N2 -fixation can sustain wastewater treatment performance of photogranules under nitrogen-limiting conditions.

Wastewater characteristics can vary significantly, and in some municipal wastewaters the N:P ratio is as low as 5 resulting in nitrogen-limiting conditions. In this study, the microbial community, function, and morphology of photogranules under nitrogen-replete (N+) and limiting (N-) conditions was assessed in sequencing batch reactors. Photogranules under N- condition were nitrogen deprived 2/3 of a batch cycle duration. Surprisingly, this nitrogen limitation had no adverse effect on biomass productivity. Moreover, phosphorus and chemical oxygen demand removal were similar to their removal under N+ conditions. Although performance was similar, the difference in granule morphology was obvious. While N+ photogranules were dense and structurally confined, N- photogranules showed loose structures with occasional voids. Microbial community analysis revealed high abundance of cyanobacteria capable of N2 -fixation. These were higher at N- (38%) than N+ (29%) treatments, showing that photogranules could adjust and maintain treatment performance and high biomass productivity by means of N2 -fixation.

Distinct glycoconjugate cell surface structures make the pelagic diatom Thalassiosira rotula an attractive habitat for bacteria.

Interactions between marine diatoms and bacteria have been studied for decades. However, the visualization of physical interactions between these diatoms and their colonizers is still limited. To enhance our understanding of these specific interactions, a new Thalassiosira rotula isolate from the North Sea (strain 8673) was characterized by scanning electron microscopy and confocal laser scanning microscopy (CLSM) after staining with fluorescently labeled lectins targeting specific glycoconjugates. To investigate defined interactions of this strain with bacteria the new strain was made axenic and co-cultivated with a natural bacterial community and in two- or three-partner consortia with different bacteria of the Roseobacter group, Gammaproteobacteria and Bacteroidetes. The CLSM analysis of the consortia identified six out of 78 different lectins as very suitable to characterize glycoconjugates of T. rotula. The resulting images show that fucose-containing threads were the dominant glycoconjugates secreted by the T. rotula cells but chitin and to a lesser extent other glycoconjugates were also identified. Bacteria attached predominantly to the fucose glycoconjugates. The colonizing bacteria showed various attachment patterns such as adhering to the diatom threads in aggregates only or attaching to both the surfaces and the threads of the diatom. Interestingly the colonization patterns of single bacteria differed strikingly from those of bacterial co-cultures, indicating that interactions between two bacterial species impacted the colonization of the diatom. Our observations help to better understand physical interactions and specific colonization patterns of distinct bacterial mono- and co-cultures with an abundant diatom of costal seas.

Production of nonulosonic acids in the extracellular polymeric substances of "Candidatus Accumulibacter phosphatis".

Nonulosonic acids (NulOs) are a family of acidic carbohydrates with a nine-carbon backbone, which include different related structures, such as sialic acids. They have mainly been studied for their relevance in animal cells and pathogenic bacteria. Recently, sialic acids have been discovered as an important compound in the extracellular matrix of virtually all microbial life and in "Candidatus Accumulibacter phosphatis", a well-studied polyphosphate-accumulating organism, in particular. Here, bioaggregates highly enriched with these bacteria (approx. 95% based on proteomic data) were used to study the production of NulOs in an enrichment of this microorganism. Fluorescence lectin-binding analysis, enzymatic quantification, and mass spectrometry were used to analyze the different NulOs present, showing a wide distribution and variety of these carbohydrates, such as sialic acids and bacterial NulOs, in the bioaggregates. Phylogenetic analysis confirmed the potential of "Ca. Accumulibacter" to produce different types of NulOs. Proteomic analysis showed the ability of "Ca. Accumulibacter" to reutilize and reincorporate these carbohydrates. This investigation points out the importance of diverse NulOs in non-pathogenic bacteria, which are normally overlooked. Sialic acids and other NulOs should be further investigated for their role in the ecology of "Ca. Accumulibacter" in particular, and biofilms in general. KEY POINTS: •"Ca. Accumulibacter" has the potential to produce a range of nonulosonic acids. •Mass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids. •The role of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail.

