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Microbial anaerobic Fe(II) oxidation at neutral pH produces poorly soluble Fe(III) which is expected to bind to cell surfaces causing cell encrustation and potentially impeding cell metabolism. The challenge for Fe(II)-oxidizing prokaryotes therefore is to avoid encrustation with Fe(III). Using different microscopic techniques we tracked Fe(III) minerals at the cell surface and within cells of phylogenetically distinct phototrophic and nitrate-reducing Fe(II)-oxidizing bacteria. While some strains successfully prevented encrustation others precipitated Fe(III) minerals at the cell surface and in the periplasm. Our results indicate differences in the cellular mechanisms of Fe(II) oxidation, transport of Fe(II)/Fe(III) ions, and Fe(III) mineral precipitation.

Ground source heat pump systems are increasingly being used to exploit the energy content of shallow geothermal resources for space heating and cooling. In this study we evaluate the potential for groundwater contamination of the different organic anti-freeze compounds (ethylene glycol, propylene glycol and betaine) used in these pumps, based on a literature review of their biodegradability and the results of our own laboratory experiments on aquifer material. Ethylene and propylene glycol were found to be readily biodegradable under both oxic and anoxic conditions, without formation of toxic or persistent intermediates. Long-term groundwater contamination by the glycols is therefore not expected. Betaine is also expected to be readily biodegradable in oxic and anoxic groundwater. The potential formation of trimethylamine, an intermediate of anaerobic betaine degradation, is, however, regarded as critical due to its unpleasant odor even at very low concentrations. Additionally, betaine has the potential to complex metal ions and thus may mobilize toxic metals in groundwater. We therefore recommend that betaine not be used in borehole heat exchanger fluids. In addition to organic anti-freeze compounds such as glycols, borehole heat exchanger fluids also contain additives such as corrosion inhibitors or biocides. We demonstrate that potentially toxic additives in these fluids inhibit biodegradation of the organic anti-freeze compounds. In order to ensure environmental compatibility of borehole heat exchanger fluids, further research should be conducted on the impact of additives on subsurface microbiological activity and on groundwater quality.

In April 2004, the German Research Foundation (DFG) and the National Science Foundation (NSF) co-organized a meeting for US and German junior geoscientists in Washington DC. As an outcome of this Research Conference titled ‘Earth, Fire, Water, Air and Life’, a follow-up workshop took place in May 2005 at the Eberhard-Karls University of Tuebingen in Germany. This workshop covered new aspects of research to improve the understanding of steep biogeochemical gradients covering pH changes, redox zones, as well as solute and particulate concentration variations in aqueous systems. Detailed understanding of biogeochemistry in this context delivers new fundamental aspects in interdisciplinary research. Such work is also urgently needed to control ever-increasing scarcity of water that is to large parts driven by decreasing water quality. Research ideas on gradients in a biogeochemical context that were discussed by a subgroup of biogeochemists during that workshop are summarized and presented here.

Geobacter metallireducens and G. sulfurreducens have been classified as strictly anaerobic bacteria which grow and thrive in subsurface and sediment environments. Hopanoids are pentacyclic triterpenoid lipids and are important for bacterial membrane stability and functioning. Hopanoids predominantly occur in aerobically growing bacteria of oxic environments. They rarely have been found in facultatively anaerobic bacteria and, to date, not at all in strict anaerobes. Our research shows that anaerobically grown G. metallireducens and G. sulfurreducens bacteria contain a range of hopanoid lipids, such as diploptene (i.e. hop-22(29)-ene) and hop-21-ene, and more complex, elongated hopanoids. In geological formations, diagenetic derivatives of hopanoids are widely used as biomarkers and are recognized as molecular fossils of bacterial origin. To date, these biomarkers have largely been interpreted as those derived from ancient oxic environments. Our findings presented here suggest that this interpretation needs to be re-evaluated. In addition to the origin in oxic environments, 'geohopanoids' may originate from ancient anaerobic environments as well.

