose as organic sources. High throughput sequencing of archaeal and bacterial 16S rRNA genes was employed to assess the abundance and composition of the active methanogenic and methanotrophic microbial groups potentially involved in Fe(III)-dependent AOM in the soil used as inoculum. Positive correlation was revealed between Fe(II) concentration and methane production, with higher accumulation of Fe(II) in incubated soil layer collected at 0–15 cm in both forest and agroforestry sites for all the three organic sources. The accumulation of Fe(II) in the incubated soil evidenced the oxidation of Fe(III) potentially by Methanobacterium, Desulfobulbus and ‘Candidatus methanoperedens nitroreducens’ living in anaerobic condition at this soil layer. The results point out to the microbial ferric iron reduction as an important potential pathway for anaerobic organic matter decomposition in Amazonian floodplain, evidencing methanogenesis suppression by Fe(III) reduction in flooded-forest and -agroforestry in Amazonian clear water river floodplain.

Fate of Labile Organic Carbon in Paddy Soil Is Regulated by Microbial Ferric Iron Reduction.

Global paddy soil is the primary source of methane, a potent greenhouse gas. It is therefore highly important to understand the carbon cycling in paddy soil. Microbial reduction of iron, which is widely found in paddy soil, is likely coupled with the oxidation of dissolved organic matter (DOM) and suppresses methanogenesis. However, little is known about the biotransformation of small molecular DOM accumulated under flooded conditions and the effect of iron reduction on the biotransformation pathway. Here, we carried out anaerobic incubation experiments using field-collected samples amended with ferrihydrite and different short-chain fatty acids. Our results showed that less than 20% of short-chain fatty acids were mineralized and released to the atmosphere. Using Fourier transform ion cyclotron resonance mass spectrometry, we further found that a large number of recalcitrant molecules were produced during microbial consumption of these short-chain fatty acids. Moreover, the biotransformation efficiency of short-chain fatty acids decreased with the increasing length of carbon chains. Ferrihydrite addition promoted microbial assimilation of short-chain fatty acids as well as enhanced the activation and biotransformation of indigenous stable carbon in the soil replenished with formate. This study demonstrates the significance of ferrihydrite in the biotransformation of labile DOM and promotes a more comprehensive understanding of the coupling of iron reduction and carbon cycling in paddy soils.

Decreases in Iron Oxide Reducibility during Microbial Reductive Dissolution and Transformation of Ferrihydrite.

Ferrous iron formed during microbial ferric iron reduction induces phase transformations of poorly crystalline into more crystalline and thermodynamically more stable iron (oxyhydr)oxides. Yet, characterizing the resulting decreases in the reactivity of the remaining oxide ferric iron toward reduction (i.e., its reducibility) has been challenging. Here, we used the reduction of six-line ferrihydrite by Shewanella oneidensis MR-1 as a model system to demonstrate that mediated electrochemical reduction (MER) allows directly following decreases in oxide ferric iron reducibility during the transformation of ferrihydrite into goethite and magnetite which we characterized by X-ray diffraction analysis and transmission electron microscopy imaging. Ferrihydrite was fully reducible in MER at both pHMER of 5.0 and 7.5. Decreases in iron oxide reducibility associated with ferrihydrite transformation into magnetite were accessible at both pHMER because the formed magnetite was not reducible under either of these conditions. Conversely, decreases in iron oxide reducibility associated with goethite formation were apparent only at the highest tested pHMER of 7.5 and thus the thermodynamically least favorable conditions for iron oxide reductive dissolution. The unique capability to adjust the thermodynamic boundary conditions in MER to the specific reducibilities of individual iron (oxyhydr)oxides makes this electrochemical approach broadly applicable for studying changes in iron oxide reducibility in heterogeneous environmental samples such as soils and sediments.