Decorating the Anammox House: Sialic Acids and Sulfated Glycosaminoglycans in the Extracellular Polymeric Substances of Anammox Granular Sludge.

Anammox (anaerobic ammonium oxidation) bacteria are important for the nitrogen cycle in both natural environments and wastewater treatment plants. These bacteria have a strong tendency to grow in aggregates like biofilms and granular sludge. To understand the formation of anammox aggregates, it is required to unravel the composition of the extracellular polymeric substances (EPS), which are produced by the bacteria to develop into aggregates and granules. Here, we investigated anionic polymers in anammox granular sludge, focussing on sialic acids and sulfated glycosaminoglycans. Quantification assays and fluorescent stains indicated that sialic acids and sulfated glycosaminoglycans were present in the anammox EPS (1.6% equivalents of sialic acids and 2.4% equivalents of sulfated glycosaminoglycans). Additionally, the potential genes for the biosynthesis of sialic acids and sulfated glycosaminoglycans were analyzed in the anammox draft genomes. The finding of these components in anammox granular sludge and previously in other nonpathogenic bacteria pointed out that sialic acids and sulfated glycosaminoglycans are worth investigating in the context of a broader function in microbial communities and biofilm systems in general.

Flatworm mucus as the base of a food web.

BACKGROUND: By altering their habitats, engineering species can improve their own fitness. However, the effect of this strategy on the fitness of coexisting species or on the structure of the respective food web is poorly understood. In this study, bacteria and bacterivorous nematodes with short (Caenorhabditis elegans) and long (Plectus acuminatus) life cycles were exposed to the mucus secreted by the freshwater flatworm Polycelis tenuis. The growth, reproduction, and feeding preferences of the nematodes in the presence/absence of the mucus were then determined. In addition, confocal laser scanning microscopy (CLSM) was used to examine the structural footprint of the mucus and the mucus colonization dynamics of bacteria and protozoans. RESULTS: Mucus exposure resulted in a greater reproductive output in P. acuminatus than in C. elegans. In a cafeteria experiment, both nematode species were attracted by bacteria-rich patches and were not deterred by mucus. CLSM showed that the flatworms spread a layer of polysaccharide-rich mucus ca. 15 µm thick from their tails. Subsequent colonization of the mucus by bacteria and protozoans resulted in an architecture that progressively resembled a complex biofilm. The presence of protozoans reduced nematode reproduction, presumably due to competition for their bacterial food supply. CONCLUSION: Animal secretions such as mucus may have broader, community-level consequences and contribute to fueling microbial food webs.

Thermodesulfobium sp. strain 3baa, an acidophilic sulfate reducing bacterium forming biofilms triggered by mineral precipitation.

Sulfate reducing prokaryotes are promising candidates for the remediation of acidic metal-rich waste waters. However, only few acidophilic species have been described to date. Chemolithoautotrophic strain 3baa was isolated from sediments of an acidic mine pit lake. Based on its 16S-rRNA gene sequence it belongs to the genus Thermodesulfobium. It was identified as an acidophile growing in artificial pore water medium in the range of pH 2.6-6.6. Though the highest sulfate reduction rates were obtained at the lower end of this range, elongated cells and extended lag phases demonstrated acid stress. Sulfate reduction at low pH was accompanied by the formation of mineral precipitates strongly adhering to solid surfaces. A structural investigation by laser scanning microscopy, electron microscopy and X-ray microanalysis revealed the formation of Al hydroxides and Fe sulfides which were densely populated by cells. Al hydroxides precipitated first, enabling initial cell attachment. Colonization of solid surfaces coincided with increased sulfate reducing activity indicating more favourable growth conditions within biofilms compared with free-living cells. These findings point out the importance of cell-mineral interaction for biofilm formation and contribute to our understanding how sulfate reducing prokaryotes thrive in both natural and engineered systems at low pH.

Plastic Alters Biofilm Quality as Food Resource of the Freshwater Gastropod Radix balthica.