Iron is the most abundant element on Earth and the most frequently utilized transition metal in the biosphere. It is a component of many cellular compounds and is involved in numerous physiological functions. Hence, iron is an essential micronutrient for all eukaryotes and the majority of prokaryotes. Prokaryotes that need iron for biosynthesis require micromolar concentrations, levels that are often not available in neutral pH oxic environments. Therefore, prokaryotes have evolved specific acquisition molecules, called siderophores, to increase iron bioavailability. Acquisition of iron by siderophores is a complex process and is discussed in detail by Kraemer et al. (2005). Here we focus on prokaryotes that generate energy for growth by oxidation or reduction of iron. In both processes single electron transfers are involved. Hence, for a significant extent of energy generation, turnover of iron in the millimolar rather than the micromolar range is necessary. Iron metabolizing organisms have therefore a strong influence on iron cycling in the environment. Microbial iron oxidation and reduction will be discussed, with emphasis on circumneutral pH environments that prevail on Earth. The active metabolic processes outlined above have to be distinguished from indirect biologically induced iron mineral formation in which prokaryotic cell surfaces simply act as passive templates (“passive iron biomineralization”) (e.g., Konhauser 1997). ### General aspects of the iron cycle On our planet, iron is ubiquitous in the hydrosphere, lithosphere, biosphere and atmosphere, either as particulate ferric [Fe(III)] or ferrous [Fe(II)] iron-bearing minerals or as dissolved ions. Redox transformations of iron, as well as dissolution and precipitation and thus mobilization and redistribution, are caused by chemical and to a significant extent by microbial processes (Fig. 1⇓). Microorganisms catalyze the oxidation of Fe(II) under oxic or anoxic conditions as well as the reduction of Fe(III) in anoxic habitats. Microbially influenced transformations of iron are often much faster than the …

Observations in enrichment cultures of ferric iron-reducing bacteria indicated that ferrihydrite was reduced to ferrous iron minerals via sulfur cycling with sulfide as the reductant. Ferric iron reduction via sulfur cycling was investigated in more detail with Sulfurospirillum deleyianum , which can utilize sulfur or thiosulfate as an electron acceptor. In the presence of cysteine (0.5 or 2 mM) as the sole sulfur source, no (microbial) reduction of ferrihydrite or ferric citrate was observed, indicating that S. deleyianum is unable to use ferric iron as an immediate electron acceptor. However, with thiosulfate at a low concentration (0.05 mM), growth with ferrihydrite (6 mM) was possible and sulfur was cycled up to 60 times. Also, spatially distant ferrihydrite in agar cultures was reduced via diffusible sulfur species. Due to the low concentrations of thiosulfate, S. deleyianum produced only small amounts of sulfide. Obviously, sulfide delivered electrons to ferrihydrite with no or only little precipitation of black iron sulfides. Ferrous iron and oxidized sulfur species were produced instead, and the latter served again as the electron acceptor. These oxidized sulfur species have not yet been identified. However, sulfate and sulfite cannot be major products of ferrihydrite-dependent sulfide oxidation, since neither compound can serve as an electron acceptor for S. deleyianum . Instead, sulfur (elemental S or polysulfides) and/or thiosulfate as oxidized products could complete a sulfur cycle-mediated reduction of ferrihydrite.

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.

Two strictly anaerobic, dissimilatory ferric-iron-reducing bacteria, strains Dfr1T and Dfr2T, were isolated from freshwater mud samples with ferrihydrite as electron acceptor. Both strains also grew by reducing Mn(IV), S0 and fumarate. Electron donors used by strains Dfr1T and Dfr2T for growth with ferric iron as electron acceptor included hydrogen, formate, acetate, pyruvate, succinate, fumarate and ethanol. An affiliation with the family Geobacteraceae was revealed by comparative analysis of 165 rRNA gene sequences. Strains Dfr1T and Dfr2T shared 92.5% sequence identity and their closest known relative was Geobacter sulfurreducens, with approximately 93% sequence identity. Cultures and colonies of strains Dfr1T and Dfr2T were intensely red in colour, due to the presence of c-type cytochromes. On the basis of physiological and phylogenetic data, strain Dfr1T (= DSM 12179T = OCM 796T) is described as Geobacter bremensis sp. nov. and strain Dfr2T (= DSM 12255T = OCM 797T) as Geobacter pelophilus sp. nov.

