Natural cleanup of heavy fuel oil on rocks: an in situ experiment.

Changes in the chemical composition of a heavy fuel oil, Bunker C, exposed to the elements for 556 days in the vicinity of Brest Harbour (France, (48 degrees 18(') N, 4 degrees 32(') W)) have been studied. Samples with exposure to full or reflected sunlight, and in the dark, were analysed by thin layer chromatography and gas chromatography coupled with mass spectrometry and compared with the initial oil. Using hopane as a conserved internal standard, an average of more than 56% of the total hydrocarbon in the residual stranded oil had been removed in the 556 days. The results indicate that dissolution, biodegradation and photooxidation all play important roles in the weathering process, with their respective contributions depending on the exposure.

In situ observation of the generation of isothiocyanates from sinigrin in horseradish and wasabi.

Sulfur K-edge X-ray absorption spectroscopy has been used to determine the chemical identity of the sulfur-containing species in horseradish (Armoracia lapthifolia) and wasabi (Wasabia japonica) in situ, before and after cell disruption. The major sulfur-containing species in the intact root is sinigrin (1-thio-beta-D-glucopyranose 1-N-(sulfoxy)-3-buteneimidate) and related congeners. Disrupting the cells by applying local pressure allowed the conversion of the sulfur moieties in sinigrin to isothiocyanates and sulfate in approximately equimolar amounts. In contrast to previous suggestions, no detectable thiocyanates were formed, but an unusual thio intermediate may have been identified for the first time.

Analysis of sulfur biochemistry of sulfur bacteria using X-ray absorption spectroscopy.

Many sulfide-oxidizing organisms, including the photosynthetic sulfur bacteria, store sulfur in "sulfur globules" that are readily detected microscopically. The chemical form of sulfur in these globules is currently the focus of a debate, because they have been described as "liquid" by some observers, although no known allotrope of sulfur is liquid at physiological temperatures. In the present work we have used sulfur K-edge X-ray absorption spectroscopy to identify and quantify the chemical forms of sulfur in a variety of bacterial cells, including photosynthetic sulfur bacteria. We have also taken advantage of X-ray fluorescence self-absorption to derive estimates of the size and density of the sulfur globules in photosynthetic bacteria. We find that the form of sulfur that most resembles the globule sulfur is simply solid S(8), rather than more exotic forms previously proposed.

Deep desulfurization of extensively hydrodesulfurized middle distillate oil by Rhodococcus sp. strain ECRD-1.

Dibenzothiophene (DBT), and in particular substituted DBTs, are resistant to hydrodesulfurization (HDS) and can persist in fuels even after aggressive HDS treatment. Treatment by Rhodococcus sp. strain ECRD-1 of a middle distillate oil whose sulfur content was virtually all substituted DBTs produced extensive desulfurization and a sulfur level of 56 ppm.

Structural basis of the antagonism between inorganic mercury and selenium in mammals.

Mercuric chloride toxicity in mammals can be overcome by co-administration of sodium selenite. We report a study of the mutual detoxification product in rabbit plasma, and of a Hg-Se-S-containing species synthesized by addition of equimolar mercuric chloride and sodium selenite to aqueous, buffered glutathione. Chromatographic purification of this Hg-Se-S species and subsequent structural analysis by Se and Hg extended X-ray absorption fine structure (EXAFS) spectroscopy revealed the presence of four-coordinate Se and Hg entities separated by 2.61 A. Hg and Se near-edge X-ray absorption spectroscopy of erythrocytes, plasma, and bile of rabbits that had been injected with solutions of sodium selenite and mercuric chloride showed that Hg and Se in plasma samples exhibited X-ray absorption spectra that were essentially identical to those of the synthetic Hg-Se-S species. Thus, the molecular detoxification product of sodium selenite and mercuric chloride in rabbits exhibits similarities to the synthetic Hg-Se-S species. The underlying molecular mechanism for the formation of the Hg-Se-S species is discussed.

Biodegradation of methyl tertiary-butyl ether (MTBE) and other fuel oxygenates.

