Dissimilatory reduction of Cr(VI), Fe(III), and U(VI) by Cellulomonas isolates.

The reduction of Cr(VI), Fe(III), and U(VI) was studied using three recently isolated environmental Cellulomonas sp. (WS01, WS18, and ES5) and a known Cellulomonas strain ( Cellulomonas flavigena ATCC 482) under anaerobic, non-growth conditions. In all cases, these cultures were observed to reduce Cr(VI), Fe(III), and U(VI). In 100 h, with lactate as electron donor, the Cellulomonas isolates (500 mg/l total cell protein) reduced nitrilotriacetic acid chelated Fe(III) [Fe(III)-NTA] from 5 mM to less than 2.2 mM, Cr(VI) from 0.2 mM to less than 0.001 mM, and U(VI) from 0.2 mM to less than 0.12 mM. All Cellulomonas isolates also reduced Cr(VI), Fe(III), and U(VI) in the absence of lactate, while no metal reduction was observed in either the cell-free or heat-killed cell controls. This is the first report of Cellulomonas sp. reducing Fe(III) and U(VI). Further, this is the first report of Cellulomonas spp. coupling the oxidation of lactate, or other unknown electron donors in the absence of lactate, to the reduction of Cr(VI), Fe(III), and U(VI).

Removal of low concentrations of carbon tetrachloride in compost-based biofilters operated under methanogenic conditions.

Research was performed to demonstrate the removal of carbon tetrachloride (CT) using compost biofilters operated under methanogenic conditions. Biofilters were operated at an empty-bed residence time of 2.8 minutes using nitrogen as the atmosphere. Hydrogen and carbon dioxide were supplied as an electron donor and carbon source, respectively, during acclimation of the bed medium microbes. Once methanogenesis was demonstrated, CT flow to the biofilter was established. Biofilters were operated over a CT concentration range from 20 to 700 ppbv for 6 months. Bed medium microbes were able to remove up to 75% of the inlet CT. At excessively high CT concentrations (> 500 ppmv), methane production and hydrogen utilization by the bed medium microbes appeared to be inhibited. CT removal by the biofilter decreased when the hydrogen supply was removed from the biofilter inlet, indicating that hydrogen acted as the electron donor for reductive dechlorination. The removal efficiency and relatively low empty bed residence times demonstrated by these laboratory-scale biofilters indicate that anaerobic biofiltration of CT may be a feasible full-scale process.

Activity-dependent fluorescent labeling of bacteria that degrade toluene via toluene 2,3-dioxygenase.

Alternative substrates for the toluene 2,3-dioxygenase pathway of several pseudomonads served as enzyme-activity-dependent fluorescent probes for the bacteria. Phenylacetylene and cinnamonitrile were transformed to fluorescent and brightly colored products by Pseudomonas putida F1, Pseudomonas fluorescens CFS215, and Burkholderia (Pseudomonas) strain JS150. Active bacteria transformed phenylacetylene, producing bright yellow solutions containing the putative product 2-hydroxy-6-oxo-7-octyn-2,4-dienoate. Transformation of cinnamonitrile resulted in bright orange solutions due to accumulation of the putative product 2-hydroxy-6-oxo-8-cyanoocta-2,4,7-trienoate. Chemical and physical properties of the products supported their identification, which indicated that the first three enzymes of the pathway catalyzed product formation. Phenylacetylene labeled bacteria with green fluorescence emission; bacteria were concentrated on black 0.2-micron-pore-size polycarbonate filters containing polyvinylpyrrolidone (PVP) as a wetting agent. Bacteria labeled with cinnamonitrile were fluorescent orange; labeling was effective with bacteria trapped on PVP-free polycarbonate filters. Production of the enzymes involved in labeling of P. putida F1 and P. fluorescens CFS215 was induced by growth (on arginine) in the presence of toluene; cells grown on arginine without toluene were not labeled. Labeling of P. putida F1 by phenylacetylene was inhibited by toluene, indicating that the same enzymatic pathway was required for transformations of both substrates. Bacteria expressing other toluene-degrading enzymatic pathways were not fluorescently labeled with phenylacetylene.

Bacterial reduction of chromium.

A mixed culture was enriched from surface soil obtained from an eastern United States site highly contaminated with chromate. Growth of the culture was inhibited by a chromium concentration of 12 mg/L. Another mixed culture was enriched from subsurface soil obtained from the Hanford reservation, at the fringe of a chromate plume. The enrichment medium was minimal salts solution augmented with acetate as the carbon source, nitrate as the terminal electron acceptor, and various levels of chromate. This mixed culture exhibited chromate tolerance, but not chromate reduction capability, when growing anaerobically on this medium. However, this culture did exhibit chromate reduction capability when growing anaerobically on TSB. Growth of this culture was not inhibited by a chromium concentration of 12 mg/L. Mixed cultures exhibited decreasing diversity with increasing levels of chromate in the enrichment medium. An in situ bioremediation strategy is suggested for chromate contaminated soil and groundwater.

