Complexation by cysteine and iron mineral adsorption limit cadmium mobility during metabolic activity of Geobacter sulfurreducens.

Cadmium (Cd) adversely affects human health by entering the food chain via anthropogenic activity. In order to mitigate risk, a better understanding of the biogeochemical mechanisms limiting Cd mobility in the environment is needed. While Cd is not redox-active, Cd speciation varies (i.e., aqueous, complexed, adsorbed), and influences mobility. Here, the cycling of Cd in relation to initial speciation during the growth of Geobacter sulfurreducens was studied. Either fumarate or ferrihydrite (Fh) was provided as an electron acceptor and Cd was present as: (1) an aqueous cation, (2) an aqueous complex with cysteine, which is often present in metal stressed soil environments, or (3) adsorbed to Fh. During microbial Fe(iii) reduction, the removal of Cd was substantial (∼80% removal), despite extensive Fe(ii) production (ratio Fe(ii)total : Fetotal = 0.8). When fumarate was the electron acceptor, there was higher removal from solution when Cd was complexed with cysteine (97-100% removal) compared to aqueous Cd (34-50%) removal. Confocal laser scanning microscopy (CLSM) demonstrated the formation of exopolymeric substances (EPS) in all conditions and that Cd was correlated with EPS in the absence of Fe minerals (r = 0.51-0.56). Most notable is that aqueous Cd was more strongly correlated with Geobacter cells (r = 0.72) compared to Cd-cysteine complexes (r = 0.51). This work demonstrates that Cd interactions with cell surfaces and EPS, and Cd solubility during metabolic activity are dependent upon initial speciation. These processes may be especially important in soil environments where sulfur is limited and Fe and organic carbon are abundant.

Role of in Situ Natural Organic Matter in Mobilizing As during Microbial Reduction of FeIII-Mineral-Bearing Aquifer Sediments from Hanoi (Vietnam).

Natural organic matter (NOM) can contribute to arsenic (As) mobilization as an electron donor for microbially-mediated reductive dissolution of As-bearing Fe(III) (oxyhydr)oxides. However, to investigate this process, instead of using NOM, most laboratory studies used simple fatty acids or sugars, often at relatively high concentrations. To investigate the role of relevant C sources, we therefore extracted in situ NOM from the upper aquitard (clayey silt) and lower sandy aquifer sediments in Van Phuc (Hanoi area, Vietnam), characterized its composition, and used 100-day microcosm experiments to determine the effect of in situ OM on Fe(III) mineral reduction, As mobilization, and microbial community composition. We found that OM extracted from the clayey silt (OMC) aquitard resembles young, not fully degraded plant-related material, while OM from the sandy sediments (OMS) is more bioavailable and related to microbial biomass. Although all microcosms were amended with the same amount of C (12 mg C/L), the extent of Fe(III) reduction after 100 days was the highest with acetate/lactate (43 ± 3.5% of total Fe present in the sediments) followed by OMS (28 ± 0.3%) and OMC (19 ± 0.8%). Initial Fe(III) reduction rates were also higher with acetate/lactate (0.53 mg Fe(II) in 6 days) than with OMS and OMC (0.18 and 0.08 mg Fe(II) in 6 days, respectively). Although initially more dissolved As was detected in the acetate/lactate setups, after 100 days, higher concentrations of As (8.3 ± 0.3 and 8.8 ± 0.8 µg As/L) were reached in OMC and OMS, respectively, compared to acetate/lactate-amended setups (6.3 ± 0.7 µg As/L). 16S rRNA amplicon sequence analyses revealed that acetate/lactate mainly enriched Geobacter, while in situ OM supported growth and activity of a more diverse microbial community. Our results suggest that although the in situ NOM is less efficient in stimulating microbial Fe(III) reduction than highly bioavailable acetate/lactate, it ultimately has the potential to mobilize the same amount or even more As.

Magnetite and Green Rust: Synthesis, Properties, and Environmental Applications of Mixed-Valent Iron Minerals.

Mixed-valent iron [Fe(II)-Fe(III)] minerals such as magnetite and green rust have received a significant amount of attention over recent decades, especially in the environmental sciences. These mineral phases are intrinsic and essential parts of biogeochemical cycling of metals and organic carbon and play an important role regarding the mobility, toxicity, and redox transformation of organic and inorganic pollutants. The formation pathways, mineral properties, and applications of magnetite and green rust are currently active areas of research in geochemistry, environmental mineralogy, geomicrobiology, material sciences, environmental engineering, and environmental remediation. These aspects ultimately dictate the reactivity of magnetite and green rust in the environment, which has important consequences for the application of these mineral phases, for example in remediation strategies. In this review we discuss the properties, occurrence, formation by biotic as well as abiotic pathways, characterization techniques, and environmental applications of magnetite and green rust in the environment. The aim is to present a detailed overview of the key aspects related to these mineral phases which can be used as an important resource for researchers working in a diverse range of fields dealing with mixed-valent iron minerals.