Methyl tertiary-butyl ether (MTBE) has been added to gasoline in the U.S. for the last decade in order to meet the mandates of the 1990 Clean Air Act. This law decreed that gasoline sold in many locations must contain oxygenates to improve combustion and minimize air pollution. Unfortunately, this widespread use has led to the contamination of some soils and aquifers, and remediation is now required. Bioremediation has proven to be an environmentally responsible and cost-effective approach to remediating petroleum spills; this article reviews the potential that bioremediation may also be appropriate for remediating MTBE contamination. There is now good evidence that MTBE can be degraded by bacteria and fungi under aerobic conditions, and promising indications that the process also occurs under methanogenic and ferric iron-reducing conditions. Yet, apparently it is not a widespread phenomenon. The challenge is to find effective bioremediation strategies that maximize this biodegradation so that it can be used reliably in cleaning contaminated sites. Both simple biostimulation and more complex bioaugmentation protocols are being developed to meet this pressing need.

Fate of selenate and selenite metabolized by Rhodobacter sphaeroides.

Cultures of a purple nonsulfur bacterium, Rhodobacter sphaeroides, amended with approximately 1 or approximately 100 ppm selenate or selenite, were grown phototrophically to stationary phase. Analyses of culture headspace, separated cells, and filtered culture supernatant were carried out using gas chromatography, X-ray absorption spectroscopy, and inductively coupled plasma spectroscopy-mass spectrometry, respectively. While selenium-amended cultures showed much higher amounts of SeO(3)(2-) bioconversion than did analogous selenate experiments (94% uptake for SeO(3)(2-) as compared to 9.6% for SeO(4)(2-)-amended cultures from 100-ppm solutions), the chemical forms of selenium in the microbial cells were not very different except at exposure to high concentrations of selenite. Volatilization accounted for only a very small portion of the accumulated selenium; most was present in organic forms and the red elemental form.

The active site structure of Thalassiosira weissflogii carbonic anhydrase 1.

X-ray absorption spectroscopy at the Zn K-edge indicates that the active site of the marine diatom Thalassiosira weissflogii carbonic anhydrase is strikingly similar to that of mammalian alpha-carbonic anhydrase enzymes. The zinc has three histidine ligands and a single water at 1.98 A. This is quite different from the beta-carbonic anhydrases of higher plants in which zinc is coordinated by two cysteine thiolates, one histidine, and a water molecule. The diatom carbonic anhydrase shows no significant sequence similarity with other carbonic anhydrases and may represent an example of convergent evolution at the molecular level.

The orf162b sequence of Rhodobacter capsulatus encodes a protein required for optimal levels of photosynthetic pigment-protein complexes.

The orf162b sequence, the second open reading frame 3' of the reaction center (RC) H protein gene puhA in the Rhodobacter capsulatus photosynthesis gene cluster, is shown to be transcribed from a promoter located 5' of puhA. A nonpolar mutation of orf162b was generated by replacing most of the coding region with an antibiotic resistance cartridge. Although the mutant strain initiated rapid photosynthetic growth, growth slowed progressively and cultures often entered a pseudostationary phase. The amounts of the RC and light harvesting complex I (LHI) in cells obtained from such photosynthetic cultures were abnormally low, but these deficiencies were less severe when the mutant was grown to a pseudostationary phase induced by low aeration in the absence of illumination. The orf162b mutation did not significantly affect the expression of a pufB::lacZ translationally in-frame gene fusion under the control of the puf promoter, indicating normal transcription and translation of RC and LHI genes. Spontaneous secondary mutations in the strain with the orf162b disruption resulted in a bypass of the photosynthetic growth retardation and reduced the level of light harvesting complex II. These results and the presence of sequences similar to orf162b in other species indicate that the Orf162b protein is required for normal levels of the photosynthetic apparatus in purple photosynthetic bacteria.

Quantitative, chemically specific imaging of selenium transformation in plants.

Quantitative, chemically specific images of biological systems would be invaluable in unraveling the bioinorganic chemistry of biological tissues. Here we report the spatial distribution and chemical forms of selenium in Astragalus bisulcatus (two-grooved poison or milk vetch), a plant capable of accumulating up to 0.65% of its shoot dry biomass as Se in its natural habitat. By selectively tuning incident x-ray energies close to the Se K-absorption edge, we have collected quantitative, 100-microm-resolution images of the spatial distribution, concentration, and chemical form of Se in intact root and shoot tissues. To our knowledge, this is the first report of quantitative concentration-imaging of specific chemical forms. Plants exposed to 5 microM selenate for 28 days contained predominantly selenate in the mature leaf tissue at a concentration of 0.3-0.6 mM, whereas the young leaves and the roots contained organoselenium almost exclusively, indicating that the ability to biotransform selenate is either inducible or developmentally specific. While the concentration of organoselenium in the majority of the root tissue was much lower than that of the youngest leaves (0.2-0.3 compared with 3-4 mM), isolated areas on the extremities of the roots contained concentrations of organoselenium an order of magnitude greater than the rest of the root. These imaging results were corroborated by spatially resolved x-ray absorption near-edge spectra collected from selected 100 x 100 microm(2) regions of the same tissues.

