Relationship between conformation and physicochemical properties of polypeptides. I. Synthesis of homo- and co-oligopeptides by the liquid-phase method

The synthesis of the following oligo- and co-oligopeptides by the liquid-phase method is described: (L -Met)₁₅ (I), [L -Glu(OBzl)]₂₀ (II), (L -Val)₈-Gly (IV), (L -Ile)₈-Gly (V), (L -Ile)₄-Gly-(L -Ile)₄ (VI), (L -Ile)₄-Pro-(L -Ile)₄ (VII), (L -Met)₅-L -Pro-(L -Met)₅ (VIII), [L -Glu(OBzl)]₇-L -Pro-[L -Glu(OBzl)]₇ (IX). The oligomers are covalently bound to bifunctional polyethylene glycol (PEG) and monofunctional PEG-M of M_r 5 × 10³?2 × 10⁴. Analytical controls were carried out after each step of synthesis in order to ensure quantitative coupling yields. All products could be obtained in high purity as indicated by amino acid analysis, thin-layer chromatography and chiroptical methods. The solubility of the oligomers was strongly enhanced by the presence of the C-terminal PEG group, enabling conformational investigations in a variety of solvents. A significant relationship between conformation and physicochemical properties of the oligopeptides was observed. Oligomers with tendencies to adopt α-helical (I, II) or unordered structures (VI–IX) showed no pronounced change in solubility or coupling kinetics during chain elongation, whereas the onset of a β-structure (IV, V) was paralleled by a drastic decrease in solubility and reactivity of the terminal amino groups. Most notably, the insertion of a proline or glycine in the middle of a β-forming peptide chain (VI, VII) resulted in a considerable increase in solubility compared to the corresponding homo-oligomers. The impact of the conformational properties of a peptide chain on strategic considerations of peptide synthesis in solution is delineated.     

Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris.

The mechanism for U(VI) reduction by Desulfovibrio vulgaris (Hildenborough) was investigated. The H2-dependent U(VI) reductase activity in the soluble fraction of the cells was lost when the soluble fraction was passed over a cationic exchange column which extracted cytochrome c3. Addition of cytochrome c3 back to the soluble fraction that had been passed over the cationic exchange column restored the U(VI)-reducing capacity. Reduced cytochrome c3 was oxidized by U(VI), as was a c-type cytochrome(s) in whole-cell suspensions. When cytochrome c3 was combined with hydrogenase, its physiological electron donor, U(VI) was reduced in the presence of H2. Hydrogenase alone could not reduce U(VI). Rapid U(VI) reduction was followed by a subsequent slow precipitation of the U(IV) mineral uraninite. Cytochrome c3 reduced U(VI) in a uranium-contaminated surface water and groundwater. Cytochrome c3 provides the first enzyme model for the reduction and biomineralization of uranium in sedimentary environments. Furthermore, the finding that cytochrome c3 can catalyze the reductive precipitation of uranium may aid in the development of fixed-enzyme reactors and/or organisms with enhanced U(VI)-reducing capacity for the bioremediation of uranium-contaminated waters and waste streams.     

Reduction of chromate by fixed films of sulfate-reducing bacteria using hydrogen as an electron source

The ability of sulfate-reducing bacteria (SRB) to reduce chromate, Cr(VI), was evaluated using fixed-film growth systems and H₂ as the electron source. A main objective of the experiment was to distinguish between direct enzymatic reduction and indirect reduction by hydrogen sulfide, in order to subsequently verify and control the synergy of these two mechanisms. In batch experiments with the sulfate-reducing consortium CH10 selected from a mining site, 50 mg l⁻¹ Cr(VI) was reduced in 15 min in the presence of 500 mg l⁻¹ hydrogen sulfide compared to 16 mg l⁻¹ reduced in 1 h without hydrogen sulfide. Fixed films of a CH10 population and Desulfomicrobium norvegicum were fed-batch grown in a column bioreactor. After development of the biofilm, hydrogen sulfide was removed and the column was fed continuously with a 13-mg l⁻¹ Cr(VI) solution. Specific Cr(VI) reduction rates on pozzolana were close to 90 mg Cr(VI) h⁻¹ per gram of protein. Exposure to Cr(VI) had a negative effect on the subsequent ability of CH10 to reduce sulfate, but the inhibited bacteria remained viable. Journal of Industrial Microbiology & Biotechnology (2002) 28, 154–159 DOI: 10.1038/sj/jim/7000226 Received 20 September 2000/ Accepted in revised form 13 November 2001     

