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Biomedical subjects

L E Macaskie

Publications and source records attributed to L E Macaskie.

At least 37 records · Page 2Linked to original sources
PubMed1999

Microbial reduction of technetium by Escherichia coli and Desulfovibrio desulfuricans: enhancement via the use of high-activity strains and effect of process parameters.

Escherichia coli and Desulfovibrio desulfuricans reduce Tc(VII) (TcO(4)(-)) with formate or hydrogen as electron donors. The reaction is catalyzed by the hydrogenase component of the formate hydrogenlyase complex (FHL) of E. coli and is associated with a periplasmic hydrogenase activity in D. desulfuricans. Tc(VII) reduction in E. coli by H(2) and formate was either inhibited or repressed by 10 mM nitrate. By contrast, Tc(VII) reduction catalyzed by D. desulfuricans was less sensitive to nitrate when formate was the electron donor, and unaffected by 10 mM or 100 mM nitrate when H(2) was the electron donor. The optimum pH for Tc(VII) reduction by both organisms was 5.5 and the optimum temperature was 40 degrees C and 20 degrees C for E. coli and D. desulfuricans, respectively. Both strains had an apparent K(m) for Tc(VII) of 0.5 mM, but Tc(VII) was removed from a solution of 300 nM TcO(4)(-) within 30 h by D. desulfuricans at the expense of H(2). The greater bioprocess potential of D. desulfuricans was shown also by the K(s) for formate (>25 mM and 0.5 mM for E. coli and D. desulfuricans, respectively), attributable to the more accessible, periplasmic localization of the enzyme in the latter. The relative rates of Tc(VII) reduction for E. coli and D. desulfuricans (with H(2)) were 12.5 and 800 micromol Tc(VII) reduced/g biomass/h, but the use of an E. coli HycA mutant (which upregulates FHL activities by approx. 50%) had a similarly enhancing effect on the rate of Tc reduction. The more rapid reduction of Tc(VII) by D. desulfuricans compared with the E. coli strains was also shown using cells immobilized in a hollow-fiber reactor, in which the flow residence times sustaining steady-state removal of 80% of the radionuclide were 24.3 h for the wild-type E. coli, 4.25 h for the upregulated mutant, and 1.5 h for D. desulfuricans.

Biomass↗
PubMed1998

Fungal volatilization of arsenic and antimony and the sudden infant death syndrome.

Fungal volatilization of antimony and other group Vb elements has been proposed to have a causal role in the sudden infant death syndrome (SIDS; cot death). The ability of fungi to produce volatile arsenic and antimony compounds in pure culture was examined using Scopulariopsis brevicaulis, reported as an inhabitant of PVC cot mattress covers, and Phaeolus schweinitzii, a wood decay fungus known to be a good volatilizer of arsenic. Volatile arsenic compounds were detected from all cultures grown on arsenic-supplemented media, but antimony volatilization was not reliably detected. Although antimony levels above the baseline sensitivity of the analytical technique were detected in four (out of 24) of the samples analyzed, the concentrations recorded were too low to be reliably interpreted as evidence for volatilization. Our results are discussed in relation to hypotheses regarding the causes of SIDS.

Antimony↗
PubMed1998

Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology.

Bacterial nonspecific acid phosphohydrolases (NSAPs) are secreted enzymes, produced as soluble periplasmic proteins or as membrane-bound lipoproteins, that are usually able to dephosphorylate a broad array of structurally unrelated substrates and exhibit optimal catalytic activity at acidic to neutral pH values. Bacterial NSAPs are monomeric or oligomeric proteins containing polypeptide components with an M(r) of 25-30 kDa. On the basis of amino acid sequence relatedness, three different molecular families of NSAPs can be distinguished, indicated as molecular class A, B and C, respectively. Members of each class share some common biophysical and functional features, but may also exhibit functional differences. NSAPs have been detected in several microbial taxa, and enzymes of different classes can be produced by the same bacterial species. Structural and phyletic relationships exist among the various bacterial NSAPs and some other bacterial and eucaryotic phosphohydrolases. Current knowledge on bacterial NSAPs is reviewed, together with analytical tools that may be useful for their characterization. An overview is also presented concerning the use of bacterial NSAPs in biotechnology.