Reduction and coordination of arsenic in Indian mustard.

The bioaccumulation of arsenic by plants may provide a means of removing this element from contaminated soils and waters. However, to optimize this process it is important to understand the biological mechanisms involved. Using a combination of techniques, including x-ray absorption spectroscopy, we have established the biochemical fate of arsenic taken up by Indian mustard (Brassica juncea). After arsenate uptake by the roots, possibly via the phosphate transport mechanism, a small fraction is exported to the shoot via the xylem as the oxyanions arsenate and arsenite. Once in the shoot, the arsenic is stored as an As(III)-tris-thiolate complex. The majority of the arsenic remains in the roots as an As(III)-tris-thiolate complex, which is indistinguishable from that found in the shoots and from As(III)-tris-glutathione. The thiolate donors are thus probably either glutathione or phytochelatins. The addition of the dithiol arsenic chelator dimercaptosuccinate to the hydroponic culture medium caused a 5-fold-increased arsenic level in the leaves, although the total arsenic accumulation was only marginally increased. This suggests that the addition of dimercaptosuccinate to arsenic-contaminated soils may provide a way to promote arsenic bioaccumulation in plant shoots, a process that will be essential for the development of an efficient phytoremediation strategy for this element.

Subcellular localization and speciation of nickel in hyperaccumulator and non-accumulator Thlaspi species.

The ability of Thlaspi goesingense Hálácsy to hyperaccumulate Ni appears to be governed by its extraordinary degree of Ni tolerance. However, the physiological basis of this tolerance mechanism is unknown. We have investigated the role of vacuolar compartmentalization and chelation in this Ni tolerance. A direct comparison of Ni contents of vacuoles from leaves of T. goesingense and from the non-tolerant non-accumulator Thlaspi arvense L. showed that the hyperaccumulator accumulates approximately 2-fold more Ni in the vacuole than the non-accumulator under Ni exposure conditions that were non-toxic to both species. Using x-ray absorption spectroscopy we have been able to determine the likely identity of the compounds involved in chelating Ni within the leaf tissues of the hyperaccumulator and non-accumulator. This revealed that the majority of leaf Ni in the hyperaccumulator was associated with the cell wall, with the remaining Ni being associated with citrate and His, which we interpret as being localized primarily in the vacuolar and cytoplasm, respectively. This distribution of Ni was remarkably similar to that obtained by cell fractionation, supporting the hypothesis that in the hyperaccumulator, intracellular Ni is predominantly localized in the vacuole as a Ni-organic acid complex.

Structure of the molybdenum site of Rhodobacter sphaeroides biotin sulfoxide reductase.

Conditions for heterologous expression of Rhodobacter sphaeroides biotin sulfoxide reductase in Escherichia coli were modified, resulting in a significant improvement in the yield of recombinant enzyme and enabling structural studies of the molybdenum center. Quantitation of the guanine and the molybdenum as compared to that found in R. sphaeroides DMSO reductase demonstrated the presence of the bis(MGD)molybdenum cofactor. UV-visible absorption spectra were obtained for the oxidized, NADPH-reduced, and dithionite-reduced enzyme. EPR spectra were obtained for the Mo(V) state of the enzyme. X-ray absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of the enzyme. The molybdenum site of the oxidized protein possesses a Mo(VI) mono-oxo site (Mo=O at 1.70 A) with additional coordination by approximately four thiolate ligands at 2.41 A and probably one oxygen or nitrogen at 1.95 A. The NADPH- and dithionite-reduced Mo(IV) forms of the enzyme are des-oxo molybdenum sites with approximately four thiolates at 2.33 A and two different Mo-O/N ligands at 2.19 and 1.94 A.

X-ray absorption spectroscopy of cadmium phytochelatin and model systems.

