Recovery of uranium by immobilized microorganisms

Summary Some attempts were made to recover uranium from sea and fresh water using immobilized Streptomyces viridochromogenes and Chlorella regularis cells. The cells immobilized in polyacrylamide gel have the most favorable features for uranium recovery; high adsorption ability, good mechanical properties, and applicability in a column system. The adsorption of uranium by the immobilized cells is not affected by the pH values between 4 and 9. These results show that uranium adsorption becomes independent of pH after immobilization. The amounts of uranium adsorbed by the immobilized cells increased linearly with temperature, suggesting that the adsorption of uranium by the immobilized cells is an endothermic reaction. The immobilized cells can recover uranium almost quantitatively from both fresh and sea water containing uranium, and almost all uranium adsorbed is desorbed with a solution of Na₂CO₃. Thus the immobilized cells of Streptomyces and Chlorella can be used repeatedly in adsorption-desorption process.Studies on the Accumulation of Heavy Metal Elements in Biological Systems. XXI     

Selective accumulation of heavy metals by microorganisms

Summary An investigation of the removal and recovery of urnnium from aqueous systems using microbial biomass has been described previously (Nakajima et al. 1982). To establish which microorganisms accumulate the most uranium, we extended our investigation of uranium uptake to 83 species of microorganisms, 32 bacteria, 15 yeasts, 16 fungi and 20 actinomycetes. Of these 83 species of microorganisms tested, extremely high uranium-absorbing ability was found in Pseudomonas stutzeri, Neurospora sitophila, Streptomyces albus and Streptomyces viridochromogenes.The selective accumulation of heavy metal ions by various microorganisms has also been examined. Uranyl, mercury and lead ions were readily accumulated by almost all the species of microorganisms tested. Actinomycetes and fungi differ from many bacteria and most yeasts in their selective accumulation of uranium and mercury.In addition to this fundamental research, uranium recovery was investigated in immobilized Streptomyces albus, a microorganism with high uranium-uptake ability. These immobilized cells adsorbed uranium readily and selectively. The immobilized cells recovered uranium almost quantitatively and almost all uranium absorbed was desorbed with 0.1 M Na₂CO₃. The dry weight of the free cells decreased by 50% during 5 adsorption-desorption cycles. However, the dry weight of the immobilized cells decreased by only 2% during 5 cycles. These results showed that microbial cells are more stable after immobilization and can be used repeatedly for the process of uranium adsorption-desorption.     

Recovery and removal of uranium by using plant wastes

The uranium-adsorbing abilities of seven plant wastes were investigated. High abilities to adsorb uranium from non-saline water containing 10 mg dm⁻³ of uranium were observed with a number of plant wastes tested. However, with seawater supplemented with 10 mg dm⁻³ of uranium, similar results were found only with chestnut residues. When the plant wastes were immobilized with formaldehyde, their ability to adsorb uranium was increased. Uranium and copper ions were more readily adsorbed by all plant wastes tested than other metal ions from a solution containing a mixture of seven different heavy metals. The selective adsorption of heavy metal ions differs with different species of plant wastes. The immobilization of peanut inner skin, orange peel and grapefruit peel increased the selectivity for uranium.     

Uranium uptake by immobilized cells of Pseudomonas strain EPS 5028

Polyacrylamide-gel-immobilized cells of Pseudomonas strain EPS 5028 were effective in the removal of uranium (U) from synthetic effluents. Metal accumulation was performed in an open system in columns filled with immobilized cells that were challenged with continuous flows containing U. Possible variables of the system were studied. Uranium uptake by the immobilized cells of this microorganism was affected by pH but not by temperature or flow rate. In addition, U binding could be interpreted in terms of the Freundlich adsorption isotherm indicating single-layer adsorption. The feasibility of reusing the immobilized cells was suggested after the recovery of U with a solution of 0.1 m sodium carbonate. Correspondence to: M. C. Fusté     