High amounts of plastic debris enter and accumulate in freshwater systems across the globe. The plastic contamination of benthic habitats in lakes and running waters poses a potential threat to freshwater ecosystems. This study investigates the effects of plastic on two trophic levels of the aquatic food web: primary production, that is, epiplastic biofilm, and primary consumption, that is, a benthic invertebrate grazer. Two plastic types, polymethyl methacrylate (PMMA) and polycarbonate (PC), and glass (control) were used as substrata for natural biofilm establishment. PMMA and PC are, for example, intensively used in the automobile, construction, and electronical industries and in cosmetics (PMMA), CDs, and DVDs (PC). These biofilms were fed to the freshwater gastropod  Radix balthica (Linnaeus 1758) in a laboratory-grazing experiment. Biofilm structure and composition were observed using confocal laser scanning microscopy before the grazing experiment. Sublethal effects on R. balthica were observed measuring consumption of biofilm and growth rates. The biofilm composition on PMMA significantly differed compared to PC and glass. The grazing experiments showed limited biofilm consumption and lower growth rates of R. balthica in both plastic treatments. Concluding, plastic in freshwaters has a direct effect on the primary production and an indirect effect on higher trophic levels.

Identification of Glycoproteins Isolated from Extracellular Polymeric Substances of Full-Scale Anammox Granular Sludge.

ANaerobic AMMonium OXidation (anammox) is an established process for efficient nitrogen removal from wastewater, relying on anammox bacteria to form stable biofilms or granules. To understand the formation, structure, and stability of anammox granules, it is important to determine the composition of the extracellular polymeric substances (EPS). The aim of this research was to elucidate the nature of the proteins, which are the major fraction of the EPS and were suspected to be glycosylated. EPS were extracted from full-scale anammox granular sludge, dominated by " Candidatus Brocadia", and subjected to denaturing polyacrylamide gel electrophoresis. By further analysis with mass spectrometry, a high abundant glycoprotein, carrying a heterogeneous O-glycan structure, was identified. The potential glycosylation sequence motif was identical to that proposed for the surface layer protein of " Candidatus Kuenenia stuttgartiensis". The heavily glycosylated protein forms a large fraction of the EPS and was also located by lectin staining. Therefore, we hypothesize an important role of glycoproteins in the structuring of anammox granules, comparable to the importance of glycans in the extracellular matrix of multicellular organisms. Furthermore, different glycoconjugates may have distinct roles in the matrix of granular sludge, which requires more in-depth characterization of different glycoconjugates in future EPS studies.

Schwertmannite formation at cell junctions by a new filament-forming Fe(II)-oxidizing isolate affiliated with the novel genus Acidithrix.

A new acidophilic iron-oxidizing strain (C25) belonging to the novel genus Acidithrix was isolated from pelagic iron-rich aggregates ('iron snow') collected below the redoxcline of an acidic lignite mine lake. Strain C25 catalysed the oxidation of ferrous iron [Fe(II)] under oxic conditions at 25 °C at a rate of 3.8 mM Fe(II) day(-1) in synthetic medium and 3.0 mM Fe(II) day(-1) in sterilized lake water in the presence of yeast extract, producing the rust-coloured, poorly crystalline mineral schwertmannite [Fe(III) oxyhydroxylsulfate]. During growth, rod-shaped cells of strain C25 formed long filaments, and then aggregated and degraded into shorter fragments, building large cell-mineral aggregates in the late stationary phase. Scanning electron microscopy analysis of cells during the early growth phase revealed that Fe(III)-minerals were formed as single needles on the cell surface, whereas the typical pincushion-like schwertmannite was observed during later growth phases at junctions between the cells, leaving major parts of the cell not encrusted. This directed mechanism of biomineralization at specific locations on the cell surface has not been reported from other acidophilic iron-oxidizing bacteria. Strain C25 was also capable of reducing Fe(III) under micro-oxic conditions which led to a dissolution of the Fe(III)-minerals. Thus, strain C25 appeared to have ecological relevance for both the formation and transformation of the pelagic iron-rich aggregates at oxic/anoxic transition zones in the acidic lignite mine lake.