Quantitative analysis of O2 and Fe2+ profiles in gradient tubes for cultivation of microaerophilic Iron(II)-oxidizing bacteria.

The classical approach for the cultivation of neutrophilic microaerophilic Fe(II)-oxidizing bacteria is agar-based gradient tubes where these bacteria find optimal growth conditions in opposing gradients of oxygen (O2) and dissolved Fe(II) (Fe2+). The goals of this study were to quantify the temporal development of O2 and Fe2+ concentrations over time, to compare abiotic and microbially inoculated tubes and to test the suitability of different Fe(II)-sources for the cultivation of freshwater and marine microaerophilic Fe(II)-oxidizers. O2 and Fe2+ gradients were monitored on a high spatial resolution as a function of time applying amperometric and voltammetric microsensors. Fe(II)-oxidizers could be cultivated well with FeS and zero-valent iron powder as Fe(II)-source, but FeCO3 and FeCl2 are extremely sensitive for this application. Fe(III) minerals accumulated in inoculated tubes within the first days in regions with an O2 concentration of 20-40 µM and were confirmed to be related to bacterial growth. Microbial Fe(II) oxidation could compete only for the first days with the abiotic reaction after which heterogeneous Fe(II) oxidation, catalyzed by Fe(III) minerals, dominated. Our results imply that transfer of cultures to fresh tubes within 48-72 h is crucial to provide optimal growth conditions for microaerophilic Fe(II)-oxidizers, particularly for the isolation of new strains.

Insights into Nitrate-Reducing Fe(II) Oxidation Mechanisms through Analysis of Cell-Mineral Associations, Cell Encrustation, and Mineralogy in the Chemolithoautotrophic Enrichment Culture KS.

Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic and depend on organic cosubstrates for growth. Encrustation of cells in Fe(III) minerals has been observed for mixotrophic NRFeOB but not for autotrophic phototrophic and microaerophilic Fe(II) oxidizers. So far, little is known about cell-mineral associations in the few existing autotrophic NRFeOB. Here, we investigate whether the designated autotrophic Fe(II)-oxidizing strain (closely related to Gallionella and Sideroxydans) or the heterotrophic nitrate reducers that are present in the autotrophic nitrate-reducing Fe(II)-oxidizing enrichment culture KS form mineral crusts during Fe(II) oxidation under autotrophic and mixotrophic conditions. In the mixed culture, we found no significant encrustation of any of the cells both during autotrophic oxidation of 8 to 10 mM Fe(II) coupled to nitrate reduction and during cultivation under mixotrophic conditions with 8 to 10 mM Fe(II), 5 mM acetate, and 4 mM nitrate, where higher numbers of heterotrophic nitrate reducers were present. Two pure cultures of heterotrophic nitrate reducers (Nocardioides and Rhodanobacter) isolated from culture KS were analyzed under mixotrophic growth conditions. We found green rust formation, no cell encrustation, and only a few mineral particles on some cell surfaces with 5 mM Fe(II) and some encrustation with 10 mM Fe(II). Our findings suggest that enzymatic, autotrophic Fe(II) oxidation coupled to nitrate reduction forms poorly crystalline Fe(III) oxyhydroxides and proceeds without cellular encrustation while indirect Fe(II) oxidation via heterotrophic nitrate-reduction-derived nitrite can lead to green rust as an intermediate mineral and significant cell encrustation. The extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen species depends on Fe(II) concentrations and is probably negligible under environmental conditions in most habitats.IMPORTANCE Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic (their growth depends on organic cosubstrates) and can become encrusted in Fe(III) minerals. Encrustation is expected to be harmful and poses a threat to cells if it also occurs under environmentally relevant conditions. Nitrite produced during heterotrophic denitrification reacts with Fe(II) abiotically and is probably the reason for encrustation in mixotrophic NRFeOB. Little is known about cell-mineral associations in autotrophic NRFeOB such as the enrichment culture KS. Here, we show that no encrustation occurs in culture KS under autotrophic and mixotrophic conditions while heterotrophic nitrate-reducing isolates from culture KS become encrusted. These findings support the hypothesis that encrustation in mixotrophic cultures is caused by the abiotic reaction of Fe(II) with nitrite and provide evidence that Fe(II) oxidation in culture KS is enzymatic. Furthermore, we show that the extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen species depends on Fe(II) concentrations and is probably negligible in most environmental habitats.