Two new strains of marine purple bacteria, N1T and N2T, were isolated from coastal sediment of the North Sea (Germany) with ferrous iron as the only electron donor for anoxygenic photosynthesis. The isolates are the first salt-dependent, ferrous-iron-oxidizing purple bacteria characterized so far. Analysis of 16S rRNA gene sequences revealed an affiliation with the genus Rhodovulum, which until now comprises only marine species. The sequence similarity of both strains was 95.2%, and their closest relative was Rhodovulum adriaticum. Like all known Rhodovulum species, the new strains had ovoid to rod-shaped cells, contained bacteriochlorophyll a and carotenoids of the spheroidene series, and were able to oxidize sulfide and thiosulfate. Like Rhodovulum adriaticum, both strains were unable to assimilate sulfate; for growth they needed a reduced sulfur source, e.g. thiosulfate. In contrast to the new strains, none of the known Rhodovulum species tested was able to oxidize ferrous iron or iron sulfide. In growth experiments, strains N1T and N2T oxidized 65 and 95%, respectively, of the ferrous iron supplied. Electron diffraction analysis revealed ferrihydrite as the main product of ferrous iron oxidation. In addition, traces of magnetite were formed. Strains N1T (= DSM 12328T) and N2T (= DSM 12329T) are described as Rhodovulum iodosum sp. nov. and Rhodovulum robiginosum sp. nov., respectively.

Summary Nitrate-reducing bacteria, which grow anaerobically with Fe(II) as electron donor, were isolated from freshwater mud samples. Since extensive phylogenetic and physiological characterization of multiple strains is very time-consuming and labour-intensive, the isolates were first screened for genetic diversity by denaturing gradient gel electrophoresis (DGGE) and whole-cell hybridization. DGGE analysis of 16S rDNA fragments amplified from 12 strains indicated that three different types of bacteria had been independently isolated (type A–C). Whole-cell hybridization with domain- and group-specific oligonucleotide probes suggested that the type-A and -C isolates were members of the β-subdivision of the Proteobacteria. The type-B isolates hybridized only with the bacterial probe but not with any of the probes specific for the α-, β- or γ-Proteobacteria. Based on these results representative strains of each type were chosen for further phylogenetic characterization using 16S rDNA sequencing. This analysis confirmed that the type-A and -C isolates were members of the β-Proteobacteria. The type-B isolate was shown to be a member of the Xanthomonas group of the γ-Proteobacteria. Our results demonstrate that probe GAM42a (specific for γ-Proteobacteria; Manz et al., 1992) does not hybridize to the 23S rRNA target sequence of this group of deep branching γ-Proteobacteria: this was confirmed by hybridization experiments with Xanthomonas fragariae , which also failed to hybridize to this probe. 23S rDNA sequence analysis revealed that probe GAM42a has one mismatch with the target sequence of the type-B isolate and two mismatches with the target sequence of X. fragariae . Furthermore it was shown that double stranded DNA fragments of 626 bp length, which differed by as many as two to three nucleotides were not separated by DGGE. This suggested that rDNA fragments of closely related bacteria (99.8% sequence similarity or more) are not resolved by DGGE. We propose that the combined use of DGGE and whole-cell hybridization provides a rapid way to distinguish distantly related microbial isolates.

We have analysed the developmental defects in Drosophila embryos lacking a gelsolin-related protein encoded by the gene flightless I. Such embryos have previously been reported to gastrulate abnormally. We now show that the most dramatic defects are seen earlier, in actin-dependent events during cellularisation of the syncytial blastoderm, a process with similarities to cytokinesis. The blastoderm nuclei migrate to the periphery of the egg normally but lose their precise cortical positioning during cellularisation. Cleavage membranes are initially formed, but invaginate irregularly and often fail to close at the basal end of the newly formed cells. The association of actin with the cellularisation membranes is irregular, suggesting a role for flightless I in the delivery of actin to the actin network, or in its stabilisation.

The concurrent use of karst aquifers as drinking water resources and receptors of combined sewer overflow lacking appropriate pre-treatment may cause conflicts between drinking water supply and storm water management. A storm water tank (SWT) for combined wastewater is identified as the source of sporadic contamination of a karst spring (Gallusquelle, "Schwabische Alb", SW Germany) used for public water supply. Spring water quality was examined by routine and event sampling and by evaluating physicochemical and microbiological parameters. The total number of microbial colonies growing at 20A degrees C and the number of Escherichia coli colonies rose to values up to four orders of magnitude higher than background, 2-5 days after overflow of the SWT. High concentrations of chloride, sodium, and total organic carbon (TOC) and high values of turbidity coincide with this increase. However, high bacterial contamination is also observed while turbidity and TOC are low. Several wastewater-related organic micro-pollutants such as chlorinated and non-chlorinated organophosphates were detected in the SWT and, depending on their K (ow) values and their biodegradability, in lower concentrations at the spring.

