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Fungi possess the biochemical and ecological capacity to degrade environmental organic chemicals and to decrease the risk associated with metals, metalloids and radionuclides, either by chemical modification or by influencing chemical bioavailability. Furthermore, the ability of these fungi to form extended mycelial networks, the low specificity of their catabolic enzymes and their independence from using pollutants as a growth substrate make these fungi well suited for bioremediation processes. However, despite dominating the living biomass in soil and being abundant in aqueous systems, fungi have not been exploited for the bioremediation of such environments. In this Review, we describe the metabolic and ecological features that make fungi suited for use in bioremediation and waste treatment processes, and discuss their potential for applications on the basis of these strengths.

The capacity of fungi to serve as vectors for the dispersion of pollutant-degrading bacteria was analyzed in laboratory model systems mimicking water-saturated (agar surfaces) and unsaturated soil environments (glass-bead-filled columns). Two common soil fungi (Fusarium oxysporum and Rhexocercosporidium sp.) forming hydrophilic and hydrophobic mycelia, respectively, and three polycyclic aromatic hydrocarbon degrading bacteria (Achromobacter sp. SK1, Mycobacterium frederiksbergense LB501TG, and Sphingomonas sp. L138) were selected based on the absence of mutual antagonistic effects. It was shown that fungal hyphae act as vectors for bacterial transport with mobilization strongly depending on the specific microorganisms chosen: The motile strain Achromobacter sp. SK1 was most efficiently spread along hyphae of hydrophilic F. oxysporum in both model systems with transport velocities of up to 1 cm d(-1), whereas no dispersion of the two nonmotile strains was observed in the presence of F. oxysporum. By contrast, none of the bacteria was mobilized along the hydrophobic mycelia of Rhexocercosporidium sp. growing on agar surfaces. In column experiments however, strain SK1 was mobilized by Rhexocercosporidium sp. It is hypothesized that bacteria may move by their intrinsic motilitythrough continuous (physiological) liquid films forming around fungal hyphae. The results of this study suggest that the specific stimulation of indigenous fungi may be a strategy to mobilize pollutant-degrading bacteria leading to their homogenization in polluted soil thereby improving bioremediation.

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Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.

Using proteins from soil or groundwater as functional biomarkers requires efficient extraction. We developed an extraction method in which the separation of proteins from the inorganic and organic constituents of the soil matrix was achieved by a combination of 0.1 m NaOH treatment and phenol extraction. Incubation with NaOH released humic acids and proteins from soil minerals, and simultaneously, disrupted microorganisms. The subsequent phenol extraction separated the proteins from the humic organic matter. Protein extracts were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 2D-electrophoresis (2-DE). Spots and bands were excised and individual proteins identified by liquid chromatography online linked to mass spectrometry (MS) via electrospray ionization source (LC-ESI-MS). To assess the suitability of the method for the functional analysis of environmental metaproteomes, it was applied to soil that had been enriched in chlorophenoxy acid-degrading bacteria by incubation with 2,4-dichlorophenoxy acetic acid (2,4-D) for 22 days. The method was also used to analyze groundwater from the aquifer of a chlorobenzene-contaminated site. The identification of enzymes such as chlorocatechol dioxygenases was consistent with bacterial metabolic pathways expected to be expressed in these samples. The protocol enabled thus the analysis of the metaproteome of soil and groundwater samples. It thereby provides a means to study the diversity of environmental microbial communities while addressing functional aspects more directly than metagenome or even metatranscriptome analysis.

Testing for arsenic pollution is commonly performed with chemical test kits of unsatisfying accuracy. Bacterial biosensors are an interesting alternative as they are easily produced, simple, and highly accurate devices. Here, we describe the development of a set of bacterial biosensors based on a nonpathogenic laboratory strain of Escherichia coli, the natural resistance mechanism of E. coli against arsenite and arsenate, and three reporter proteins: bacterial luciferase, beta-galactosidase and Green Fluorescent Protein (GFP). The biosensors were genetically optimized to reduce background expression in the absence of arsenic. In calibration experiments with the biosensors and arsenite-amended potable water, arsenite concentrations at 4 microg of As/L (0.05 microM) were routinely and accurately measured. The currently most quantitative system expressed the bacterial luciferase as reporter protein, responding proportional with a concentration range between 8 and 80 microg of As/L. Sensor cells could be stored as frozen batches, resuspended in plain media, and exposed to the aqueous test sample, and light emission was measured after 30-min incubation. Field testing for arsenite was achieved with a system that contained beta-galactosidase, producing a visible blue color at arsenite concentrations above 8 microg/L. For this sensor, a protocol was developed in which the sensor cells were dried on a paper strip and placed in the aqueous test solution for 30 min after which time color development was allowed to take place. The GFP sensor showed good potential for continuous rather than end point measurements. In all cases, growth of the biosensors and production of the strip test was achieved by very simple means with common growth media, and quality control of the sensors was performed by isolating the respective plasmids with the genetic constructs according to simple standard genetic technologies. Therefore, the biosensor cells and protocols may offer a realistic alternative for measuring arsenic contamination in potable water.

Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram 2 Sphingomonas sp. LB126 and the gram 1 Mycobacterium frederiksbergense LB501T. Bacterial proliferation activities observed via DAPI and Hoechst 33342 staining were linked to the energy charge and the proportion of dead cells as obtained by diOC 6 (3)-staining and PI-uptake, respectively. Calibration and verification experiments were performed using batch cultures grown on different substrates. PI uptake depended on the physiological state of the bacterial cells. Unexpectedly, up to 40% of both strains were stained by PI during early exponential growth on glucose when compared to 2-5% of cells in the early stationary phase of growth. The results question the utility of PI as a universal indicator for the viability of (environmental) bacteria. It rather appears that in addition to nonviable cells, PI also stains growing cells of Sphingomonas sp. and M. frederiksbergense during a short period of their life cycle. ' 2007 International Society for Analytical Cytology