Physiological adsorption of Sulfolobus acidocaldarius on coal surfaces

Summary The adsorption of Sulfolobus acidocaldarius on bituminous coal surfaces and the respiration rate during adsorption at 70° C were enhanced at pH 1.0–2.0, in comparison with those at pH 3.0–5.0. The maximum number of bacterial cells adsorbed per unit area of coal attained a maximum (1.4 × 10¹¹ cells/m²) at pH 2.0. The rate of desulphurization at pH 2.2–2.5 was higher than at other pHs tested. Micrographs of S. acidocaldarius obtained by TEM and SEM indicated that the cells were adsorbed to the coal surfaces by extracellular slime. Specific inhibitors of membrane-bound ATPase (NaF, 20 mm) and respiration (NaN₃, 1 mm; KCN, 1 mm) had pronounced effects on suppressing adsorption. The amount of S. acidocaldarius adsorbed decreased when the coal particles were leached in advance with 2.0 m HNO₃. These facts lead to the conclusion that the adsorption of S. acidocaldarius on coal surfaces requires physiological activity relatd to respiration or energy conversion. Offprint requests to: V. B. Vitaya     

Accumulation of gold by microorganisms

Thirty species of microorganisms (8 bacteria, 9 actinomycetes, 8 fungi and 5 yeasts) were screened for maximal gold accumulation. Extremely high abilities to accumulate gold from a solution containing hydrogen tetrachloroaurate(III) were found in bacterial strains, such as Escherichia coli and Pseudomonas maltophilia. Most of the actinomycetes, fungi and yeasts had lower ability to accumulate gold than bacteria. Some microorganisms could accumulate similar amounts of gold from a solution containing sodium gold(I) thiomalate as those from gold(III) solution. However, most microorganisms tested accumulated far lesser amounts of gold from a solution containing sodium dicyanoaurate(I) than from the other two gold solutions. The accumulation of gold from the solution containing hydrogen tetrachloroaurate(III) by Pseudomonas maltophilia was very rapid, was affected by the pH of the solution, and obeyed the Langmuir adsorption isotherm. Pseudomonas maltophilia cells immobilized in polyacrylamide gel adsorbed gold effectively from the solution containing hydrogen tetrachloroaurate(III). The gold adsorbed on the cells was easily desorbed with 0.1 M thiourea solution. The immobilized Pseudomonas cells could be used repeatedly in the adsorption–desorption cycle using 0.1 M thiourea solution as desorbent.     

Improvement of production of l-aspartic acid using immobilized microbial cells

Summary In our laboratory, EAPc-7 a strain having higher aspartase activity was derived from Escherichia coli ATCC 11303. For the improvement of l-aspartic acid productivity using EAPc-7 cells immobilized in -carrageenan, it was necessary to eliminate the fumarase activity which converts fumaric acid to l-malic acid. Several treatments for specifically eliminating fumarase activity from EAPc-7 cells were tested and it was found that when EAPc-7 cells were treated in a culture broth (pH 4.9) containing 50 mM l-aspartic acid at 45° C for 1 h, fumarase activity was almost completely eliminated without inactivation of the aspartase.The treated cells, immobilized in -carrageenan, were used for continuous production of l-aspartic acid from ammonium fumarate. The formation of l-malic acid was negligible and the half-life of the immobilized preparation was 126 days.Productivity of immobilized preparation of treated EAPc-7 cells in l-aspartic acid production was six times of that of the parent cell preparation.     

Degradation of Soil Humic Extract by Wood- and Soil-Associated Fungi, Bacteria, and Commercial Enzymes

Abstract An alkaline humic extract (HE) of a black calcareous forest mull was exposed to 36 fungal and 9 eubacterial isolates in liquid standing culture. At 21 d in fungi, and 4 d in bacteria, the groups of wood-degrading basidiomycetes, terricolous basidiomycetes, ectomycorrhizal fungi, soil-borne microfungi, and eubacteria had reduced the absorbance (A ₃₄₀) of HE media by 57, 28, 19, 26 and 5%, respectively. Gel permeation chromatography revealed that the large humic acid molecules were more readily degraded than the smaller fulvic acid molecules and served as a sole source of carbon and energy. The more active HE degraders reduced the overall molecular weight of humic and fulvic acids by 0.25 to 0.47 kDa. They also reduced the chemical reactivity of HE to tetrazotized o-dianisidine, indicating the degradation of hydroxylated aromatic molecules (which are responsible for this reaction). Decreases in absorbance, molecular weight, and reactivity were caused by fungal manganese peroxidase, horseradish peroxidase, β-glucosidase, and abiotic oxidants such as H₂O₂ and Mn(III) acetate. It is concluded that fungi, some of which are propagated in contaminated soils to control xenobiotics, metabolize HE compounds enzymatically. They use enzymes which are also involved in the degradation of soil xenobiotics. Because of reductions in the molecular weight of HE, which is a potential carrier of heavy metal ions and xenobiotics, solubility and motility of humic substances in soil and surface waters are increased. Received: 4 March 1998; Accepted: 1 June 1998     