Bioremediation of Cr(VI) from Chromium-Contaminated Wastewater by Free and Immobilized Cells of Cellulosimicrobium cellulans KUCr3

In this report, possible utilization of a chromium-reducing bacterial strain Cellulosimicrobium cellulans KUCr3 for effective bioremediation of hexavalent chromium (Cr(VI))-containing wastewater fed with tannery effluents has been discussed. Cr(VI) reduction and bioremediation were found to be related to the growth supportive conditions in wastewater, which is indicative of cell mass dependency for Cr(VI) reduction. Cr(VI) reduction was determined by measuring the residual Cr(VI) in the cell-free supernatant using colorimetric reagent S-diphenylcarbazide. Nutrient availability and initial cell density showed a positive relation with Cr(VI) reduction, but it was inhibited with increasing concentration of Cr(VI) under laboratory condition. The optimum temperature and pH for effective Cr(VI) reduction in wastewater were found to be 35°C and 7.5, respectively. The viable cells of KUCr3 were successfully entrapped in an agarose bead that was used in continuous column and batch culture for assaying Cr(VI) reduction. In packed bed column (continuous flow) experiment, approximately 25% Cr(VI) reduction occurred after 144 h. Cr(VI) was almost 75% and 52% reduced at concentrations of 0.5 mM and 2 mM Cr(VI), respectively, after 96 h in batch culture experiment in peptone-yeast extract-glucose medium, whereas it could decrease the Cr(VI) content up to 40% from the water containing tannery waste. This study suggests that KUCr3 could be used as a candidate for possible environmental clean up operation with respect to Cr(VI) bioremediation.     

Online column preconcentration for speciation and selective determination of Cr(III) in natural water samples using flow injection with chemiluminescence detection

A simple, rapid, sensitive and inexpensive approach is described in this work based on a combination of solid-phase extraction of 8-hydroxyquinoline (8HQ), for speciation and preconcentration of Cr(III) and Cr(VI) in river water, and the direct determination of these species using a flow injection system with chemiluminescence detection (FI–CL) and a 4-diethylamino phenyl hydrazine (DEAPH)–hydrogen peroxide system. At different pH, the two forms of chromium [Cr(III) and Cr(VI)] have different exchange capacities for 8HQ, therefore two columns were constructed; the pH of column 1 was adjusted to pH 3 for retaining Cr(III) and column 2 was adjusted to pH 1 for retaining of Cr(VI). The sorbed Cr(III) and Cr(VI) species were eluted from columns using 3.0 ml of 0.1 N of HCl and 3.0 ml of 0.1 N of NaOH, respectively. The flow injection–chemiluminescence (FI–CL) method is based on light emitted due to the oxidation of DEAPH by the H₂O₂ in the presence of Cr(III), which catalyzes the reaction. The flow cell is a transparent coiled tube made from glass (2.0 × 4.0, inner and outer diameter) and located close to the photodetector. The flow parameters: flow rate, sample volume, flow cell length, and distance to the CL detector were studied and optimized. Under optimum flow conditions, the Cr(III) concentration can be determined over the range 5–350 μg L⁻¹ with a limit of detection of 1.2 μg L⁻¹, as the Cr(III) concentration is proportional to the intensity of the CL signal. The relative standard deviations (%) for 10 and 50 μg L⁻¹ Cr(III) were 1.2% and 3.2%, respectively. The effects of Al(III), Cd(II), Zn(II), Hg(II), Pb(II), Co(II), Cu(II), Ni(II), Mn(II), Ca(II), and Fe(III) were investigated. The proposed method is highly selective and sensitive, enabling a rapid determination of the Cr(III) amount in the presence of other interfering metals. Finally, the FI–CL method was examined in five river water samples with excellent recoveries.     

Hexavalent chromium (VI) stress induces mitogen-activated protein kinase activation mediated by distinct signal molecules in roots of Zea mays L.