Acid Phosphatase↗
PubMed1998

Purification and characterization of acid-type phosphatases from a heavy-metal-accumulating Citrobacter sp.

An acid phosphatase from a heavy-metal-accumulating strain of a Citrobacter sp. was resolved into two forms on the basis of their nonbinding (phosphatase I) or binding (phosphatase II) behaviour on the cation-exchange resin SP-Sephadex C50. Both holoenzymes had a molecular mass of 103-108 kDa as determined by Superose Q-6 column chromatography in the presence of 150 mM KCl and a subunit molecular mass of 27 kDa as determined by SDS-PAGE; the enzyme was tetrameric. Both enzymes had a pI approximately 9.0 and were immunologically cross-reactive. There were minor differences in amino acid composition and in peptide maps following tryptic digest. The pH optimum for phosphatases I and II was 5.5 and 6.25, respectively; phosphatase II alone retained activity at pH values up to 9.0. Phosphatase I was more resistant to mechanical shear, gamma-irradiation, high temperature, and toxins (F- and formaldehyde). Glycerol increased the thermostability of both enzymes, particularly the more thermosensitive phosphatase II. Phosphatase II had a lower Km and a lower Vmax for glycerol 2-phosphate hydrolysis. The production of enzyme isoforms is a phenomenon similar to that described previously for the alkaline phosphatase of Escherichia coli, where the isoforms relate to precursive and final processed forms of the enzyme. Acid phosphatase is physiologically distinct, with a role that is still obscure but that may relate to cellular stress responses.

Acid Phosphatase↗
PubMed1998

The effect of growth conditions on the biodegradation of tributyl phosphate and potential for the remediation of acid mine drainage waters by a naturally-occurring mixed microbial culture.

The biodegradation of tributyl phosphate (Bu3-P, TBP), releasing phosphate at a high enough concentration locally to precipitate uranium from solution, was demonstrated by a mixed culture consisting primarily of pseudomonads. The effect of various parameters on Bu3-P biodegradation by growing cells is described. Growth at the expense of Bu3-P as the carbon and phosphorus source occurred over a pH range from 6.5 to 8, and optimally at pH 7. Bu3-P biodegradation was optimal at 30 degrees C, reduced at 20 degrees C and negligible at 4 degrees C and 37 degrees C. Incorporation of Cu or Cd inhibited, and Ni, Co and Mn reduced its degradation. Inorganic phosphate (above 10 mM) and kerosene (up to 1 g/l) reduced Bu3-P biodegradation significantly, but nitrate had no effect. Sulphate (10-100 mM) was inhibitory. When pregrown biomass was used the fastest rates of tributyl and dibutyl phosphate biodegradation were 25 mumol h-1 mg protein-1 and 37 mumol h-1 mg protein-1 respectively. Microcarrier-immobilised biomass decontaminated uranium-bearing acid mine waste water by uranium phosphate precipitation at the expense of Bu3-P hydrolysis in the presence of 35 mM SO(4)2-. At pH 4.5, 79% of the UO2(2)+ was removed at a flow rate of 1.4 ml/h on a 7-ml.test column.

Anions↗
PubMed1998

The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel from aqueous flows.

A Citrobacter sp. originally isolated from metal-polluted soil accumulates heavy metals via metalphosphate deposition utilizing inorganic phosphate liberated via PhoN phosphatase activity. Further strain development was limited by the non-transformability of this environmental isolate. Recombinant Escherichia coli DH5 alpha bearing cloned phoN or the related phoC acquired metal-accumulating ability, which was compared with that of the Citrobacter sp. with respect to removal of uranyl ion (UO2(2+)) from dilute aqueous flows and its deposition in the form of polycrystalline hydrogen uranyl phosphate (HUO2PO4). Subsequently, HUO2PO4-laden cells removed Ni2+ from dilute aqueous flows via intercalation of Ni2+ into the HUO2PO4 lattice. Despite comparable acid phosphatase activity in all three strains, the E. coli DH5 alpha (phoN) construct was superior to Citrobacter N14 in both uranyl and nickel accumulation, while the E. coli DH5 alpha (phoC) construct was greatly inferior in both respects. Expression of phosphatase activity alone is not the only factor that permits efficient and prolonged metal phosphate accumulation, and the data highlight possible differences in the PhoN and PhoC phosphatases, which are otherwise considered to be related in many respects.