Iron Isotope Fractionation during Fe(II) Oxidation Mediated by the Oxygen-Producing Marine Cyanobacterium Synechococcus PCC 7002.

In this study, we couple iron isotope analysis to microscopic and mineralogical investigation of iron speciation during circumneutral Fe(II) oxidation and Fe(III) precipitation with photosynthetically produced oxygen. In the presence of the cyanobacterium Synechococcus PCC 7002, aqueous Fe(II) (Fe(II)aq) is oxidized and precipitated as amorphous Fe(III) oxyhydroxide minerals (iron precipitates, Feppt), with distinct isotopic fractionation (ε56Fe) values determined from fitting the δ56Fe(II)aq (1.79‰ and 2.15‰) and the δ56Feppt (2.44‰ and 2.98‰) data trends from two replicate experiments. Additional Fe(II) and Fe(III) phases were detected using microscopy and chemical extractions and likely represent Fe(II) and Fe(III) sorbed to minerals and cells. The iron desorbed with sodium acetate (FeNaAc) yielded heavier δ56Fe compositions than Fe(II)aq. Modeling of the fractionation during Fe(III) sorption to cells and Fe(II) sorption to Feppt, combined with equilibration of sorbed iron and with Fe(II)aq using published fractionation factors, is consistent with our resulting δ56FeNaAc. The δ56Feppt data trend is inconsistent with complete equilibrium exchange with Fe(II)aq. Because of this and our detection of microbially excreted organics (e.g., exopolysaccharides) coating Feppt in our microscopic analysis, we suggest that electron and atom exchange is partially suppressed in this system by biologically produced organics. These results indicate that cyanobacteria influence the fate and composition of iron in sunlit environments via their role in Fe(II) oxidation through O2 production, the capacity of their cell surfaces to sorb iron, and the interaction of secreted organics with Fe(III) minerals.

Microaerophilic Fe(II)-Oxidizing Zetaproteobacteria Isolated from Low-Fe Marine Coastal Sediments: Physiology and Composition of Their Twisted Stalks.

Microaerophilic Fe(II) oxidizers are commonly found in habitats containing elevated Fe(II) and low O2 concentrations and often produce characteristic Fe mineral structures, so-called twisted stalks or tubular sheaths. Isolates originating from freshwater habitats are all members of the Betaproteobacteria, while isolates from marine habitats belong almost exclusively to the Zetaproteobacteria So far, only a few isolates of marine microaerophilic Fe(II) oxidizers have been described, all of which are obligate microaerophilic Fe(II) oxidizers and have been thought to be restricted to Fe-rich systems. Here, we present two new isolates of marine microaerophilic Fe(II)-oxidizing Zetaproteobacteria that originate from typical coastal marine sediments containing only low Fe concentrations (2 to 11 mg of total Fe/g of sediment [dry weight]; 70 to 100 µM dissolved Fe2+ in the porewater). The two novel Zetaproteobacteria share characteristic physiological properties of the Zetaproteobacteria group, even though they come from low-Fe environments: the isolates are obligate microaerophilic Fe(II) oxidizers and, like most isolated Zetaproteobacteria, they produce twisted stalks. We found a low organic carbon content in the stalks (∼0.3 wt%), with mostly polysaccharides and saturated aliphatic chains (most likely lipids). The Fe minerals in the stalks were identified as lepidocrocite and possibly ferrihydrite. Immobilization experiments with Ni2+ showed that the stalks can function as a sink for trace metals. Our findings show that obligate microaerophilic Fe(II) oxidizers belonging to the Zetaproteobacteria group are not restricted to Fe-rich environments but can also be found in low-Fe marine environments, which increases their overall importance for the global biogeochemical Fe cycle.IMPORTANCE So far, only a few isolates of benthic marine microaerophilic Fe(II) oxidizers belonging to the Zetaproteobacteria exist, and most isolates were obtained from habitats containing elevated Fe concentrations. Consequently, it was thought that these microorganisms are important mainly in habitats with high Fe concentrations. The two novel isolates of Zetaproteobacteria that are presented in the present study were isolated from typical coastal marine sediments that do not contain elevated Fe concentrations. This increases the knowledge about possible habitats in which Zetaproteobacteria can exist. Furthermore, we show that the physiology and the typical organo-mineral structures (twisted stalks) that are produced by the isolates do not notably differ from the physiology and the cell-mineral structures of isolates from environments with high Fe concentrations. We also showed that the organo-mineral structures can function as a sink for trace metals.