Hexavalent chromium (VI) is a cytotoxic metal ion in plants. However, the mechanisms involved in the cellular response to the metal have not yet been well established. In plants, mitogen-activated protein kinase (MAPK) cascades play an important role in signal transduction related to biotic and abiotic stresses. In the present study, we investigated the Cr(VI)-induced MAPKs activation and the correlative mechanism of activation in maize (Zea mays L.) roots. Cr(VI) elicited a remarkable increase in a 45-kDa myelin basic protein (MBP) kinase activity with MAPK-like characteristics, which was identified as ZmMPK5 by immunokinase and immunoblot assays. Pretreatment with DMTU, a peroxide hydrogen (H₂O₂) scavenger, and DPI, a NADPH oxidase inhibitor, the Cr(VI)-induced ZmMPK5 activation was almost completely suppressed, suggesting that Cr(VI)-activated ZmMPK5 requires for H₂O₂. Application of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, could activate ZmMPK5. Pretreatment with cPTIO and l-NAME, a NO scavenger and a nitric oxide synthase (NOS) inhibitor, respectively, Cr(VI)-induced ZmMPK5 activation was attenuated effectively, implying that NO is involved in Cr(VI)-activated ZmMPK5. Furthermore, a calcium-dependent protein kinase (CDPK) antagonist, W7, abolished Cr(VI)-stimulated ZmMPK5 activation, indicating that CDPKs may participate in the ZmMPK5 activation. The results obtained suggest that Cr(VI)-induced activation of ZmMPK5, a candidate for MAPK signaling cascades, can be modulated by other distinct signaling pathways.     

Concurrent Removal of Mn(II) and Cr(VI) by Achromobacter sp. TY3-4

In this study, we report a bacterium, Achromobacter sp. TY3-4, capable of concurrently removing Mn (II) and Cr (VI) under oxic condition. TY3-4 reduced as much as 2.31?mM of Cr (VI) to Cr (III) in 70?h, and oxidized as much as 20?mM of Mn(II) to Mn oxides in 80?h. When 0.58?mM Cr (VI) and 10?mM Mn(II) were present together, both Cr(VI) and Mn(II) were completely removed by TY3-4 and the generated precipitates are Mn^IIIOOH, Mn^III,IV₃O₄, Mn^IVO₂ and Cr^III(OH)₃. Experiments also show that both biosroption and bioreduction of Mn(II) are the driving forces for Mn(II) removal, whereas bioreduction of Cr(VI) is the driving force for Cr(VI) removal. On the basis of these results, a possible reaction was proposed that TY3-4 concurrently reduces Cr(VI) and oxidizes Mn(II). This study is fundamental for Mn and Cr cycles. The strain shows potential for practical application.     

Uptake and Translocation of Tri- and Hexa-Valent Chromium and Their Effects on Black Gram (Vigna mungo L. Hepper cv. Co4) Roots

An equal concentration (100 μM) of Cr(III)- and Cr(VI)-induced changes in activities of antioxidative enzymes and metabolites of ascorbate-glutathione cycle was studied in 7-d-old black gram (Vigna mungo L Hepper cv. Co4) seedlings for 5-d after infliction of Cr stress. Seeds were germinated and grown in the presence or absence of Cr under controlled environmental conditions. Uptake and translocation of Cr rate was relatively higher during first 12 h of treatment with both speciation of Cr, Cr(III)- and Cr(VI)-treated black gram roots retained 15 times more Cr than the shoots. Significantly increased lipid peroxidation was observed in the form of accumulation of malondialdehyde (MDA) and production of hydrogen peroxide (H₂O₂) molecule and superoxide (O₂ ) radical after 6 h of infliction with Cr(VI) and after 12 h in Cr(III)-treated black gram roots. Superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities were significantly increased under Cr(VI)-treatment after 12 and 6 h, respectively. However, catalase (CAT) and monodehydroascorbate reductase (MDHAR) activities were not significantly increased under Cr(Ill)-treatment. There was a steep increase of 2.71 μmol g^-1 FW in ascorbic acid (AA) content was observed between 6 and 24 h of Cr(VI)-treatment. Oxidized glutathione (GSSG) content was steadily increased through the course of Cr(III)- and Cr(VI)-treatments, where as reduced glutathione (GSH) level was decreased after 24 h of treatment. GSH/GSSG ratio was rapidly decreased in treatment with Cr(III) than the Cr(VI). There was significant increase of 99 nmol g^-1 FW in non-protein thiol (NPT) content was recorded between 6 and 24 h of Cr(VI)-treatment. The present results showed differential response to AA and H₂O₂ signaling by Cr(III) and Cr(VI), AA in combination with APX was more effective in mitigating oxidative stress as against the role of GSH as an antioxidant.     