Microbial sensor for selective determination of sulphide

A microbial sensor consisting of immobilized Thiobacillus thiooxidans, a gas-permeable membrane, and an O₂ electrode was prepared for the determination of sulphide. When a sample solution containing sulphide was passed into the flow cell, the output of the microbial sensor decreased markedly with time until a steady state was reached. The total time required for an assay was 20–30 min by the steady-state method. In the pulse method, the total time required for an assay was about 5 min. A linear relationship was obtained between the sensor output and the concentration of sodium sulphide below 0.40 mm. The minimum detectable concentration of sodium sulphide was 0.02 mm. Selectivity of the sensor was satisfactory. The microbial sensor was applied to the determination of sulphide in spring water. A good agreement was obtained between the microbial sensor and the methylene blue method. The regression coefficient was 0.97 for five experiments. The activity of the microbial membrane was stable for more than 25 days. The response was reproducible with 2.5% of the relative standard deviation when a sample solution containing 0.2 mm sodium sulphide was employed. *** DIRECT SUPPORT *** AG903053 00005     

Evaluation ofDrosophila melanogaster as an alternative animal for studying the neurotoxicity of heavy metals

Heavy metals cause irreversible neurobehavioral damage in many developing mammals, but the mechanisms of this damage are unknown. The influence of three heavy metal compounds, triethyllead chloride, lead acetate and cadmium chloride, on lethality, development, behavior and learning was studied using the fruit fly,Drosophila melanogaster. This animal was used because it allows hundreds of subjects to be assayed very easily in individual experiments and because it is a system in which toxicological questions might be answered by using the techniques of modern molecular genetics. When triethyllead chloride, lead acetate or cadmium chloride was placed in the medium, the larval LC₅₀ (± standard error) was found to be 0.090±0.004, 6.60±0.64 and 0.42±0.04mm, respectively. Each of the tested compounds produced a dose-related delay in development. In particular, they caused a delay in the development of larvae to pupae. When larvae were reared on medium containing triethyllead chloride (0.06mm), lead acetate (3.07mm) or cadmium chloride (0.11mm), phototaxis, locomotion and learning were not inhibited. Since significant neurobehavioral effects were not observed under the experimental conditions used,Drosophila does not appear to be an appropriate animal for the genetic dissection of such effects of heavy metals during development.     

Production of l-glutamate by immobilized protoplasts

Summary Protoplasts of Brevibacterium flavum cultured in a medium containing 50 g·l^-1 of biotin were prepared with lysozyme and immobilized in matrices of agar-acetylcellulose filters. The immobilized protoplasts were applied to l-glutamate production from glucose and urea in a batch system. The productivity of l-glutamate by the immobilized protoplasts was 2.5 times higher than that by immobilized whole cells under optimal conditions. Maximal productivity initially reached 1.5 mg·ml^-1. The immobilized protoplasts of B. flavum could be used six times for l-glutamate production with retention of about 70% of the initial productivity.     

Conversion of phenylpyruvate to l-phenylalanine by immobilized Clostridium butyricum-alanine dehydrogenase-Micrococcus luteus under hydrogen high pressure

Summary Alanine was the best amino donor among various amino acids and NH₄Cl for the phenylalanine production of Micrococus luteus. l-Alanine was regenerated at the rate of 9.2 moles/min/g dry cells from NH₄Cl and pyruvate by immobilized Clostridium butyricum-alanine dehydrogenase. l-Phenylalanine was continuously produced from hydrogen, NH₄Cl and phenylpyruvate by coupling immobilized C. butyricum, alanine dehydrogenase and M. luteus. The rate of phenylalanine production was 1.74 moles/min/g dry cells.     