Geochemistry and Mineralogy of Western Australian Salt Lake Sediments: Implications for Meridiani Planum on Mars.

UNLABELLED: Hypersaline lakes are characteristic for Western Australia and display a rare combination of geochemical and mineralogical properties that make these lakes potential analogues for past conditions on Mars. In our study, we focused on the geochemistry and mineralogy of Lake Orr and Lake Whurr. While both lakes are poor in organic carbon (<1%), the sediments' pH values differ and range from 3.8 to 4.8 in Lake Orr and from 5.4 to 6.3 in Lake Whurr sediments. Lake Whurr sediments were dominated by orange and red sediment zones in which the main Fe minerals were identified as hematite, goethite, and tentatively jarosite and pyrite. Lake Orr was dominated by brownish and blackish sediments where the main Fe minerals were goethite and another paramagnetic Fe(III)-phase that could not be identified. Furthermore, a likely secondary Fe(II)-phase was observed in Lake Orr sediments. The mineralogy of these two salt lakes in the sampling area is strongly influenced by events such as flooding, evaporation, and desiccation, processes that explain at least to some extent the observed differences between Lake Orr and Lake Whurr. The iron mineralogy of Lake Whurr sediments and the high salinity make this lake a suitable analogue for Meridiani Planum on Mars, and in particular the tentative identification of pyrite in Lake Whurr sediments has implications for the interpretation of the Fe mineralogy of Meridiani Planum sediments. KEY WORDS: Western Australia-Salt lakes-Jarosite-Hematite-Pyrite-Mars analogue. Astrobiology 16, 525-538.

Limited influence of Si on the preservation of Fe mineral-encrusted microbial cells during experimental diagenesis.

The reconstruction of the history of microbial life since its emergence on early Earth is impaired by the difficulty to prove the biogenicity of putative microfossils in the rock record. While most of the oldest rocks on Earth have been exposed to different grades of diagenetic alterations, little is known about how the remains of micro-organisms evolve when exposed to pressure (P) and temperature (T) conditions typical of diagenesis. Using spectroscopy and microscopy, we compared morphological, mineralogical, and chemical biosignatures exhibited by Fe mineral-encrusted cells of the bacterium Acidovorax sp. BoFeN1 after long-term incubation under ambient conditions and after experimental diagenesis. We also evaluated the effects of Si on the preservation of microbial cells during the whole process. At ambient conditions, Si affected the morphology but not the identity (goethite) of Fe minerals that formed around cells. Fe-encrusted cells were morphologically well preserved after 1 week at 250 °C-140 MPa and after 16 weeks at 170 °C-120 MPa in the presence or in the absence of Si. Some goethite transformed to hematite and magnetite at 250 °C-140 MPa, but in the presence of Si more goethite was preserved. Proteins-the most abundant cellular components-were preserved over several months at ambient conditions but disappeared after incubations at high temperature and pressure conditions, both in the presence and in the absence of Si. Other organic compounds, such as lipids and extracellular polysaccharides seemed well preserved after exposure to diagenetic conditions. This study provides insights about the composition and potential preservation of microfossils that could have formed in Fe- and Si-rich Precambrian oceans.

Formation of chloroform and tetrachloroethene by Sinorhizobium meliloti strain 1021.

UNLABELLED: The mechanisms and organisms involved in the natural formation of volatile organohalogen compounds (VOX) are largely unknown. We provide evidence that the common and widespread soil bacterium Sinorhizobium meliloti strain 1021 is capable of producing up to 3338·6 ± 327·8 ng l(-1) headspace volume of chloroform (CHCl3 ) and 807·8 ± 13·5 ng l(-1)  headspace volume of tetrachloroethene (C2 Cl4 ) within 1 h when grown in soil extract medium. Biotic VOX formation has been suggested to be linked to the activity of halogenating enzymes such as haloperoxidases. We tested if the observed VOX formation by S. meliloti can be attributed to one of its chloroperoxidases (Smc01944) that is highly expressed in the presence of H2 O2. However, addition of 10 mmol l(-1) H2 O2 to the S. meliloti cultures decreased VOX formation by 52% for chloroform and 25% for tetrachloroethene, while viable cell numbers decreased by 23%. Interestingly, smc01944 gene expression increased 450-fold. The quantification of extracellular chlorination activity in cell suspension experiments did not provide evidence for a role of S. meliloti chloroperoxidases in the observed VOX formation. This suggests that a momentarily unknown mechanism which requires no H2 O2 might be responsible for the VOX formation by S. meliloti. Regardless of the underlying mechanism our results suggest that the soil bacterium S. meliloti might be an important source of VOX in soils. SIGNIFICANCE AND IMPACT OF THE STUDY: Volatile organohalogen compounds (VOX) strongly influence atmospheric chemistry and Earth's climate. Besides anthropogenic emissions they are naturally produced by either abiotic or biotic pathways in various environments. Particularly in soils, microbial processes drive the natural halogen cycle but the direct link to microbial VOX formation has not been studied in detail yet. In this study we provide evidence that the common and widespread soil bacterium Sinorhizobium meliloti strain 1021 forms chloroform and tetrachloroethene. The potential contribution of S. meliloti to soil VOX release could significantly influence soil and atmospheric chemistry.