Metal Reduction at Low pH by a Desulfosporosinus species: Implications for the Biological Treatment of Acidic Mine Drainage

We isolated an acid-tolerant sulfate-reducing bacterium, GBSRB4.2, from coal mine-derived acidic mine drainage (AMD)-derived sediments. Sequence analysis of partial 16S rRNA gene of GBSRB4.2 revealed that it was affiliated with the genus Desulfosporosinus. GBSRB4.2 reduced sulfate, Fe(III) (hydr)oxide, Mn(IV) oxide, and U(VI) in acidic solutions (pH 4.2). Sulfate, Fe(III), and Mn(IV) but not U(VI) bioreduction led to an increase in the pH of acidic solutions and concurrent hydrolysis and precipitation of dissolved Al³⁺. Reduction of Fe(III), Mn(IV), and U(VI) in sulfate-free solutions revealed that these metals are enzymatically reduced by GBSRB4.2. GBSRB4.2 reduced U(VI) in groundwater from a radionuclide-contaminated aquifer more rapidly at pH 4.4 than at pH 7.1, possibly due to the formation of poorly bioreducible Ca-U(VI)-CO₃ complexes in the pH 7.1 groundwater.     

Using the Selective Ion Exchange Resin Extraction and XANES Methods to Evaluate the Effect of Compost Amendments on Soil Chromium(VI) Phytotoxicity

The Cu-saturated selective ion exchange resin (DOWEX M4195) extraction method was used to investigate the effects of two amendments, 5 and 15% organic matter in the form of hog-dung compost (HC) or cattle-dung compost (CC), on Cr(VI) bioavailability in three soils spiked with various levels of Cr(VI). The results showed that addition of composts could decrease the amounts of resin-extractable Cr(VI) in Cr(VI)-spiked soils, and the CC amendment decreased resin-extractable Cr(VI) more than the HC amendment. The X-ray Absorption Near-edge Structure spectroscopy (XANES) method was used to examine the distribution of Cr(III) and Cr(VI) species in Cr(VI)-spiked soils that were affected by compost amendments, and to elucidate the mechanisms for the decrease of resin-extractable Cr(VI) due to the application of composts. The XANES results suggested that the decrease in the amounts of resin-extractable Cr(VI) after compost addition was mainly due to the reduction of Cr(VI) to Cr(III). The amounts of soil resin-extractable Cr(VI) were also correlated with wheat seedling growth in order to evaluate the effect of compost amendments on decreasing the phytotoxicity of soil Cr(VI). The results showed that there was a sigmoidal relationship between soil resin-extractable Cr(VI) and the plant height of wheat seedlings and the obtained effective concentrations of resin-extractable Cr(VI) resulting in 10 and 50% growth inhibition (EC₁₀ and EC₅₀) were 76 and 191 mg kg⁻¹ respectively. The above results suggested that the resin extraction method was a useful tool for assessing Cr(VI) phytotoxicity and that addition of composts would enhance Cr(VI) reduction to Cr(III) in soils and thus relieve Cr(VI) phytotoxicity.     

Effect of Competing Electron Acceptors on the Reduction of U(VI) by Desulfotomaculum reducens

The biological reduction of soluble U(VI) to the less soluble U(IV) has been proposed as a strategy to remediate uranium-contaminated sites. However, the majority of the contaminated sites contain, in addition to U(VI), competing electron acceptors (CEAs) that can either enhance or inhibit U(VI) reduction. Desulfotomaculum reducens MI-1 is a sulfate-reducing bacterium able to reduce a variety of electron acceptors including U(VI). We characterized U(VI) reduction by D. reducens in the presence of pyruvate and three CEAs: sulfate, nitrate or soluble ferric iron. In the presence of sulfate or ferric iron and U(VI), cell growth was driven by respiration of the CEA. Nitrate was not used as an electron acceptor for growth and vegetative cells grew instead by fermenting pyruvate. Sulfate remaining after sulfate reduction has ceased or the presence of nitrate did not affect U(VI) reduction. However, in the case of sulfate, the addition of H₂ after the depletion of pyruvate greatly enhanced U(VI) reduction. Contrary to sulfate and nitrate, the presence of Fe(II), the product of Fe(III) reduction, abolished U(VI) reduction. The results from this investigation suggest that this microorganism and others with similar characteristics may play a role in U(VI) bioremediation efforts but only after the soluble Fe(II) produced by Fe(III) reduction has been advected away.     