Biodegradation, Environmental↗
PubMed1998

Biodegradation of metal-EDTA complexes by an enriched microbial population.

A mixed culture utilizing EDTA as the sole carbon source was isolated from a mixed inoculum of water from the River Mersey (United Kingdom) and sludge from an industrial effluent treatment plant. Fourteen component organisms were isolated from the culture, including representatives of the genera Methylobacterium, Variovorax, Enterobacter, Aureobacterium, and Bacillus. The mixed culture biodegraded metal-EDTA complexes slowly; the biodegradability was in the order Fe > Cu > Co > Ni > Cd. By incorporation of inorganic phosphate into the medium as a precipitant ligand, heavy metals were removed in parallel to EDTA degradation. The mixed culture also utilized a number of possible EDTA degradation intermediates as carbon sources.

Biodegradation, Environmental↗
PubMed1997

The biodegradation of tributyl phosphate by naturally occurring microbial isolates.

The biodegradation of tributyl phosphate by a mixed culture of Pseudomonads was demonstrated. Growth and the rate of tributyl phosphate consumption were variable and divisible into rapid and slow rates. Rapidly growing, rapidly tributyl phosphate-utilising cultures contained a 22-24 kb DNA fragment isolated by two methods, which was not visible in the cultures growing slowly. The mixed culture gave five periods of rapid growth interspersed with periods of poor growth during 7 months of weekly subculture, with the 22-24 kb DNA fragment detectable during the rapidly growing periods only. Seven Pseudomonads isolated from the culture grew at the expense of tributyl phosphate as the sole phosphorus source but spontaneously and irreversibly lost this ability after eight serial subcultures.

Biodegradation, Environmental↗
PubMed1997

Immobilisation of whole bacterial cells for anaerobic biotransformations.

Anaerobically grown cells of Escherichia coli were immobilised within a range of entrapment matrices and packed into a column under standard conditions, and the ability of the immobilised cells to reduce nitrite (0.5 mM) was measured at a range of flow rates using sodium formate (20 mM) as the electron donor for nitrite reduction. A flow-rate/activity plot was constructed for each flow-through reactor and RA1/2 values (residence time corresponding to 50% nitrite removal) calculated for each reactor type. Cells immobilised in flat and hollow-fibre membranes were the most effective (RA1/2 = 0.35 h and 0.47 h respectively), with cells entrapped by dialysis membrane (1.53 h), alginate beads (1.93 h), Hypol foam (2.31 h) and polyacrylamide gel (50% nitrite not removed at maximum residence time tested: 4.9 h) performing progressively less effectively. Cells grown as a biofilm on a range of support materials were also tested in comparable packed-bed reactors. Cell loss from these supports was extensive and contributed to poor performance of the reactors despite high initial biomass loadings (RA1/2 values using raschig rings, coke and activated-carbon supports: 1.6 h, 2.3 h and 1.0 h respectively). Biofilms grown on Pharmacia microcarrier supports and used in packed and also fluidised beds were more stable and the performance of these reactors was superior to that of biofilm reactors using other supports, and comparable to that of the membrane reactors (RA1/2 values for Cytoline 2, Cytopore 2 and Cytodex 3: 0.76 h, 0.56 h, 0.68 h respectively).

Acrylic Resins↗
PubMed1997

Reduction and removal of heptavalent technetium from solution by Escherichia coli.