Effect of elevated pressure on ferric iron reduction coupled to sulfur oxidation by biomining microorganisms

Acidophiles, including a mixed mesophilic, iron-oxidizing culture (FIGB) and a thermophilic enrichment culture (TK65) from a black shale and sandstone copper ore (Kupferschiefer), were used to evaluate microbial ferric iron reduction coupled to oxidation of reduced inorganic sulfur compounds (RISCs) at elevated pressure to simulate potential biogeochemical processes in deep in situ leaching operations. Experiments were performed from atmospheric pressure (0.1 MPa) up to 36 MPa in different incubation chambers and high-pressure reactors. Microbial abundance and activity were determined by quantifying iron and sulfur species concentrations, by direct cell counting, terminal restriction fragment length polymorphism (T-RFLP) analysis and quantitative PCR (qPCR). The data indicate that the tested acidophilic cultures were able to reduce soluble ferric iron coupled to oxidation of sulfur compounds under anaerobic conditions. Even at high pressure (up to 36 MPa) these acidophiles were capable of growing, and microbial ferric iron reduction was only partially inhibited. These results show that the tested acidophiles are barotolerant and their activities may contribute to sulfur and iron cycling in anaerobic environments including deep ore deposits, which is relevant for in situ leaching operations.

Reduction of Iron(III) Ions at Elevated Pressure by Acidophilic Microorganisms

A composed mixed acidophilic, iron-oxidizing culture (FIGB) and a thermo-acidophilic enrichment culture (TK65) were used to evaluate microbial iron(III) reduction coupled to oxidation of reduced inorganic sulfur compounds (RISCs) under high pressure. Experiments were done in batch culture in high pressure vessels at 1 and 100 bar. Microbial abundance and activity were determined by measuring iron(II) concentration, direct cell counting, T-RFLP and quantitative real-time PCR. The data indicate that both cultures are able to reduce soluble iron(III) by oxidation of sulfur compounds under anaerobic conditions. At high pressure (100 bar) these acidophiles were capable of growing and microbial ferric iron reduction was only partially inhibited. These results indicate that acidophiles can be barotolerant and their activities are contributing to sulfur and iron cycling in anaerobic environments including deep ore deposits which is highly relevant for in situ leaching operations.

Citrate influences microbial Fe hydroxide reduction via a dissolution–disaggregation mechanism

Microbial reduction of ferric iron is partly dependent on Fe hydroxide particle size: nanosized Fe hydroxides greatly exceed the bioavailability of their counterparts larger than 1μm. Citrate as a low molecular weight organic acid can likewise stabilize colloidal suspensions against aggregation by electrostatic repulsion but also increase Fe bioavailability by enhancing Fe hydroxide solubility. The aim of this study was to see whether adsorption of citrate onto surfaces of large ferrihydrite aggregates results in the formation of a stable colloidal suspension by electrostatic repulsion and how this effect influences microbial Fe reduction. Furthermore, we wanted to discriminate between citrate-mediated colloid stabilization out of larger aggregates and ferrihydrite dissolution and their influence on microbial Fe hydroxide reduction. Dissolution kinetics of ferrihydrite aggregates induced by different concentrations of citrate and humic acids were compared to microbial reduction kinetics with Geobacter sulfurreducens. Dynamic light scattering results showed the formation of a stable colloidal suspension and colloids with hydrodynamic diameters of 69 (±37) to 165 (± 65) nm for molar citrate:Fe ratios of 0.1 to 0.5 and partial dissolution of ferrihydrite at citrate:Fe ratios⩾0.1. No dissolution or colloid stabilization was detected in the presence of humic acids. Adsorption of citrate, necessary for dissolution, reversed the surface charge and led to electrostatic repulsion between sub-aggregates of ferrihydrite and colloid stabilization when the citrate:Fe ratio was above a critical value (⩽ 0.1). Lower ratios resulted in stronger ferrihydrite aggregation instead of formation of a stable colloidal suspension, owing to neutralization of the positive surface charge. At the same time, microbial ferrihydrite reduction increased from 0.029 to 0.184mMh−1 indicating that colloids stabilized by citrate addition enhanced microbial Fe reduction.Modelling of abiotic dissolution kinetics revealed that colloid stabilization was most pronounced at citrate:Fe ratios of 0.1 – 0.5, whereas higher ratios led to enhanced dissolution of both colloidal and larger aggregated fractions. Mathematical simulation of the microbial reduction kinetics under consideration of partial dissolution and colloid stabilization showed that the bioaccessibility increases in the order large aggregates<stable colloids<Fe-citrate.These findings indicate that much of the organic acid driven mobilization of Fe oxy(hydr)oxides is most likely due to colloid formation and stabilization rather than solubilisation.