Reduction of Hexavalent Chromium by Cell-Free Extract of Bacillus sphaericus AND 303 Isolated from Serpentine Soil

Cell-free extracts (CFEs) of chromium-resistant bacterium Bacillus sphaericus AND 303 isolated from serpentine soil of Andaman, India reduced Cr(VI) in in vitro condition, and the reductase activity was solely localized in the soluble cell-fractions (S₁₂, S₃₂, and S₁₅₀). The enzyme was constitutive as the CFEs from cells grown in Cr(VI)-free and Cr(VI)-containing media reduced a more or less equal amount of Cr(VI). Optimum Cr(VI) reductase activity was obtained at an enzyme (S₁₅₀) concentration equivalent to 4.56 mg protein/mL, 300 μM Cr(VI) and pH 6.0 after 30 min incubation at 30°C. The enzyme was heat labile; 80% of its activity was lost when exposed at 70°C for 15 min. Kinetics of Cr(VI) reductase activity fit well with the linearized Lineweaver-Burk plot and showed a V_max of 1.432 μmol Cr(VI)/mg protein/min and K_m of 158.12 μM Cr(VI). The presence of additional electron donors accelerated Cr(VI) reductase activity of CFE, and an increase of 28% activity over control was recorded with 1.0 μM NADH. Heavy metal ions such as Ni(II), Cu(II), and Cd(II) were strong inhibitors of Cr(VI) reductase unlike that of 100 μM Co(II), which retained 93% activity over control.     

Chromium(VI)-reducing <Emphasis Type="Italic">Chlorella</Emphasis> spp. isolated from disposal sites of paper-pulp and electroplating industry

We isolated four cultures of chromate resistant, unicellular, non-motile green algae from disposal sites of the paper-pulp and electroplating industries. These algae were maintained in Tris-acetate-glycerophosphate medium containing 30 μM K₂Cr₂O₇. The morphological features as well as analysis of the 500-bp fragment of 18S rDNA (NS 12 region) showed that these isolates belong to Chlorella spp. These isolates showed EC₅₀ values for chromate ranging from 60 to 125 μM. Uptake studies with radioactive ⁵¹Cr(VI) showed that 10–19% of total radioactivity was intracellular, and 1–2% was bound to the cell wall. The rest of the activity remained in the medium, suggesting that resistance was not related to accumulation of Cr(VI) in the cells. Interestingly, when these isolates were grown in the presence of 30 μM of K₂Cr₂O₇, a decrease in the Cr(VI) concentration in the medium was observed. Only live cells could deplete Cr(VI) from the supernatant, suggesting the presence of chromium reduction activity in these Chlorella isolates. Cr(VI) reduction activity of the cells of Chlorella was stimulated by light as well as by acetate and glycerophosphate. Treatment of Chlorella cells with 3-(3,4 dichlorophenyl),1,1dimethyl urea (DCMU) did not affect the Cr(VI) reduction. However, if the cells were treated with sodium azide, Cr(VI) reduction was severely affected. Though chromate resistance has been well documented in algae, the information on chromate reduction by algae is scant. This paper discusses the Cr(VI) reduction by Cr(VI) resistant Chlorella, which may find a use in the effective bioremediation of Cr(VI).     

Can microbially-generated hydrogen sulfide account for the rates of U(VI) reduction by a sulfate-reducing bacterium?

In situ remediation of uranium contaminated soil and groundwater is attractive because a diverse range of microbial and abiotic processes reduce soluble and mobile U(VI) to sparingly soluble and immobile U(IV). Often these processes are linked. Sulfate-reducing bacteria (SRB), for example, enzymatically reduce U(VI) to U(IV), but they also produce hydrogen sulfide that can itself reduce U(VI). This study evaluated the relative importance of these processes for Desulfovibrio aerotolerans, a SRB isolated from a U(VI)-contaminated site. For the conditions evaluated, the observed rate of SRB-mediated U(VI) reduction can be explained by the abiotic reaction of U(VI) with the microbially-generated H₂S. The presence of trace ferrous iron appeared to enhance the extent of hydrogen sulfide-mediated U(VI) reduction at 5 mM bicarbonate, but had no clear effect at 15 mM. During the hydrogen sulfide-mediated reduction of U(VI), a floc formed containing uranium and sulfur. U(VI) sequestered in the floc was not available for further reduction.     