Anaerobic, but not aerobic, cultures of Escherichia coli accumulated Tc(VII) and reduced it to a black insoluble precipitate. Tc was the predominant element detected when the precipitate was analyzed by proton-induced X-ray emission. Electron microscopy in combination with energy-dispersive X-ray analysis showed that the site of Tc deposition was intracellular. It is proposed that Tc precipitation was a result of enzymatically mediated reduction of Tc(VII) to an insoluble oxide. Formate was an effective electron donor for Tc(VII) reduction which could be replaced by pyruvate, glucose, or glycerol but not by acetate, lactate, succinate, or ethanol. Mutants defective in the synthesis of the transcription factor FNR, in molybdenum cofactor (molybdopterin guanine dinucleotide [MGD]) synthesis, or in formate dehydrogenase H synthesis were all defective in Tc(VII) reduction, implicating a role for the formate hydrogenlyase complex in Tc(VII) reduction. The following observations confirmed that the hydrogenase III (Hyc) component of formate hydrogenlyase in both essential and sufficient for Tc(VII) reduction: (i) dihydrogen could replace formate as an effective electron donor for Tc(VII) reduction by wild-type bacteria and mutants defective in MGD synthesis; (ii) the inability of fnr mutants to reduce Tc(VII) can be suppressed phenotypically by growth with 250 microM Ni2+ and formate; (iii) Tc(VII) reduction is defective in a hyc mutant; (iv) the ability to reduce Tc(VII) was repressed during anaerobic growth in the presence of nitrate, but this repression was counteracted by the addition of formate to the growth medium; (v) H2, but not formate, was an effective electron donor for a Sel- mutant which is unable to incorporate selenocysteine into any of the three known formate dehydrogenases of E. coli. This appears to be the first report of Hyc functioning as an H2-oxidizing hydrogenase or as a dissimilatory metal ion reductase in enteric bacteria.

Anaerobiosis↗
PubMed1996

Bioaccumulation of nickel by intercalation into polycrystalline hydrogen uranyl phosphate deposited via an enzymatic mechanism.

A Citrobacter sp. accumulates uranyl ion (UO2(2+)) as crystalline HUO2PO4.4H2O (HUP), using enzymatically generated inorganic phosphate. Ni was not removed by this mechanism, but cells already loaded with HUP removed Ni2+ by intercalative ion-exchange, forming Ni(UO2PO4)2.7H2O, as concluded by x-ray diffraction (XRD) and proton induced x-ray emission (PIXE) analyses. The loaded biomass became saturated with Ni rapidly, with a molar ratio of Ni:U in the cellbound deposit of approx. 1:6; Ni penetration was probably surface-localized. Cochallenge of the cells with Ni2+ and UO2(2+), and glycerol 2-phosphate (phosphate donor for phosphate release and metal bioprecipitation) gave sustained removal of both metals in a flow through bioreactor, with more extensively accumulated Ni. We propose 'Microbially Enhanced Chemisorption of Heavy Metals' (MECHM) to describe this hybrid mechanism of metal bioaccumulation via intercalation into preformed, biogenic crystals, and note also that MECHM can promote the removal of the transuranic radionuclide neptunium, which is difficult to achieve by conventional methods.

Biomass↗
PubMed1996

A Novel PhosphorImager-Based Technique for Monitoring the Microbial Reduction of Technetium.

A novel PhosphorImager-based technique which can be used to quantify low concentrations of radionuclides is described. The technique offers several benefits, combining very high sensitivity with containment of the radioisotope in the solid state, thus minimizing disposal procedures. In this study, it was used in conjunction with paper chromatography to quantify different oxidation states of (sup99)Tc in solution. The technique was used to evaluate the potential of anaerobic cultures of Shewanella putrefaciens and Geobacter metallireducens (bacteria with known metal-reducing capabilities) to reduce highly soluble Tc(VII) to insoluble lower-valence species, facilitating its removal from solution. Both organisms reduced Tc(VII), but profiles of Tc species produced in culture supernatants were strain specific. S. putrefaciens produced Tc(V), Tc(IV), and one unidentified species, but no Tc was removed from solution. G. metallireducens removed 70% of the 250 (mu)M Tc added in solution, with trace amounts of Tc(V) and the unidentified species detected in culture supernatants. Possible uses for these organisms in the bioremediation of Tc-contaminated waters are discussed, and other uses of the PhosphorImager technique are highlighted.

Journal Article↗
PubMed1995

Phosphatase production and activity in Citrobacter freundii and a naturally occurring, heavy-metal-accumulating Citrobacter sp.