The effects of immobilization on the biochemical,physiological and morphological features of Anabaena azollae

Anabaena azollae, a presumptive isolate from Azolla filiculoides, was immobilized in polyurethane foam, hydrophilic polyvinyl foam and alginate. When viewed by low-temperature scanning electron microscopy a thick mucilage layer covered the surface of both cells and matrix; this closely resembles the mode of attachment of the symbiont Anabaena in the Azolla leaf cavity. The heterocyst frequency of the immobilized A. azollae doubled relative to free-living cells and reached a level of 14–17%. Immobilization induced increases in both hydrogen production via nitrogenase or hydrogenase and in the rates and stabilization of acetylene reduction (N₂-fixation). Ammonia production by immobilized cells with L-methionine-D,L-sulfoximine (MSX) is greater than that of freeliving cells. Immobilized cells without MSX were, however, able to excrete ammonium at lower rates thus emulating the characteristic of the symbiotic cyanobacteria (A. azollae) in the leaf cavity of Azolla.Abbreviations Chl chlorophyll - GS glutamine synthetase - MSX L-methionine-D,L-sulfoximine - SEM scanning electron microscopy - PU polyurethane - PV polyvinyl     

Immobilization of Cu(II) and Cr(IV) in basidiomycete-colonized sawdust

An investigation into the feasibility of removing Cu(II) and Cr(IV) from solution with basidiomycete (Gloeophylum sepiarium, Pleurotus sp.)-colonized sawdust was undertaken. Obeche (Triplochyton scleroxylon) sawdust exposed to the basidiomycetes for 1–3 months reduced the concentration of the metals in the solution to 22.0–84.4 mg/l. The supernatant from the centrifuged mixture of a solution of 100 mg metal ions/l and aqueous extract of a 3-month basidiomycete-degraded obeche sawdust contained lower concentration of the metal ions (38.6–75.4 mg/l). Unextracted sawdust of pigmented tropical timbers, African mahogany (Khaya ivorensis), black afara (Terminalia ivorensis) and camwood (Baphia nitida) exposed to the test basidiomycetes, removed Cu and Cr significantly better than the extracted sawdust. It is hypothesised that some products of basidiomycete wood-degradative activities were ligands which immobilized the test metals.     

Interaction between uranium and the cytochrome c 3 of Desulfovibrio desulfuricans strain G20

Cytochrome c₃ of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c₃ in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c₃ (cycA) to lacZ. Instead, cytochrome c₃ protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c₃ with U(IV) was interpreted to be non-specific, since pure cytochrome c₃ adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe₂O₃), and commercially available U(IV) oxide.An erratum to this article can be found at     

Role and Source of ATP for Activation of Nonselective Cation Channels by AlF Complex in Guinea Pig Chromaffin Cells

Intracellular dialysis with the solution containing the G protein activator, AlF complex, induced an inward nonselective cation current (I _NS) at −55 mV in chromaffin cells. Amplitudes of I _NS induced by dialysis with ATP-free AlF solutions progressively diminished as cells were pretreated with cyanide, a mitochondrial inhibitor. After a 10-min pretreatment, generation of I _NS by the AlF complex depended on exogenous ATP delivered from pipette solution. The relationship between amplitudes of I _NS and concentrations of MgATP was well expressed by a rectangular hyperbola with an EC₅₀ of 0.265 mm. This result suggests that the cyanide treatment almost depleted ATP near the plasma membrane. On the other hand, a similar cyanide treatment of adrenal medullary preparations did not induce a marked decrease in cellular ATP content. GTP, ITP, or UTP could not substitute for ATP in generation of I _NS by the AlF complex. Similarly, the substitution of ATP with non- or poorly hydrolyzable ATP analogues did not aid in generating I _NS. Bath application of the kinase inhibitor, H-7 (100 μm), suppressed AlF-induced I _NS in a manner depending on intracellular Mg²⁺. We conclude that ATP is a prerequisite for generation of I _NS as a phosphoryl donor and that mitochondria is the main source of ATP. Received: 17 April 1996/Revised: 26 July 1996     