Microbial mediated formation of Fe-carbonate minerals under extreme acidic conditions

Discovery of Fe-carbonate precipitation in Rio Tinto, a shallow river with very acidic waters, situated in Huelva, South-western Spain, adds a new dimension to our understanding of carbonate formation. Sediment samples from this low-pH system indicate that carbonates are formed in physico-chemical conditions ranging from acid to neutral pH. Evidence for microbial mediation is observed in secondary electron images (Fig. 1), which reveal rod-shaped bacteria embedded in the surface of siderite nanocrystals. The formation of carbonates in Rio Tinto is related to the microbial reduction of ferric iron coupled to the oxidation of organic compounds. Herein, we demonstrate for the first time, that Acidiphilium sp. PM, an iron-reducing bacterium isolated from Rio Tinto, mediates the precipitation of siderite (FeCO3) under acidic conditions and at a low temperature (30°C). We describe nucleation of siderite on nanoglobules in intimate association with the bacteria cell surface. This study has major implications for understanding carbonate formation on the ancient Earth or extraterrestrial planets.

Diversity and Characterization of Potential H2-Dependent Fe(III)-Reducing Bacteria in Paddy Soils

Microbial ferric iron reduction, with organic carbon or hydrogen as the electron donor, is one of the most important biogeochemical processes in anoxic paddy soils; however, the diversity and community structure of hydrogen-dependent dissimilatory iron-reducers remain unknown. Potential H2-dependent Fe(III)-reducing bacteria in paddy soils were explored using enrichment cultures with ferrihydrite or goethite as the electron acceptor and hydrogen as the electron donor. Terminal restriction fragment length polymorphism (T-RFLP) analysis and cloning/sequencing were conducted to reveal bacterial community structure. Results showed that Geobacter and Clostridium were the dominant bacteria in the enrichment cultures. Fe(III) oxide mineral phases showed a strong effect on the community structure; Geobacter and Clostridium were dominant in the ferrihydrite treatment, while Clostridium spp. were dominant in the goethite treatment. These suggested that H2-dependent Fe(III)-reducing bacteria might be widely distributed in paddy soils and that besides Geobacter, Clostridium spp. might also be an important group of H2-dependent Fe(III)-reducing microorganisms.

Phylogenetic diversity of Fe(III)-reducing microorganisms in rice paddy soil: enrichment cultures with different short-chain fatty acids as electron donors

Microbial ferric iron reduction is an important biogeochemical process in nonsulfidogenic anoxic environments, yet the structure of microbial communities involved is poorly understood because of the lack of a functional gene marker. Here, with ferrihydrite as the iron source, we characterized the potential Fe(III)-reducing bacteria from the paddy soil in the presence of different short-chain fatty acids, formate, acetate, propionate, pyruvate, succinate, and citrate. Enrichment culture was used to characterize the potential Fe(III)-reducing bacteria in the present study. Clone library and terminal restriction fragment length polymorphism (T-RFLP) analyses of bacterial 16S rRNA gene sequence fragments were conducted to reveal the bacterial community structure. T-RFLP and cloning/sequence analysis showed that Firmicutes were the predominant phylotype in all the enrichment cultures (more than 81% of total peak height, more than 75% of all clones), whereas Geobacter spp. represented a small fraction of the bacterial community. Specifically, distinct bacterial families in the phylum Firmicutes were enriched depending on the short-chain fatty acid amended. Clostridium spp. were the predominant microorganisms in pyruvate treatment, while both Bacillus and Clostridium were enriched in formate, acetate, and propionate treatments. Sequences related to Veillonellaceae and Alkaliphilus were predominant in succinate and citrate treatment, respectively. The results indicated Fe(III)-reducing microorganisms in rice paddy soil are phylogenetically diverse. Besides the well-known Geobacter species, Firmicutes-related Fe(III)-reducing bacteria might also be an important group of Fe(III) reducers in paddy soil. To confirm the populations which play an important role in situ, further studies with culture-independent mRNA-based analysis are needed.