In this study we determined the composition and biogeochemistry of novel, brightly colored, white and orange microbial mats at the surface of a brine seep at the outer rim of the Chefren mud volcano. These mats were interspersed with one another, but their underlying sediment biogeochemistries differed considerably. Microscopy revealed that the white mats were granules composed of elemental S filaments, similar to those produced by the sulfide-oxidizing epsilonproteobacterium “ Candidatus Arcobacter sulfidicus.” Fluorescence in situ hybridization indicated that microorganisms targeted by a “ Ca . Arcobacter sulfidicus”-specific oligonucleotide probe constituted up to 24% of the total the cells within these mats. Several 16S rRNA gene sequences from organisms closely related to “ Ca . Arcobacter sulfidicus” were identified. In contrast, the orange mat consisted mostly of bright orange flakes composed of empty Fe(III) (hydr)oxide-coated microbial sheaths, similar to those produced by the neutrophilic Fe(II)-oxidizing betaproteobacterium Leptothrix ochracea . None of the 16S rRNA gene sequences obtained from these samples were closely related to sequences of known neutrophilic aerobic Fe(II)-oxidizing bacteria. The sediments below both types of mats showed relatively high sulfate reduction rates (300 nmol·cm −3 ·day −1 ) partially fueled by the anaerobic oxidation of methane (10 to 20 nmol·cm −3 ·day −1 ). Free sulfide produced below the white mat was depleted by sulfide oxidation within the mat itself. Below the orange mat free Fe(II) reached the surface layer and was depleted in part by microbial Fe(II) oxidation. Both mats and the sediments underneath them hosted very diverse microbial communities and contained mineral precipitates, most likely due to differences in fluid flow patterns.

In anoxic habitats, ferric iron oxides and humic acids are widespread, and ferric‐iron‐ and humic‐acid‐reducing microorganisms presumably play an important role in the oxidation of organic matter. Representative strains of ferric‐iron‐ or humic‐acid‐reducing bacteria were isolated from a wide range of freshwater or marine environments. Most of them are strict anaerobes, and facultatively anaerobic microorganisms reduce ferric iron oxides or humic acids only after oxygen has been consumed. Hence, anaerobic techniques have to be used for the preparation of media as well as for the cultivation of microorganisms. Furthermore, special caution is needed in the preparation of ferric iron oxides and humic acids.

Geobacter species such as G. bremensis, G. pelophilus, and G. sulfurreducens are obligately anaerobic and grow in anoxic, non-reduced medium by fast reduction of soluble ferric citrate. In contrast, insoluble ferrihydrite was either only slowly or not reduced when supplied as electron acceptor in similar growth experiments. Ferrihydrite reduction was stimulated by addition of a reducing agent or by concomitant growth of secondary bacteria that were physiologically and phylogenetically as diverse as Escherichia coli, Lactococcus lactis, or Pseudomonas stutzeri. In control experiments with heat-inactivated Geobacter cells and viable secondary bacteria, no ( E. coli, P. stutzeri) or only little ( L. lactis) ferrihydrite was reduced. Redox indicator dyes showed that growing E. coli, P. stutzeri, or L. lactis cells lowered the redox potential of the medium in a similar way as a reducing agent did. The lowered redox potential was presumably the key factor that stimulated ferrihydrite reduction by all three Geobacter species. The observed differences in anoxic non-reduced medium with ferric citrate versus ferrihydrite as electron acceptor indicated that reduction of these electron acceptors involved different cellular components or different biochemical strategies. Furthermore, it appears that redox-sensitive components are involved, and/or that gene expression of components needed for ferrihydrite reduction is controlled by the redox state.