The biofilm matrix of Campylobacter jejuni determined by fluorescence lectin-binding analysis.

Campylobacter jejuni is responsible for the most common bacterial foodborne gastroenteritis. Despite its fastidious growth, it can survive harsh conditions through biofilm formation. In this work, fluorescence lectin-binding analysis was used to determine the glycoconjugates present in the biofilm matrix of two well-described strains. Screening of 72 lectins revealed strain-specific patterns with six lectins interacting with the biofilm matrix of both strains. The most common sugar moiety contained galactose and N-acetylgalactosamine. Several lectins interacted with N-acetylglucosamine and sialic acid, probably originated from the capsular polysaccharides, lipooligosaccharides and N-glycans of C. jejuni. In addition, glycoconjugates containing mannose and fucose were detected within the biofilm, which have not previously been found in the C. jejuni envelope. Detection of thioflavin T and curcumin highlighted the presence of amyloids in the cell envelope without association with specific cell appendages. The lectins ECA, GS-I, HMA and LEA constitute a reliable cocktail to detect the biofilm matrix of C. jejuni.

Visualization and analysis of EPS glycoconjugates of the thermoacidophilic archaeon Sulfolobus metallicus.

Biofilms are surface-associated colonies of microorganisms embedded in a matrix of extracellular polymeric substances (EPS). As EPS mediate the contact between cells and surfaces, an understanding of their composition and production is of particular interest. In this study, the EPS components of Sulfolobus metallicus DSM 6482(T) forming biofilms on elemental sulfur (S(0)) were investigated by confocal laser scanning microscopy (CLSM). In order to visualize cell and EPS distributions, biofilm cells were stained with various dyes specific for glycoconjugates, proteins, nucleic acids and lipids. Biofilm cells on S(0) were heterogeneously distributed and characterized as individual cells, microcolonies, and large clusters up to a hundred micrometers in diameter. The glycoconjugates in biofilms were detected by fluorescence lectin-binding analysis (FLBA). Screening of 72 commercially available lectins resulted in the selection of 21 lectins useful for staining biofilms of S. metallicus (T). Capsular EPS from planktonic cells were mainly composed of carbohydrates and proteins. In contrast, colloidal EPS from planktonic cells were dominated by carbohydrates. Proteins were found to be major components in EPS from biofilms on S(0). Using specific probes combined with CLSM, we showed that extracellular proteins and nucleic acids were present in the EPS matrix. Finally, we showed that S. metallicus (T) cells were embedded in a flexible EPS matrix. This study provides new insights into archaeal biofilms and EPS composition and properties with respect to their interactions with S(0).

Selective elimination of bacterial faecal indicators in the Schmutzdecke of slow sand filtration columns.

Slow sand filtration (SSF) is an effective low-tech water treatment method for pathogen and particle removal. Yet despite its application for centuries, it has been uncertain to which extent pathogenic microbes are removed by mechanical filtration or due to ecological interactions such as grazing and competition for nutrients. In this study, we quantified the removal of bacterial faecal indicators, Escherichia coli and Enterococcus faecalis, from secondary effluent of a wastewater treatment plant and analysed the microbial community composition in compartments of laboratory model SSF columns. The columns were packed with different sand grain sizes and eliminated 1.6-2.3 log units of faecal indicators, which translated into effluents of bathing water quality according to the EU directive (<500 colony forming units of E. coli per 100 ml) for columns with small grain size. Most of that removal occurred in the upper filter area, the Schmutzdecke. Within that same zone, total bacterial numbers increased however, thus suggesting a specific elimination of the faecal indicators. The analysis of the microbial communities also revealed that some taxa were removed more from the wastewater than others. These results accentuate the contribution of biological mechanisms to water purification in SSF.

Arsenic-rich acid mine water with extreme arsenic concentration: mineralogy, geochemistry, microbiology, and environmental implications.