Oxidation of Fe(II)-EDTA by nitrite and by two nitrate-reducing Fe(II)-oxidizing Acidovorax strains.

The enzymatic oxidation of Fe(II) by nitrate-reducing bacteria was first suggested about two decades ago. It has since been found that most strains are mixotrophic and need an additional organic co-substrate for complete and prolonged Fe(II) oxidation. Research during the last few years has tried to determine to what extent the observed Fe(II) oxidation is driven enzymatically, or abiotically by nitrite produced during heterotrophic denitrification. A recent study reported that nitrite was not able to oxidize Fe(II)-EDTA abiotically, but the addition of the mixotrophic nitrate-reducing Fe(II)-oxidizer, Acidovorax sp. strain 2AN, led to Fe(II) oxidation (Chakraborty & Picardal, 2013). This, along with other results of that study, was used to argue that Fe(II) oxidation in strain 2AN was enzymatically catalyzed. However, the absence of abiotic Fe(II)-EDTA oxidation by nitrite reported in that study contrasts with previously published data. We have repeated the abiotic and biotic experiments and observed rapid abiotic oxidation of Fe(II)-EDTA by nitrite, resulting in the formation of Fe(III)-EDTA and the green Fe(II)-EDTA-NO complex. Additionally, we found that cultivating the Acidovorax strains BoFeN1 and 2AN with 10 mM nitrate, 5 mm acetate, and approximately 10 mM Fe(II)-EDTA resulted only in incomplete Fe(II)-EDTA oxidation of 47-71%. Cultures of strain BoFeN1 turned green (due to the presence of Fe(II)-EDTA-NO) and the green color persisted over the course of the experiments, whereas strain 2AN was able to further oxidize the Fe(II)-EDTA-NO complex. Our work shows that the two used Acidovorax strains behave very differently in their ability to deal with toxic effects of Fe-EDTA species and the further reduction of the Fe(II)-EDTA-NO nitrosyl complex. Although the enzymatic oxidation of Fe(II) cannot be ruled out, this study underlines the importance of nitrite in nitrate-reducing Fe(II)- and Fe(II)-EDTA-oxidizing cultures and demonstrates that Fe(II)-EDTA cannot be used to demonstrate unequivocally the enzymatic oxidation of Fe(II) by mixotrophic Fe(II)-oxidizers.

Atmospheric hydrogen peroxide and Eoarchean iron formations.

It is widely accepted that photosynthetic bacteria played a crucial role in Fe(II) oxidation and the precipitation of iron formations (IF) during the Late Archean-Early Paleoproterozoic (2.7-2.4 Ga). It is less clear whether microbes similarly caused the deposition of the oldest IF at ca. 3.8 Ga, which would imply photosynthesis having already evolved by that time. Abiological alternatives, such as the direct oxidation of dissolved Fe(II) by ultraviolet radiation may have occurred, but its importance has been discounted in environments where the injection of high concentrations of dissolved iron directly into the photic zone led to chemical precipitation reactions that overwhelmed photooxidation rates. However, an outstanding possibility remains with respect to photochemical reactions occurring in the atmosphere that might generate hydrogen peroxide (H2 O2 ), a recognized strong oxidant for ferrous iron. Here, we modeled the amount of H2 O2 that could be produced in an Eoarchean atmosphere using updated solar fluxes and plausible CO2 , O2 , and CH4 mixing ratios. Irrespective of the atmospheric simulations, the upper limit of H2 O2 rainout was calculated to be <10(6) molecules cm(-2) s(-1) . Using conservative Fe(III) sedimentation rates predicted for submarine hydrothermal settings in the Eoarchean, we demonstrate that the flux of H2 O2 was insufficient by several orders of magnitude to account for IF deposition (requiring ~10(11) H2 O2 molecules cm(-2) s(-1) ). This finding further constrains the plausible Fe(II) oxidation mechanisms in Eoarchean seawater, leaving, in our opinion, anoxygenic phototrophic Fe(II)-oxidizing micro-organisms the most likely mechanism responsible for Earth's oldest IF.