Microbe-clay mineral interactions

Clays and clay minerals are common components in soils, sediments, and sedimentary rocks, and they play an important role in many environmental processes. Iron is ubiquitous in clays and clay minerals and its oxidation state, in part, controls the physical and chemical properties of these fine-grained minerals. The structural ferric iron in clay minerals can be reduced either chemically or biologically. Biological reductants include mesophilic and thermophilic microorganisms from diverse environments such as soils, sediments, sedimentary rocks, and hydrothermal hot springs. Multiple clay minerals have been used for microbial reduction studies, including dioctahedral smectite-illite series, palygorskite, chlorite, and their various mixtures in natural soils and sediments. All of these clay minerals are reducible by microorganisms under various conditions with smectite (nontronite) being the most reducible and illite the least. The rate and extent of bioreduction depends on many experimental factors, such as the type of microorganisms and clay minerals, solution chemistry, and temperature. Despite significant efforts, current understanding of the mechanisms of microbial reduction of ferric iron in clay minerals is still limited. Whereas some studies have presented evidence for a solid-state reduction mechanism, others argue that the clay mineral structure partially dissolves when the extent of reduction is high. This inconsistency may be related to several experimental conditions, and their specific effects are discussed in this paper. Whereas past experiments have been largely conducted in well-controlled laboratory systems, recent efforts have attempted to transfer knowledge to the field to improve our understanding of more complex soil systems for better agricultural practices. Biologically reduced clay minerals are also important agents in remediating inorganic and organic contaminants in soil and groundwater systems. This paper reviews the most recent developments and suggests some directions for future research

Evaluation of electron-shuttling compounds in microbial ferric iron reduction

Iron-reducing bacteria can transfer electrons to ferric iron oxides which are barely soluble at neutral pH, and electron-shuttling compounds or chelators are discussed to be involved in this process. Experiments using semipermeable membranes for separation of ferric iron-reducing bacteria from ferric iron oxides do not provide conclusive results in this respect. Here, we used ferrihydrite embedded in 1% agar to check for electron-shuttling compounds in pure and in enrichment cultures. Geobacter sulfurreducens reduced spatially distant ferrihydrite only in the presence of anthraquinone-2,6-disulfonate, a small molecule known to shuttle electrons between the bacterial cell and ferrihydrite. However, indications for the production and excretion of electron-shuttling compounds or chelators were found in ferrihydrite-containing agar dilution cultures that were inoculated with ferric iron-reducing enrichment cultures.

The roles of natural organic matter in chemical and microbial reduction of ferric iron

Although natural organic matter (NOM) is known to be redox reactive, the roles and effectiveness of specific functional groups of NOM in metal reduction are still a subject of intense investigation. This study entails the investigation of the Fe(III) reduction kinetics and capacity by three fractionated NOM subcomponents in the presence or absence of the dissimilatory metal reducing bacterium Shewanella putrefaciens CN32. Results indicate that NOM was able to reduce Fe(III) abiotically; the reduction was pH-dependent and varied greatly with different fractions of NOM. The polyphenolic-rich NOM-PP fraction exhibited the highest reactivity and oxidation capacity at a low pH (<4) as compared with the carbohydrate-rich NOM-CH fraction and a soil humic acid (soil HA) in reducing Fe(III). However, at a pH>4, soil HA showed a relatively high oxidation capacity, probably resulting from its conformational and solubility changes with an increased solution pH. In the presence of S. putrefaciens CN32, all NOM fractions were found to enhance the microbial reduction of Fe(III) under anaerobic, circumneutral pH conditions. Soil HA was found to be particularly effective in mediating the bioreduction of Fe(III) as compared with the NOM-PP or NOM-CH fractions. NOM-CH was the least effective because it was depleted in both aromatic and polyphenolic organic contents. However, because both soil HA and NOM-PP contain relatively high amounts of aromatic and phenolic compounds, results may indicate that low-molecular-weight polyphenolic organics in NOM-PP were less effective in mediating the bioreduction of Fe(III) at circumneutral pH than the high-molecular-weight polycondensed, conjugated aromatics present in soil HA. These research findings may shed additional light in understanding of the roles and underlying mechanisms of NOM reactions with contaminant metals, radionuclides, and other toxic chemicals in the natural environment.