Extremely arsenic-rich acid mine waters have developed by weathering of native arsenic in a sulfide-poor environment on the 10th level of the Svornost mine in Jáchymov (Czech Republic). Arsenic rapidly oxidizes to arsenolite (As2O3), and there are droplets of liquid on the arsenolite crust with high As concentration (80,000-130,000 mg·L(-1)), pH close to 0, and density of 1.65 g·cm(-1). According to the X-ray absorption spectroscopy on the frozen droplets, most of the arsenic is As(III) and iron is fully oxidized to Fe(III). The EXAFS spectra on the As K edge can be interpreted in terms of arsenic polymerization in the aqueous solution. The secondary mineral that precipitates in the droplets is kaatialaite [Fe(3+)(H2AsO4)3·5H2O]. Other unusual minerals associated with the arsenic lens are behounekite [U(4+)(SO4)2·4H2O], stepite [U(4+)(AsO3OH)2·4H2O], vysokýite [U(4+)[AsO2(OH)2]4·4H2O], and an unnamed phase (H3O)(+)2(UO2)2(AsO4)2·nH2O. The extremely low cell densities and low microbial biomass have led to insufficient amounts of DNA for downstream polymerase chain reaction amplification and clone library construction. We were able to isolate microorganisms on oligotrophic media with pH ∼ 1.5 supplemented with up to 30 mM As(III). These microorganisms were adapted to highly oligotrophic conditions which disabled long-term culturing under laboratory conditions. The extreme conditions make this environment unfavorable for intensive microbial colonization, but our first results show that certain microorganisms can adapt even to these harsh conditions.

Unique architecture of microbial snottites from a methane driven biofilm revealed by confocal microscopy.

Microbial biofilms occur in many shapes and different dimensions. In natural and semi-artificial caves they are forming pendulous structures of 10 cm and more. In this study a methane driven microbial community of a former medicinal spring was investigated. The habitat was completely covered by massive biofilms and snottites with a wobbly, gelatinous appearance. By using fluorescence techniques in combination with confocal laser scanning microscopy the architecture of these so far unknown snottites was examined. The imaging approaches applied comprised reflection of geogenic and cellular origin, possible autofluorescence, nucleic acid staining for bacterial cells, protein staining for bacteria and extracellular fine structures, calcofluor white for ß 1 → 3, ß 1 → 4 polysaccharide staining for possible fungi as well as lectin staining for the extracellular biofilm matrix glycoconjugates. The results showed a highly complex, intricate structure with voluminous, globular, and tube-like glycoconjugates of different dimensions and densities. In addition, filamentous bacteria seem to provide additional strength to the snottites. After screening with all commercially available lectins, by means of fluorescence lectin barcoding and subsequent fluorescence lectin binding analysis, the AAL, PNA, LEA, and Ban lectins identified α-Fuc, ß-Gal, ß-GlcNAc, and α-Man with α-Fuc as a major component. Examination of the outer boundary with fluorescent beads revealed a potential outer layer which could not be stained by any of the fluorescent probes applied. Finally, suggestions are made to further elucidate the characteristics of these unusual microbial biofilms in form of snottites. RESEARCH HIGHLIGHTS: The gelatinous snottites revealed at the microscale a highly complex structure not seen before. The extracellular matrix of the snottite biofilm was identified as clusters of different shape and density. The matrix of snottites was examined by taking advantage of 78 fluorescently-labeled lectins. The extracellular matrix glycoconjugates of snottites identified comprised: α-Fuc, ß-Gal, ß-GlcNAc, and α-Man. Probing the snottite outer surface indicated an additional unknown stratum.

Microbial extracellular polymeric substances in the environment, technology and medicine.