Risk assessment of submicron PM-bound hexavalent chromium during wintertime

This study reports health risk assessment of PM₁-bound carcinogenic hexavalent chromium [Cr(VI)] from central part of Indo-Gangetic plain (IGP) (PM₁: particulate matter with aerodynamic diameter ≤1µm). Cr(VI) concentration has been estimated utilizing spectrophotometer with a modified novel method. Average ratio of Cr(VI)/Cr_T was 0.39 ± 0.07 (Cr_T: Total chromium) in the central IGP (Kanpur). Our study reports that mass fraction of Cr(VI) averaging at 0.39 is ～3 times higher than that assumed conventionally [Cr(VI)/Cr_T: 1/7]. Cancer risk assessment has been performed by assessing excess cancer risk (ECR) for the Cr(VI). ECR determined due to Cr(VI) was 57 and 14.3 (in one million) for adults and children, respectively. Our study suggests that risk due to Cr(VI) reported in previous studies were being underestimated by a factor of three. The Cr(VI)/Cr_T average ratio of 0.39 determined in this study was utilized to calculate risk assessment due to Cr(VI) from other locations in the IGP. Owing to large population of India (～125 million), the cancer risk due to Cr(VI) inhalation itself would become very significant. Thus, future research should focus on metal speciation of PM-bound samples from different locations to better constraint the toxicological risk assessment on a regional-to-global scale.     

Short-term chromium(VI) stress induces different photosynthetic responses in two duckweed species, <Emphasis Type="Italic">Lemna gibba</Emphasis> L. and <Emphasis Type="Italic">Lemna mino</Emphasis>r L.

Physiological responses of two duckweed species, Lemna gibba and Lemna minor, to hexavalent chromium [Cr(VI)] were studied in axenic cultures using short-term (48 h) treatments by K₂Cr₂O₇ (0–200 μM). Chlorophyll (Chl) fluorescence parameters and photosynthetic pigment composition of plants were screened to determine the effects of Cr(VI) exposures. The two duckweed species exhibited different sensitivity in the applied Cr(VI) concentration range. Chl fluorescence parameters of dark-adapted and light-adapted plants and electron transport inducibility were more sensitive to Cr(VI) in L. minor than in L. gibba. We also found fundamental differences in quantum yield of regulated, Y(NPQ), and nonregulated, Y(NO), non-photochemical quenching between the two species. As Cr(VI) concentration increased in the growth medium, L. minor responded with considerable increase of Y(NPQ) with a parallel significant increase of Y(NO). By contrast, in L. gibba only 200 μM Cr(VI) in the growth medium resulted in elevation of Y(NPQ) while Y(NO) remained more or less constant within the regarding Cr(VI) concentration range during 48 h. Photosynthetic pigment content did not change considerably during the short-term Cr(VI) treatment but decrease of Chl a/b and increase of Car/Chl ratios were observed in good accordance with the changes in Chl fluorescence parameters. The data suggest that various duckweed species respond with different sensitivity to the same ambient concentrations of Cr(VI) in the growth medium, and presumably to other environmental stresses too, which may have an influence on their competitive relations when heavy metal pollution occurs in aquatic ecosystem.     

Distribution and speciation of chromium accumulated in Gynura pseudochina (L.) DC.