Higher plants, algae and some yeasts respond to potentially toxic heavy metals such as cadmium by synthesizing phytochelatins and related cysteine-rich polypeptides. We have used X-ray absorption spectroscopy to study the nature of cadmium binding in such peptides isolated from maize (Zea mays) exposed to low levels of cadmium, and in two synthetic cadmium-peptide complexes, Cd-(gamma-Glu-Cys)3Gly and Cd-(alpha-Glu-Cys)3Gly. We have used the synthetic ions [Cd(SPh)4]2-, [Cd4(SPh)10]2- and [S4Cd10(SPh)16]4-as crystallographically defined models for the cadmium site. The Cd K-edge extended X-ray absorption fine structure (EXAFS) data, together with the Cd K, LI, LII and LIII near-edge spectra, reveal a predominantly tetrahedral coordination of cadmium by sulfur in both the phytochelatin and synthetic peptide complexes. In particular, the Cd LIII-edge lacks a peak at 3534.9 e V which was found to be prominent for oxygen- or nitrogen-coordinated species. The Cd-S distance in the phytochelatin complex is 2.54 A. The Cd K-edge EXAFS does not show any isolated, well-defined Cd-Cd interactions; however, contrary to the conclusion of previous work, their absence is not necessarily indicative of isolated cadmium-thiolate ligation. Evidence from other studies suggests that high static disorder, combined with a large vibrational component, serve to effectively wash out this contribution to the EXAFS. The sulfur K-edge, moreover, shows a low-energy feature both in the phytochelatin and in the synthetic cadmium-peptide complexes which is consistent with sulfide bound in a cluster with cadmium as found for [S4Cd10(SPh)16]4-. This feature strongly suggests the presence of a polynuclear cadmium cluster in maize phytochelatin.

Microbial desulfurization of a crude oil middle-distillate fraction: analysis of the extent of sulfur removal and the effect of removal on remaining sulfur.

Rhodococcus sp. strain ECRD-1 was evaluated for its ability to desulfurize a 232 to 343 degrees C middle-distillate (diesel range) fraction of Oregon basin (OB) crude oil. OB oil was provided as the sole source of sulfur in batch cultures, and the extent of desulfurization and the chemical fate of the residual sulfur in the oil after treatment were determined. Gas chromatography (GC), flame ionization detection, and GC sulfur chemiluminesce detection analysis were used to qualitatively evaluate the effect of Rhodococcus sp. strain ECRD-1 treatment on the hydrocarbon and sulfur content of the oil, respectively. Total sulfur was determined by combustion of samples and measurement of released sulfur dioxide by infrared absorption. Up to 30% of the total sulfur in the middle distillate cut was removed, and compounds across the entire boiling range of the oil were affected. Sulfur K-edge X-ray absorption-edge spectroscopy was used to examine the chemical state of the sulfur remaining in the treated OB oil. Approximately equal amounts of thiophenic and sulfidic sulfur compounds were removed by ECRD-1 treatment, and over 50% of the sulfur remaining after treatment was in an oxidized form. The presence of partially oxidized sulfur compounds indicates that these compounds were en route to desulfurization. Overall, more than two-thirds of the sulfur had been removed or oxidized by the microbial treatment.

X-ray absorption spectroscopy of selenium-containing amino acids.

The selenium K-edge X-ray absorption spectra of selenomethionine, selenocysteine, selenocystine, and sulfo-selenocystine in solution are compared with the corresponding sulfur K-edge spectra of the sulfur analogues of these compounds. The selenium and sulfur spectra follow similar trends, although the latter are significantly sharper owing to the longer core hole lifetime at the lower energies where sulfur absorbs. The spectra of the selenium compounds are sufficiently distinct that it is reasonable to expect that curve fitting will allow the speciation of the forms of selenium in complex biological samples.

Sulfur K-edge X-ray absorption spectroscopy for determining the chemical speciation of sulfur in biological systems.

Sulfur is an essential biological element, yet its biochemistry is only partially understood because there are so few tools for studying this element in biological systems. X-ray absorption spectroscopy provides a unique approach to determining the chemical speciation of sulfur in intact biological samples. Different biologically relevant sulfur compounds show distinctly different sulfur K-edge X-ray absorption spectra, and we show here, as an example, that this allows the deconvolution of the sulfur species in equine blood.