Predominance of biotic over abiotic formation of halogenated hydrocarbons in hypersaline sediments in Western Australia.

Volatile halogenated organic compounds (VOX) contribute to ozone depletion and global warming. There is evidence of natural VOX formation in many environments ranging from forest soils to salt lakes. Laboratory studies have suggested that VOX formation can be chemically stimulated by reactive Fe species while field studies have provided evidence for direct biological (enzymatic) VOX formation. However, the relative contribution of abiotic and biotic processes to global VOX budgets is still unclear. The goals of this study were to quantify VOX release from sediments from a hypersaline lake in Western Australia (Lake Strawbridge) and to distinguish between the relative contributions of biotic and abiotic VOX formation in microbially active and sterilized microcosms. Our experiments demonstrated that the release of organochlorines from Lake Strawbridge sediments was mainly biotic. Among the organochlorines detected were monochlorinated, e.g., chloromethane (CH3Cl), and higher chlorinated VOX compounds such as trichloromethane (CHCl3). Amendment of sediments with either Fe(III) oxyhydroxide (ferrihydrite) or a mixture of lactate/acetate or both ferrihydrite and lactate/acetate did not stimulate VOX formation. This suggests that although microbial Fe(III) reduction took place, there was no stimulation of VOX formation via Fe redox transformations or the formation of reactive Fe species under our experimental conditions.

3-D analysis of bacterial cell-(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate-reducing Acidovorax sp. strain BoFeN1 using complementary microscopy tomography approaches.

The formation of cell-(iron)mineral aggregates as a consequence of bacterial iron oxidation is an environmentally widespread process with a number of implications for processes such as sorption and coprecipitation of contaminants and nutrients. Whereas the overall appearance of such aggregates is easily accessible using 2-D microscopy techniques, the 3-D and internal structure remain obscure. In this study, we examined the 3-D structure of cell-(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate-reducing Acidovorax sp. strain BoFeN1 using a combination of advanced 3-D microscopy techniques. We obtained 3-D structural and chemical information on different cellular encrustation patterns at high spatial resolution (4-200 nm, depending on the method): more specifically, (1) cells free of iron minerals, (2) periplasm filled with iron minerals, (3) spike- or platelet-shaped iron mineral structures, (4) bulky structures on the cell surface, (5) extracellular iron mineral shell structures, (6) cells with iron mineral filled cytoplasm, and (7) agglomerations of extracellular globular structures. In addition to structural information, chemical nanotomography suggests a dominant role of extracellular polymeric substances (EPS) in controlling the formation of cell-(iron)mineral aggregates. Furthermore, samples in their hydrated state showed cell-(iron)mineral aggregates in pristine conditions free of preparation (i.e., drying/dehydration) artifacts. All these results were obtained using 3-D microscopy techniques such as focused ion beam (FIB)/scanning electron microscopy (SEM) tomography, transmission electron microscopy (TEM) tomography, scanning transmission (soft) X-ray microscopy (STXM) tomography, and confocal laser scanning microscopy (CLSM). It turned out that, due to the various different contrast mechanisms of the individual approaches, and due to the required sample preparation steps, only the combination of these techniques was able to provide a comprehensive understanding of structure and composition of the various Fe-precipitates and their association with bacterial cells and EPS.

Microbially enhanced dissolution of HgS in an acid mine drainage system in the California Coast Range.