In previous studies, three different strains (BrG1, BrG2, and BrG3) of ferrous iron-oxidizing, nitrate-reducing bacteria were obtained from freshwater sediments. All three strains were facultative anaerobes and utilized a variety of organic substrates and molecular hydrogen with nitrate as electron acceptor. In this study, analyses of 16S rDNA sequences showed that strain BrG1 was affiliated with the genus Acidovorax, strain BrG2 with the genus Aquabacterium, and strain BrG3 with the genus Thermomonas. Previously, bacteria similar to these three strains were detected with molecular techniques in MPN dilution series for ferrous iron-oxidizing, nitrate-reducing bacteria inoculated with different freshwater sediment samples. In the present study, further molecular analyses of these MPN cultures indicated that the ability to oxidize ferrous iron with nitrate is widespread amongst the Proteobacteria and may also be found among the Gram-positive bacteria with high GC content of DNA. Nitrate-reducing bacteria oxidized ferrous iron to poorly crystallized ferrihydrite that was suitable as an electron acceptor for ferric iron-reducing bacteria. Biologically produced ferrihydrite and synthetically produced ferrihydrite were both well suited as electron acceptors in MPN dilution cultures. Repeated anaerobic cycling of iron was shown in a coculture of ferrous iron-oxidizing bacteria and the ferric iron-reducing bacterium Geobacter bremensis. The results indicate that iron can be cycled between its oxidation states +II and +III by microbial activities in anoxic sediments.

Anaerobic dissimilatory ferric iron-reducing and ferrous iron-oxidizing bacteria gain energy through reduction or oxidation of iron minerals and presumably play an important role in catalyzing iron transformations in anoxic environments. Numerous ferric iron-reducing bacteria have been isolated from a great diversity of anoxic environments, including sediments, soils, deep terrestrial subsurfaces, and hot springs. In contrast, only few ferrous iron-oxidizing bacteria are known so far. At neutral pH, iron minerals are barely soluble, and the mechanisms of electron transfer to or from iron minerals are still only poorly understood. In natural habitats, humic substances may act as electron carriers for ferric iron-reducing bacteria. Also fermenting bacteria were shown to channel electrons to ferric iron via humic acids.Whether quinones or cytochromes released from cells act as electron transfer components in ferric iron reduction is still a matter of debate. Anaerobic ferrous iron-oxidizing phototrophic bacteria, on the other hand, appear to excrete complexing agents to prevent precipitation of ferric iron oxides at their cell surfaces. The present review evaluates recent findings on the physiology of ferric iron-reducing and ferrous iron-oxidizing bacteria with respect to their relevance to microbial iron transformations in nature.

Anaerobic, nitrate-dependent microbial oxidation of ferrous iron was recently recognized as a new type of metabolism. In order to study the occurrence of three novel groups of ferrous iron-oxidizing, nitrate-reducing bacteria (represented by strains BrG1, BrG2, and BrG3), 16S rRNA-targeted oligonucleotide probes were developed. In pure-culture experiments, these probes were shown to be suitable for fluorescent in situ hybridization, as well as for hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns. However, neither enumeration by in situ hybridization nor detection by the DGGE-hybridization approach was feasible with sediment samples. Therefore, the DGGE-hybridization approach was combined with microbiological methods. Freshwater sediment samples from different European locations were used for enrichment cultures and most-probable-number (MPN) determinations. Bacteria with the ability to oxidize ferrous iron under nitrate-reducing conditions were detected in all of the sediment samples investigated. At least one of the previously described types of bacteria was detected in each enrichment culture. MPN studies showed that sediments contained from 1 × 10 5 to 5 × 10 8 ferrous iron-oxidizing, nitrate-reducing bacteria per g (dry weight) of sediment, which accounted for at most 0.8% of the nitrate-reducing bacteria growing with acetate. Type BrG1, BrG2, and BrG3 bacteria accounted for an even smaller fraction (0.2% or less) of the ferrous iron-oxidizing, nitrate-reducing community. The DGGE patterns of MPN cultures suggested that more organisms than those isolated thus far are able to oxidize ferrous iron with nitrate. A comparison showed that among the anoxygenic phototrophic bacteria, organisms that have the ability to oxidize ferrous iron also account for only a minor fraction of the population.

Enrichment and pure cultures of nitrate-reducing bacteria were shown to grow anaerobically with ferrous iron as the only electron donor or as the additional electron donor in the presence of acetate. The newly observed bacterial process may significantly contribute to ferric iron formation in the suboxic zone of aquatic sediments.