Microbial biofilms exhibit a self-produced matrix of extracellular polymeric substances (EPS), including polysaccharides, proteins, extracellular DNA and lipids. EPS promote interactions of the biofilm with other cells and sorption of organics, metals and chemical pollutants, and they facilitate cell adhesion at interfaces and ensure matrix cohesion. EPS have roles in various natural environments, such as soils, sediments and marine habitats. In addition, EPS are relevant in technical environments, such as wastewater and drinking water treatment facilities, and water distribution systems, and they contribute to biofouling and microbially influenced corrosion. In medicine, EPS protect pathogens within the biofilm against the host immune system and antimicrobials, and emerging evidence suggests that EPS can represent potential virulence factors. By contrast, EPS yield a wide range of valuable products that include their role in self-repairing concrete. In this Review, we aim to explore EPS as a functional unit of biofilms in the environment, in technology and in medicine.

Chip-calorimetric monitoring of biofilm eradication with antibiotics provides mechanistic information.

Increased antibiotic resistance of pathogenic bacteria dwelling in biofilm structures has motivated the development of various monitoring tools specifically designed for biofilm investigations. In this study, the potential of the recently emerging chip calorimetry for this purpose was analysed. The activity of biofilms of Pseudomonas putida PaW340 was monitored chip-calorimetrically and compared with counts of colony forming units (CFU), bioluminescence-based ATP measurements, and quantitative confocal laser scanning microscopy (CLSM). The biofilms were treated with antibiotics differing in their mechanisms of action (bactericidal kanamycin vs. bacteriostatic tetracycline) and referenced to untreated biofilms. For untreated biofilms, all methods gave comparable results. Calorimetric killing curves, however, reflecting metabolic responses to biofilm eradication non-invasively in real time, differed from those obtained with the established methods. For instance, heat signals increased right after addition of the antibiotics. This transient increase of activity was not detected by the other methods, since only calorimetry delivers specific information about the catabolic part of the metabolism. In case of the bactericidal antibiotic, CFU misleadingly indicated successful biofilm eradication, whereas calorimetry revealed enduring activity. Our results show that calorimetry holds promise to provide valuable mechanistic information, thereby complementing other methods of biofilm analysis.

Insights into the structure and metabolic function of microbes that shape pelagic iron-rich aggregates ("iron snow").

Microbial ferrous iron [Fe(II)] oxidation leads to the formation of iron-rich macroscopic aggregates ("iron snow") at the redoxcline in a stratified lignite mine lake in east-central Germany. We aimed to identify the abundant Fe-oxidizing and Fe-reducing microorganisms likely to be involved in the formation and transformation of iron snow present in the redoxcline in two basins of the lake that differ in their pH values. Nucleic acid- and lipid-stained microbial cells of various morphologies detected by confocal laser scanning microscopy were homogeneously distributed in all iron snow samples. The dominant iron mineral appeared to be schwertmannite, with shorter needles in the northern than in the central basin samples. Total bacterial 16S rRNA gene copies ranged from 5.0 × 10(8) copies g (dry weight)(-1) in the acidic central lake basin (pH 3.3) to 4.0 × 10(10) copies g (dry weight)(-1) in the less acidic (pH 5.9) northern basin. Total RNA-based quantitative PCR assigned up to 61% of metabolically active microbial communities to Fe-oxidizing- and Fe-reducing-related bacteria, indicating that iron metabolism was an important metabolic strategy. Molecular identification of abundant groups suggested that iron snow surfaces were formed by chemoautotrophic iron oxidizers, such as Acidimicrobium, Ferrovum, Acidithiobacillus, Thiobacillus, and Chlorobium, in the redoxcline and were rapidly colonized by heterotrophic iron reducers, such as Acidiphilium, Albidiferax-like, and Geobacter-like groups. Metaproteomics yielded 283 different proteins from northern basin iron snow samples, and protein identification provided a glimpse into some of their in situ metabolic processes, such as primary production (CO2 fixation), respiration, motility, and survival strategies.

Detection and quantification of a mycorrhization helper bacterium and a mycorrhizal fungus in plant-soil microcosms at different levels of complexity.