Mercury sulfides (cinnabar and metacinnabar) are the main ores of Hg and are relatively stable under oxic conditions (Ksp = 10⁻54 and 10⁻5² , respectively). However, until now their stability in the presence of micro-organisms inhabiting acid mine drainage (AMD) systems was unknown. We tested the effects of the AMD microbial community from the inoperative Hg mine at New Idria, CA, present in sediments of an AMD settling pond adjacent to the main waste pile and in a microbial biofilm on the surface of this pond, on the solubility of crystalline HgS. A 16S rRNA gene clone library revealed that the AMD microbial community was dominated by Fe-oxidizing (orders Ferritrophicales and Gallionellas) and S-oxidizing bacteria (Thiomonas sp.), with smaller amounts (≤ 6%) being comprised of the orders Xanthomondales and Rhodospirillales. Though the order Ferritrophicales dominate the 16S rRNA clones (>60%), qPCR results of the microbial community indicate that the Thiomonas sp. represents ~55% of the total micro-organisms in the top 1 cm of the AMD microbial community. Although supersaturated with respect to cinnabar and metacinnabar, microcosms inoculated with the AMD microbial community were capable of releasing significantly more Hg into solution compared to inactivated or abiotic controls. Four different Hg-containing materials were tested for bacterially enhanced HgS dissolution: pure cinnabar, pure metacinnabar, mine tailings, and calcine material (processed ore). In the microcosm with metacinnabar, the presence of the AMD microbial community resulted in an increase of dissolved Hg concentrations up to 500 µg L ⁻¹during the first 30 days of incubation. In abiotic control microcosms, dissolved Hg concentrations did not increase above 100 ng L⁻¹ . When Hg concentrations were below 50 µg L⁻¹ , the Fe-oxidizing bacteria in the AMD microbial community were still capable of oxidizing Fe(II) to Fe(III) in the AMD solution, whereas concentrations above 50 µg L⁻¹ resulted in inhibition of microbial iron oxidation. Our experiments show that the AMD microbial community contributes to the dissolution of mercury sulfide minerals. These findings have major implications for risk assessment and future management of inoperative Hg mines worldwide.

Authigenic iron oxide proxies for marine zinc over geological time and implications for eukaryotic metallome evolution.

Here, we explore enrichments in paleomarine Zn as recorded by authigenic iron oxides including Precambrian iron formations, ironstones, and Phanerozoic hydrothermal exhalites. This compilation of new and literature-based iron formation analyses track dissolved Zn abundances and constrain the magnitude of the marine reservoir over geological time. Overall, the iron formation record is characterized by a fairly static range in Zn/Fe ratios throughout the Precambrian, consistent with the shale record (Scott et al., 2013, Nature Geoscience, 6, 125-128). When hypothetical partitioning scenarios are applied to this record, paleomarine Zn concentrations within about an order of magnitude of modern are indicated. We couple this examination with new chemical speciation models to interpret the iron formation record. We present two scenarios: first, under all but the most sulfidic conditions and with Zn-binding organic ligand concentrations similar to modern oceans, the amount of bioavailable Zn remained relatively unchanged through time. Late proliferation of Zn in eukaryotic metallomes has previously been linked to marine Zn biolimitation, but under this scenario the expansion in eukaryotic Zn metallomes may be better linked to biologically intrinsic evolutionary factors. In this case, zinc's geochemical and biological evolution may be decoupled and viewed as a function of increasing need for genome regulation and diversification of Zn-binding transcription factors. In the second scenario, we consider Archean organic ligand complexation in such excess that it may render Zn bioavailability low. However, this is dependent on Zn-organic ligand complexes not being bioavailable, which remains unclear. In this case, although bioavailability may be low, sphalerite precipitation is prevented, thereby maintaining a constant Zn inventory throughout both ferruginous and euxinic conditions. These results provide new perspectives and constraints on potential couplings between the trajectory of biological and marine geochemical coevolution.

Oxidation of Fe(II) leads to increased C-2 methylation of pentacyclic triterpenoids in the anoxygenic phototrophic bacterium Rhodopseudomonas palustris strain TIE-1.

Hopanoids are among the most widespread biomarkers of bacteria that are used as indicators for past and present bacterial activity. Our understanding of the production, function, and distribution of hopanoids in bacteria has improved greatly, partly due to genetic, culture-independent studies. Culture-based studies are important to determine hopanoid function and the environmental conditions under which these compounds are produced. This study compares the lipid inventory of Rhodopseudomonas palustris strain TIE-1 under anoxic photoautotrophic conditions using either H2 or Fe(II) as electron donor. The high amount to which adenosylhopane is produced irrespective of the used electron donor suggests a specific function of this compound rather than its exclusive role as an intermediate in bacteriohopanepolyol biosynthesis. C-2 methylated hopanoids and tetrahymanol account for as much as 59% of the respective C-2 methylated/non-methylated homologs during growth with Fe(II) as electron donor, as compared with 24% C-2 methylation for growth with H2 . This observation reveals that C-2 methylated hopanoids have a specific function and are preferentially synthesized in response to elevated Fe(II) concentrations. The presence of C-2 methylated pentacyclic triterpenoids has commonly been used as a biosignature for the interpretation of paleoenvironments. These new findings suggest that increased C-2 methylation may indicate anoxic ferrous conditions, in addition to other environmental stressors that have been previously reported.

Abiotic oxidation of Fe(II) by reactive nitrogen species in cultures of the nitrate-reducing Fe(II) oxidizer Acidovorax sp. BoFeN1 - questioning the existence of enzymatic Fe(II) oxidation.