Intrinsic biodegradation of toluene coupled to the microbial reduction of ferric iron: laboratory column experiments

Intrinsic biodegradation of toluene coupled with the microbial reduction of ferric iron (Fe(III)) as the terminal electron acceptor was studied by using laboratory column experiments under continuous flow conditions. Columns were packed with contaminated aquifer sediment and N2-purged groundwater taken from the western part of the Gardermoen aquifer. The columns were operated anaerobically at 8 °C (in-situ temperature). Chloride was initially used to characterize flow properties of the columns. Intrinsic biodegradation of toluene, including abiotic loss and biological loss, was estimated by comparing breakthrough curves of toluene for live columns and sterilized control columns based on mass balance in steady-state conditions. The column experiments were run at two different flow velocities. The estimated average intrinsic rate was –0.73 and –0.53 mM day–1 for pore-water velocities of 1.75 and 2.68 cm h–1, respectively, corresponding to –0.27 and –0.22 mM day–1 in biological loss rate. The results indicate that intrinsic biodegradation of toluene could be used as an efficient remediation approach for contaminated groundwater at the Gardermoen fire-fighting training site.

Fulvic acid oxidation state detection using fluorescence spectroscopy.

Humic substances are a heterogeneous class of moderate molecular weight, yellow-colored biomolecules present in all soils, sediments, and natural waters. Although humic substances are generally resistant to microbial degradation under anaerobic conditions, some microorganisms in soils and sediments can use quinone moieties in humic substances as electron acceptors. Laboratory experiments have shown that humic substances can act as electron shuttles in the microbial reduction of ferric iron. Field studies of electron shuttling processes have been constrained by the lack of methods to characterize the oxidation state of quinone moieties in humic substances at natural concentrations. All humic substances have fluorescent properties, and fluorescence spectroscopy can indicate differences in precursor organic source of humic substances. Here we show that the quinone moieties responsible for electron transfer reactions contribute significantly to the fluorescence of humic substances. Further we use fluorescence spectroscopy to elucidate the oxidation state of quinone moieties in humic substances at natural concentrations found in sediment interstitial waters.

Sorption and anaerobic biodegradation of soluble aromatic compounds during groundwater transport. 1. Laboratory column experiments

This paper deals with sorption and anaerobic biodegradation of the soluble aromatic fraction of jet fuel and how it is influenced by pore-water velocity during transport in a groundwater aquifer. The study was carried out as controlled laboratory column experiments. A binary mixture of toluene and 1,2,4-trimethylbenzene with a concentration ratio of 2:1 was used through the entire investigations. The column experiments were conducted with contaminated sediments and groundwater, taken from wells at a field research site. The columns were operated anaerobically under continuous-flow conditions at 10 °C in a temperature-controlled refrigerator. Two percent sodium azide was added to the injection solution of two of the columns to prevent biodegradation of the studied organic mixture. Chloride was used as a conservative tracer to characterize the hydrodynamic parameters such as dispersivity and porosity of the columns. The results showed that both compounds in the mixture were attenuated because of sorption and biodegradation processes in the columns. 1,2,4-trimethylbenzene was attenuated more significantly than toluene. Biodegradation of toluene was coupled mainly with the microbial reduction of ferric iron, whereas 1,2,4-trimethylbenzene, in contrast, was mostly sorbed. Their sorption and biodegradation were studied with different pore-water velocities, and a mass balance approach was applied to calculate biodegradation rates. The biodegradation rates of toluene were –0.16, –0.21, and –0.26 (unit: mM day–1) for pore-water velocities of 96, 82.4, and 54.9 (unit: cm day–1), respectively. This indicates that a decrease in the pore-water velocity significantly enhanced the biodegradation of toluene, consistent with other reports in the literature. For 1,2,4-trimethylbenzene the biodegradation rates were –0.05, –0.13 (unit: mM day–1) for pore-water velocities of 96 and 82.4 (unit: cm day–1), respectively. The biodegradation rate of 1,2,4-trimethylbenzene did not increase at the lowest pore water velocity as expected. This might be a result of substrate competition.