The ability of a naturally occurring Citrobacter sp. to accumulate cadmium has been attributed to cellular precipitation of CdHPO4, utilizing HPO4(2-) liberated via the activity of an overproduced, Cd-resistant acid-type phosphatase. Phosphatase production and heavy metal accumulation by batch cultures of this strain (N14) and a phosphatase-deficient mutant were compared with two reference strains of Citrobacter freundii. Only strain N14 expressed a high level of acid phosphatase and accumulated lanthanum and uranyl ion enzymically. Acid phosphatase is regulated via carbon-starvation; although the C. freundii strains overexpressed phosphatase activity in carbon-limiting continuous culture, this was approximately 20-fold less than the activity of strain N14 grown similarly. Citrobacter strain N14 was originally isolated from a metal-contaminated soil environment; phosphatase overproduction and metal accumulation were postulated as a detoxification mechanism. However, application of Cd-stress, and enrichment for Cd-resistant C. freundii ('training'), reduced the phosphatase activity of this organism by about 50% as compared to Cd-unstressed cultures. The acid phosphatase of C. freundii and Citrobacter N14 had a similar pattern of resistance to some diagnostic reagents. The enzyme of the latter is similar to the PhoN acid phosphatase of Salmonella typhimurium described by other workers; the results are discussed with respect to the known phosphatases of the enterobacteria.

Acid Phosphatase↗
PubMed1994

Phosphatase-mediated heavy metal accumulation by a Citrobacter sp. and related enterobacteria.

A Citrobacter sp. was reported previously to accumulate heavy metals as cell-bound heavy metal phosphates. Metal uptake is mediated by the activity of a periplasmic acid-type phosphatase that liberates inorganic phosphate to provide the precipitant ligand for heavy metals presented to the cells. Amino acid sequencing of peptide fragments of the purified enzyme revealed significant homology to the phoN product (acid phosphatase) of some other enterobacteria. These organisms, together with Klebsiella pneumoniae, previously reported to produce acid phosphatase, were tested for their ability to remove uranium and lanthanum from challenge solutions supplemented with phosphatase substrate. The coupling of phosphate liberation to metal bioaccumulation was limited to the metal accumulating Citrobacter sp.; therefore the participation of species-specific additional factors in metal bioaccumulation was suggested.

Acid Phosphatase↗
PubMed1994

Enzymically accelerated biomineralization of heavy metals: application to the removal of americium and plutonium from aqueous flows.

A biological process for the removal of heavy metals from the aqueous flows is described. Metals are precipitated on the surface of immobilized cells of a Citrobacter sp. as cell-bound metal phosphates. This uses phosphate liberated by the activity of a cell-bound phosphatase. Some radionuclides (e.g. 241americium) form metal phosphates readily; efficient removal of 241Am on a continuous basis is demonstrated. At low phosphatase activities, the efficiency of uranium removal correlates with enzyme activity. High phosphatase activities are not realised as an increase in metal removal, suggesting that chemical events become rate-limiting. Studies have suggested that maximal metal uptake occurs only after nucleation and the formation of precipitation foci. A model is presented to illustrate how nucleation and crystallization processes could enhance the removal of plutonium and neptunium from dilute solutions.

Acrylic Resins↗
PubMed1992

Uranium bioaccumulation by a Citrobacter sp. as a result of enzymically mediated growth of polycrystalline HUO2PO4.

A Citrobacter sp. accumulates heavy deposits of metal phosphate, derived from an enzymically liberated phosphate ligand. The cells are not subject to saturation constraints and can accumulate several times their own weight of precipitated metal. This high capacity is attributable to biomineralization; uranyl phosphate accumulates as polycrystalline HUO2PO4 at the cell surface. The precipitated metal is indistinguishable from crystalline HUO2PO4.4H2O grown by chemical methods.

Cell Membrane↗
PubMed1991

Phosphatase production by a Citrobacter sp. growing in batch cultures retarded by anaerobic or osmotic stress, and the effect of the osmoprotectant glycine betaine.

The effect of 500 mM NaCl on the growth, and phosphatase production of a Citrobacter sp. was investigated. Although growth was retarded, phosphatase production was enhanced by 50%. Relief from osmotic stress using the osmoprotectant glycine betaine gave normal growth, but phosphatase activity was reduced. The Citrobacter sp. ceased to grow following a shift to anaerobic conditions, but anaerobically-incubated cells continued to produce phosphatase after a transient lag.

Anaerobiosis↗