BACKGROUND: Host plant roots, mycorrhizal mycelium and microbes are important and potentially interacting factors shaping the performance of mycorrhization helper bacteria (MHB). We investigated the impact of a soil microbial community on the interaction between the extraradical mycelium of the ectomycorrhizal fungus Piloderma croceum and the MHB Streptomyces sp. AcH 505 in both the presence and the absence of pedunculate oak microcuttings. RESULTS: Specific primers were designed to target the internal transcribed spacer of the rDNA and an intergenic region between two protein encoding genes of P. croceum and the intergenic region between the gyrA and gyrB genes of AcH 505. These primers were used to perform real-time PCR with DNA extracted from soil samples. With a sensitivity of 10 genome copies and a linear range of 6 orders of magnitude, these real-time PCR assays enabled the quantification of purified DNA from P. croceum and AcH 505, respectively. In soil microcosms, the fungal PCR signal was not affected by AcH 505 in the absence of the host plant. However, the fungal signal became weaker in the presence of the plant. This decrease was only observed in microbial filtrate amended microcosms. In contrast, the PCR signal of AcH 505 increased in the presence of P. croceum. The increase was not significant in sterile microcosms that contained plant roots. CONCLUSIONS: Real-time quantitative PCR assays provide a method for directly detecting and quantifying MHB and mycorrhizal fungi in plant microcosms. Our study indicates that the presence of microorganisms and plant roots can both affect the nature of MHB-fungus interactions, and that mycorrhizal fungi may enhance MHB growth.

Benzene and sulfide removal from groundwater treated in a microbial fuel cell.

Sulfidic benzene-contaminated groundwater was used to fuel a two-chambered microbial fuel cell (MFC) over a period of 770 days. We aimed to understand benzene and sulfide removal processes in the anoxic anode chamber and describe the microbial community enriched over the operational time. Operated in batch feeding-like circular mode, supply of fresh groundwater resulted in a rapid increase in current production, accompanied by decreasing benzene and sulfide concentrations. The total electron recoveries for benzene and sulfide were between 18% and 49%, implying that benzene and sulfide were not completely oxidized at the anode. Pyrosequencing of 16S rRNA genes from the anode-associated bacterial community revealed the dominance of δ-Proteobacteria (31%), followed by ß-Proteobacteria, Bacteroidetes, ϵ-Proteobacteria, Chloroflexi, and Firmicutes, most of which are known for anaerobic metabolism. Two-dimensional compound-specific isotope analysis demonstrated that benzene degradation was initiated by monohydroxylation, probably triggered by small amounts of oxygen which had leaked through the cation exchange membrane into the anode chamber. Experiments with [(13)C(6) ]-benzene revealed incorporation of (13)C into fatty acids of mainly Gram-negative bacteria, which are therefore candidates for benzene degradation. Our study demonstrated simultaneous benzene and sulfide removal by groundwater microorganisms which use an anode as artificial electron acceptor, thereby releasing an electrical current.

Impact of mycelia on the accessibility of fluorene to PAH-degrading bacteria.

Mycelia have been recently shown to actively transport polycyclic aromatic hydrocarbons (PAH) in water-unsaturated soil over the range of centimeters, thereby efficiently mobilizing hydrophobic PAH beyond their purely diffusive transport in air and water. However, the question if mycelia-based PAH transport has an effect on PAH biodegradation was so far unsolved. To address this, we developed a laboratory model microcosm mimicking air-water interfaces in soil. Chemical analyses demonstrated transport of the PAH fluorene (FLU) by the mycelial oomycete Pythium ultimum that was grown along the air-water interfaces. Furthermore, degradation of mycelia-transported FLU by the bacterium Burkholderia sartisoli RP037-mChe was indicated. Since this organism expresses eGFP in response to a FLU flux to the cell, it was also as a bacterial reporter of FLU bioavailability in the vicinity of mycelia. Confocal laser scanning microscopy (CLSM) and image analyses revealed a significant increase of eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU. Hence, we could show that physically separated FLU becomes bioavailable to bacteria after transport by mycelia. Experiments with silicon coated glass fibers capturing mycelia-transported FLU guided us to propose a three-step mechanism of passive uptake, active transport and diffusion-driven release. These experiments were also used to evaluate the contributions of these individual steps to the overall mycelial FLU transport rate.