The lack of microbial degradation of polycyclic aromatic hydrocarbons from coal-rich soils.

Analytical techniques used to assess the environmental risk of contamination from polycyclic aromatic hydrocarbons (PAHs) typically consider only abiotic sample parameters. Supercritical fluid extraction and sorption enthalpy experiments previously suggested slow desorption rates for PAH compounds in two coal-contaminated floodplain soils. In this study, the actual PAH availability for aerobic soil microorganisms was tested in two series of soil-slurry experiments. The experimental conditions supported microbial degradation of phenanthrene if it was weakly sorbed onto silica gel. Native coals and coal-derived particles in two soils effectively acted as very strong sorbents and prevented microbial PAH degradation. The long history of PAH exposure and degree of coal contamination apparently had no influence on the capability of the microbial soil community to overcome constraints of PAH availability. Within the context of the experimental conditions and the compounds chosen, our results confirm that coal-bound PAHs are not bioavailable and hence of low environmental concern.

Biogeochemistry and community composition of iron- and sulfur-precipitating microbial mats at the Chefren mud volcano (Nile Deep Sea Fan, Eastern Mediterranean).

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 x cm(-3) x day(-1)) partially fueled by the anaerobic oxidation of methane (10 to 20 nmol x cm(-3) x 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.

Iron-mediated microbial oxidation and abiotic reduction of organic contaminants under anoxic conditions.

In anoxic environments, the oxidation of organic compounds, such as BTEX fuel components, by dissimilatory Fe(III) reduction can generate reactive mineral-bound Fe(II) species, which in turn are able to reduce other classes of organic and inorganic groundwater contaminants. In this study, we designed and evaluated an anaerobic batch reactor that mimicks iron-reducing conditions to investigate the factors that favor the coupling of microbial toluene oxidation and abiotic reduction of nitroaromatic contaminants. We investigated the influence of different Fe(III)-bearing minerals and combinations thereof on the coupling of these two processes. Results from laboratory model systems show that complete oxidation of toluene to CO2 by Geobacter metallireducens in the presence of Fe(III)-bearing minerals leads to the formation of mineral-bound Fe(II) species capable of the reduction of 4-nitroacetophenone. Whereas significant microbial toluene oxidation was only observed in the presence of amorphous Fe(III) phases, reduction of nitroaromatic compounds only proceeded with Fe(II) species bound to crystalline Fe(III) oxides. Our results suggest that in anoxic soils and sediments containing amorphous and crystalline iron phases simultaneously, coupling of microbial oxidation and abiotic reduction of organic compounds may allow for concurrent natural attenuation of different contaminant classes.

Enrichment and isolation of ferric-iron- and humic-acid-reducing bacteria.

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.

Occurrence of hopanoid lipids in anaerobic Geobacter species.

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.

Ferrihydrite-dependent growth of Sulfurospirillum deleyianum through electron transfer via sulfur cycling.

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.

Ferrihydrite reduction by Geobacter species is stimulated by secondary bacteria.

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.

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.