Soil and water contamination with chromium is an issue of recent concern in Thailand due to increases in industrial activity. Gynura pseudochina (L.) DC., a chromium tolerance plant, could be employed to address this problem via phytoremediation. To understand the tolerance mechanism, this study investigated the speciation and distribution of chromium accumulated in G. pseudochina (L.) DC. using AAS, XAFS, μ-XANES, μ-XRF imaging and EPR. The plants were separately treated with K₂Cr₂O₇ and Cr₂(SO₄)₃ in a hydroponic system. μ-XRF imaging clarified the distributions of Cr, Fe, Zn, Ca, Cl, K and S within the samples. In G. pseudochina (L.) DC. treated with Cr(VI) solution, the Cr was mainly distributed in the vascular bundle and periderm of the tuber, the stem xylem, the vein and the epidermis, including the trichome of the leaf tissues. This Cr distribution corresponded to those of Cu, Fe and Zn. In G. pseudochina (L.) DC. treated with Cr(III) solution, the Cr was distributed in the periderm of the tuber, the stem cortex, and the epidermis and parenchyma of the leaf tissues. μ-XANES and XAFS indicated that highly toxic Cr(VI) was reduced to the intermediate Cr(V) and accumulated as less toxic Cr(III), and EXAFS spectra showed that the reduced Cr(III) was bound to oxygen ligands. The coordination number (N) and the interatomic distance (R) to the first shell were approximately 3–4 (N) and 2 Å (R), respectively. EPR spectra of the plant samples treated with Cr(VI) revealed the presence of Cr(V) and Cr(III). Thus, Cr(III) and Cr(VI) were taken up into the vascular system and transported from the roots to the leaves. Cr(III) was distributed via the symplast system to the ground tissue and accumulated mainly in the stem cortex. Cr(VI) was transported to the xylem via the apoplast system, and the adsorption of Cr(VI) and its reduction to Cr(V) and Cr(III) occurred on oxygen ligands in the lignocellulosic structure of the xylem and vein.     

Responses of the Anaerobic Bacterial Community to Addition of Organic C in Chromium(VI)- and Iron(III)-Amended Microcosms

Chromium (VI) is toxic to microorganisms and can inhibit the biodegradation of organic pollutants in contaminated soils. We used microcosms amended with either glucose or protein (to drive bacterial community change) and Fe(III) (to stimulate iron-reducing bacteria) to study the effect of various concentrations of Cr(VI) on anaerobic bacterial communities. Microcosms were destructively sampled based on microbial activity (measured as evolution of CO₂) and analyzed for the following: (i) dominant bacterial community by PCR-denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene; (ii) culturable Cr-resistant bacteria; and (iii) enrichment of iron-reducing bacteria of the Geobacteraceae family by real-time PCR. The addition of organic C stimulated the activities of anaerobic communities. Cr(VI) amendment resulted in lower rates of CO₂ production in glucose microcosms and a slow mineralization phase in protein-amended microcosms. Glucose and protein amendments selected for different bacterial communities. This selection was modified by the addition of Cr(VI), since some DGGE bands were intensified and new bands appeared in Cr(VI)-amended microcosms. A second dose of Cr(VI), added after the onset of activity, had a strong inhibitory effect when higher levels of Cr were added, indicating that the developing Cr-resistant communities had a relatively low tolerance threshold. Most of the isolated Cr-resistant bacteria were closely related to previously studied Cr-resistant anaerobes, such as Pantoea, Pseudomonas, and Enterobacter species. Geobacteraceae were not enriched during the incubation. The studied Cr(VI)-contaminated soil contained a viable anaerobic bacterial community; however, Cr(VI) altered its composition, which could affect the soil biodegradation potential.     

Hydrogenases in sulfate-reducing bacteria function as chromium reductase

The ability of sulfate-reducing bacteria (SRB) to reduce chromate VI has been studied for possible application to the decontamination of polluted environments. Metal reduction can be achieved both chemically, by H₂S produced by the bacteria, and enzymatically, by polyhemic cytochromes c₃. We demonstrate that, in addition to low potential polyheme c-type cytochromes, the ability to reduce chromate is widespread among [Fe], [NiFe], and [NiFeSe] hydrogenases isolated from SRB of the genera Desulfovibrio and Desulfomicrobium. Among them, the [Fe] hydrogenase from Desulfovibrio vulgaris strain Hildenborough reduces Cr(VI) with the highest rate. Both [Fe] and [NiFeSe] enzymes exhibit the same K_m towards Cr(VI), suggesting that Cr(VI) reduction rates are directly correlated with hydrogen consumption rates. Electron paramagnetic resonance spectroscopy enabled us to probe the oxidation by Cr(VI) of the various metal centers in both [NiFe] and [Fe] hydrogenases. These experiments showed that Cr(VI) is reduced to paramagnetic Cr(III), and revealed inhibition of the enzyme at high Cr(VI) concentrations. The significant decrease of both hydrogenase and Cr(VI)-reductase activities in a mutant lacking [Fe] hydrogenase demonstrated the involvement of this enzyme in Cr(VI) reduction in vivo. Experiments with [3Fe-4S] ferredoxin from Desulfovibrio gigas demonstrated that the low redox [Fe-S] (non-heme iron) clusters are involved in the mechanism of metal reduction by hydrogenases.