Comparative study of d-xylose conversion to ethanol by immobilized growing or non-growing cells of the yeast Pachysolen tannophilus

Summary The direct conversion of d-xylose to ethanol was investigated using immobilized growing and non-growing cells of the yeast Pachysolen tannophilus. Both preparations produced ethanol from d-xylose, however the d-xylose conversion to ethanol was much better with immobilized growing cells. Ethanol concentration up to 22.9 g/l and ethanol yield of 0.351 g/g of d-xylose were obtained in batch fermentation by immobilized growing cells whereas only 17.0 g/l and 0.308 g/g of d-xylose were obtained by immobilized non-growing cells. With continuous systems, immobilized growing cells were necessary for the long-term operation, since a steady state ethanol concentration of 17.7 g/l was maintained for only one week by immobilized non-growing cell reactor. With simultaneous control of aeration rate and concentrations of nitrogen sources in feed medium, immobilized growing cells of P. tannophilus showed excellent performance. At a residence time of 25 h, the immobilized cell reactor produced 26.9 g/l of ethanol from 65 g/l of d-xylose in feed medium.     

Recycle Use of Sphingomonas sp. CDH-7 Cells for Continuous Degradation of Carbazole in the Presence of MgCl2

Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.₆₆₀ 3.3) were shaken in 50 mM K₂HPO₄-KH₂PO₄ buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly. However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium solution or in the buffer containing 20 mM MgCl₂. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 mM EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 mM MgCl₂. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 mM) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 mM) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation reaction by the resting cells. Received: 28 June 2001 / Accepted: 30 July 2001     

Alleviation of heavy metal stress in Spilanthes calva L. (antimalarial herb) by exogenous application of glutathione

To evaluate the role of exogenous application of a phytochelating agent glutathione in increasing resistance against different heavy metals stress, nodal explants excised from 28-day-old in vitro seedlings of Spilanthes calva L. were cultured on Murashige and Skoog’s medium supplemented with 10 μM benzyl adenine and five different concentrations (1, 5, 50, 100, or 200 mg/l) of four heavy metals: As₂O₃, CuSO₄, ZnSO₄, or Pb(NO₃)₂. Data were recorded for percent survival, shoot number, and shoot length after 28 d of heavy metal treatment. All four heavy metals severely inhibited growth and morphogenesis. Pb proved most inhibitory whereas Zn was least effective. Pb was further selected to study the reversal effect of glutathione on morphogenesis. The addition of different concentrations (1, 5, 10, or 25 mg/l) of glutathione to media containing the Pb resulted in a significant improvement in almost all growth parameters. Inclusion of glutathione at 10 mg/l was optimum for maximum reversal of the negative effects of heavy metals on morphogenesis.     

Volatilization of Mercury by Immobilized Bacteria (Klebsiella pneumoniae) in Different Support by Using Fluidized Bed Bioreactor

Klebsiella pneumoniae, a mercury-resistant bacterial strain able to reduce ionic mercury to metallic mercury, was isolated from wastewater of Casablanca. This strain exhibits high minimal inhibition concentrations for heavy metals such as mercury 2400 μM, lead 8000 μM, silver 2400 μM, and cadmium 1000 μM. This bacterium was immobilized in alginate, polyacrylamide, vermiculite, and cooper beech and was used for removing mercury from a synthetic water polluted by mercury by using a fluidized bead bioreactor. Immobilized bacterial cells of Klebsiella pneumoniae could effectively volatilize mercury and detoxify mercury compounds. Moreover, the efficiency of mercury volatilization was much greater than with the native cells. The highest cleanup and volatilization rates were obtained when Klebsiella pneumoniae was entrapped in alginate beads, with a cleanup rate of 100% and a volatilization rate of 89%. Immobilized cells in alginate continuously volatilized mercury even after 10 days without loss of activity. Received: 21 February 2001 / Accepted: 13 March 2001