Nitrate-reducing, Fe(II)-oxidizing bacteria were suggested to couple with enzymatic Fe(II) oxidation to nitrate reduction. Denitrification proceeds via intermediates (NO2 -, NO) that can oxidize Fe(II) abiotically at neutral and particularly at acidic pH. Here, we present a revised Fe(II) quantification protocol preventing artifacts during acidic Fe extraction and evaluate the contribution of abiotic vs. enzymatic Fe(II) oxidation in cultures of the nitrate-reducing, Fe(II) oxidizer Acidovorax sp. BoFeN1. Sulfamic acid used instead of HCl reacts with nitrite and prevents abiotic Fe(II) oxidation during Fe extraction. Abiotic experiments without sulfamic acid showed that acidification of oxic Fe(II) nitrite samples leads to 5.6-fold more Fe(II) oxidation than in anoxic samples because the formed NO becomes rapidly reoxidized by O(2) , therefore leading to abiotic oxidation and underestimation of Fe(II). With our revised protocol using sulfamic acid, we quantified oxidation of approximately 7 mm of Fe(II) by BoFeN1 within 4 days. Without addition of sulfamic acid, the same oxidation was detected within only 2 days. Additionally, abiotic incubation of Fe(II) with nitrite in the presence of goethite as surface catalyst led to similar abiotic Fe(II) oxidation rates as observed in growing BoFeN1 cultures. BoFeN1 growth was observed on acetate with N(2) O as electron acceptor. When adding Fe(II), no Fe(II) oxidation was observed, suggesting that the absence of reactive N intermediates (NO2 -, NO) precludes Fe(II) oxidation. The addition of ferrihydrite [Fe(OH)(3) ] to acetate/nitrate BoFeN1 cultures led to growth stimulation equivalent to previously described effects on growth by adding Fe(II). This suggests that elevated iron concentrations might provide a nutritional effect rather than energy-yielding Fe(II) oxidation. Our findings therefore suggest that although enzymatic Fe(II) oxidation by denitrifiers cannot be fully ruled out, its contribution to the observed Fe(II) oxidation in microbial cultures is probably lower than previously suggested and has to be questioned in general until the enzymatic machinery-mediating Fe(II) oxidation is identified.

Microbial Iron(II) Oxidation in Littoral Freshwater Lake Sediment: The Potential for Competition between Phototrophic vs. Nitrate-Reducing Iron(II)-Oxidizers.

The distribution of neutrophilic microbial iron oxidation is mainly determined by local gradients of oxygen, light, nitrate and ferrous iron. In the anoxic top part of littoral freshwater lake sediment, nitrate-reducing and phototrophic Fe(II)-oxidizers compete for the same e(-) donor; reduced iron. It is not yet understood how these microbes co-exist in the sediment and what role they play in the Fe cycle. We show that both metabolic types of anaerobic Fe(II)-oxidizing microorganisms are present in the same sediment layer directly beneath the oxic-anoxic sediment interface. The photoferrotrophic most probable number counted 3.4·10(5) cells·g(-1) and the autotrophic and mixotrophic nitrate-reducing Fe(II)-oxidizers totaled 1.8·10(4) and 4.5·10(4) cells·g(-1) dry weight sediment, respectively. To distinguish between the two microbial Fe(II) oxidation processes and assess their individual contribution to the sedimentary Fe cycle, littoral lake sediment was incubated in microcosm experiments. Nitrate-reducing Fe(II)-oxidizing bacteria exhibited a higher maximum Fe(II) oxidation rate per cell, in both pure cultures and microcosms, than photoferrotrophs. In microcosms, photoferrotrophs instantly started oxidizing Fe(II), whilst nitrate-reducing Fe(II)-oxidizers showed a significant lag-phase during which they probably use organics as e(-) donor before initiating Fe(II) oxidation. This suggests that they will be outcompeted by phototrophic Fe(II)-oxidizers during optimal light conditions; as phototrophs deplete Fe(II) before nitrate-reducing Fe(II)-oxidizers start Fe(II) oxidation. Thus, the co-existence of the two anaerobic Fe(II)-oxidizers may be possible due to a niche space separation in time by the day-night cycle, where nitrate-reducing Fe(II)-oxidizers oxidize Fe(II) during darkness and phototrophs play a dominant role in Fe(II) oxidation during daylight. Furthermore, metabolic flexibility of Fe(II)-oxidizing microbes may play a paramount role in the conservation of the sedimentary Fe cycle.