Reduction of Cr(6+) to Cr(3 +) in a packed-bed bioreactor.

Hexavalent chromium, Cr(6+), is a common and toxic pollutant in soils and waters. Reduction of the mobile Cr(6+) to the less mobile and less toxic trivalent chromium, Cr(3+), can be achieved with conventional chemical reduction technologies. Alternatively, Cr(6+) can be biochemically reduced to Cr(3+) by anaerobic microbial consortia which appear to use Cr(6+) as a terminal electron acceptor. A bioprocess for Cr(6+) reduction has been demonstrated using a packed-bed bioreactor containing ceramic packing, and then compared to a similar bioreactor containing DuPont Bio-Sep beads. An increase in volumetric productivity (from 4 mg Cr(6+)/L/h to 260 mg Cr(6+)/L/h, probably due to an increase in biomass density, was obtained using Bio-Sep beads. The beads contain internal macropores which were shown by scanning electron microscopy to house dense concentrations of bacteria. Comparisons to conventional Cr(6+) treatment technologies indicate that a bioprocess has several economic and operational advantages.

Isolation of hexavalent chromium-reducing anaerobes from hexavalent-chromium-contaminated and noncontaminated environments.

Hexavalent chromium [Cr(VI)], is a toxic, water-soluble contaminant present in many soils and industrial effluents. Bacteria from various soils were examined for Cr(VI) resistance and reducing potential. Microbes selected from both Cr(VI)-contaminated and -noncontaminated soils and sediments were capable of catalyzing the reduction of Cr(VI) to Cr(III) a less toxic, less water-soluble form of Cr, demonstrating the utility of using a selection strategy for indigenous Cr(VI)-reducing bacteria in a bioprocess. As a result, indigenous Cr(VI)- reducing microbes from contaminated sites should provide the means for developing a bioprocess to reduce Cr(VI) to Cr(III) in nonsterile effluents such as those from soil washes. This approach also avoids the contamination problems associated with pure cultures of allochthonous microorganisms. In addition the apparent ubiquity of Cr(VI)-reducing bacteria in soil and sediments indicates potential for in situ bioremediation of Cr(VI)-contaminated soils and ground water.

Bacteria and acidic drainage from coal refuse: inhibition by sodium lauryl sulfate and sodium benzoate.

The application of an aqueous solution of sodium lauryl sulfate and sodium benzoate to the surface of high-sulfur coal refuse resulted in the inhibition of iron-and sulfur-oxidizing chemoautotrophic bacteria and in the decrease of acidic drainage from the refuse, suggesting that acid drainage can be abated in the field by inhibiting iron- and sulfur-oxidizing bacteria.

Adenosine 5'-triphosphate formation in Thiobacillus ferrooxidans vesicles by H+ ion gradients comparable to those of environmental conditions.

Vesicles prepared from iron-grown Thiobacillus ferrooxidans, and subsequently loaded with adenosine 5'-diphosphate and inorganic phosphate, produced adenosine 5'-triphosphate when subjected to H+ gradients comparable to those in the cells' normal environment (i.e., an internal pH in the range of 6.0 to 8.0 with an optimum of 7.0 to 7.8 and an external pH in the range of 2.1 to 4.1 with an optimum of 2.8). Nigericin, dicyclohexylcarbodiimide, and pentachlorophenol decreased adenosine 5'-triphosphate synthesis. Valinomycin at concentrations of 2.5 and 5.0 micrograms/ml increased adenosine 5'-triphosphate formation by 25 and 30%, respectively.

Leakage of cellular material from Thiobacillus ferrooxidans in the presence of organic acids.

Thiobacillus ferroodixans cells released varying amounts of iron, phosphate, sugar, ribonucleic acid, deoxyribonucleic acid, and substances that absorbed light at both 260 and 280 nm, when exposed to 10(-2) to 10(-1) M concentrations of these organic acids: propionic, butyric, valeric, hexanoic, and oxalacetic. These acids also retarded iron oxidation by the cells. Electron microscope observation of cells after exposure to the organic acids showed varying degrees of cell envelope disruption, suggesting that the mode of inhibition of autotrophic iron oxidation in the cell involves interference with the function of the cell envelope, possibly the cell membrane.

Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.

An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique.