Lynne E. Macaskie

CADMIUM ACCUMULATION BY A CITROBACTER SP

Cadmium accumulation by a Citrobacter sp. growing in the presence of the metal occurred as a sharp peak during the mid-exponential phase of growth, but cultures showed considerable inhibition of growth compared to cadmium-free controls. This problem was overcome by pregrowing the cells in cadmium-free medium and subsequently exposing them to the metal in the resting state, under which conditions higher concentrations of cadmium were tolerated and metal uptake was enhanced. This ability was retained when the cells were immobilized and then challenged with a flow containing Cd2+; 65% of the metal presented was removed from solution. The influence on uptake of the composition of the exposure buffer and of various cell treatments were investigated and the results are discussed with respect to the anticipated speciation of the cadmium presented to the cells and also with respect to the probable mechanism of metal uptake. This is thought to occur through the activity of a cell-bound phosphatase, induced during pre-growth by the provision of glycerol 2-phosphate as sole phosphorus source. Continued enzyme function in resting cells would then precipitate the metal as cell-bound cadmium phosphate.

Metal reduction by sulphate-reducing bacteria: physiological diversity and metal specificity

Abstract The reduction of Tc(VII), Cr(VI) Se(IV) and Te(IV) by representatives of three genera of sulphate-reducing bacteria was studied with respect to the specificity of electron donor and acceptor. Tc(VII) and Cr(VI) were reduced by different mechanisms involving a hydrogenase. Cr(VI) reduction was achieved using a new isolate with lactate as the electron donor or by using H 2 in the presence of bicarbonate ion. Te(IV) and Se(IV) were reduced to base metals. The removal of Se(IV) was enhanced under sulphidogenic conditions, with metal sulphide identified by energy dispersive X-ray microanalysis. The order of preference of the electron acceptors was Te(IV)>S(VI)>Se(IV), which is in sharp contrast to that predicted by the redox potentials alone.

Technetium reduction and precipitation by sulfate‐reducing bacteria

Resting cells of the sulfate‐reducing bacterium Desulfovibrio desulfuricans ATCC 29577 were able to precipitate the radionuclide technetium, supplied as the pertechnetate anion (TcO4 ‐), under anaerobic conditions by two discrete mechanisms. Sulfidogenic cultures, supplied with sulfate and lactate as an electron acceptor and donor, respectively, precipitated the radionuclide as an insoluble sulfhide. Using electron microscopy in combination with energy‐dispersive x‐ray analysis (EDAX), the precipitate was shown to be extracellular, and contained S as the major element at a fivefold stoichiometric excess to Tc as quantified by proton‐induced x‐ray emission analysis (PIXE). With hydrogen supplied as the electron donor, the pertechnetate anion was utilized as an alternative electron acceptor in the absence of sulfate. The radionuclide was removed from solution, but in these cultures the precipitate was cell associated, with Tc as the major element detected by PIXE (Tc:S ratio of 2:1). Reduction of the radion...

Involvement of hydrogenases in the formation of highly catalytic Pd(0) nanoparticles by bioreduction of Pd(II) using Escherichia coli mutant strains

Escherichia coli produces at least three [NiFe] hydrogenases (Hyd-1, Hyd-2 and Hyd-3). Hyd-1 and Hyd-2 are membrane-bound respiratory isoenzymes with their catalytic subunits exposed to the periplasmic side of the membrane. Hyd-3 is part of the cytoplasmically oriented formate hydrogenlyase complex. In this work the involvement of each of these hydrogenases in Pd(II) reduction under acidic (pH 2.4) conditions was studied. While all three hydrogenases could contribute to Pd(II) reduction, the presence of either periplasmic hydrogenase (Hyd-1 or Hyd-2) was required to observe Pd(II) reduction rates comparable to the parent strain. An E. coli mutant strain genetically deprived of all hydrogenase activity showed negligible Pd(II) reduction. Electron microscopy suggested that the location of the resulting Pd(0) deposits was as expected from the subcellular localization of the particular hydrogenase involved in the reduction process. Membrane separation experiments established that Pd(II) reductase activity is membrane-bound and that hydrogenases are required to initiate Pd(II) reduction. The catalytic activity of the resulting Pd(0) nanoparticles in the reduction of Cr(VI) to Cr(III) varied according to the E. coli mutant strain used for the initial bioreduction of Pd(II). Optimum Cr(VI) reduction, comparable to that observed with a commercial Pd catalyst, was observed when the bio-Pd(0) catalytic particles were prepared from a strain containing an active Hyd-1. The results are discussed in the context of economic production of novel nanometallic catalysts.

Reduction of chromate by microorganisms isolated from metal contaminated sites of Karachi, Pakistan

Three bacterial strains, two identified as Pseudomonas stutzeri and one as a strain of cucurbit yellow vine disease bacterium, isolated from a foundry soil and a tannery, respectively, in Pakistan, were resistant to up to 1 mM chromate and anaerobically reduced Cr(VI) up to 100 μM. The highest removal was by P. stutzeri CMG463: 88 μmol l−1 (88% of that supplied; specific rate was 3.0 nmol mg−1 protein h−1), while 58 and 76 μmol l−1 (58% and 76%) were removed by P. stutzeri CMG462 and cucurbit yellow vine disease bacterium CMG480, respectively. These isolates were compared to strains isolated from an uncontaminated coastal site in the UK and designated as K2 (Pseudomonas synxantha) K3 (Bacillus sp.), and J3 (unidentified Gram-positive strain). Strain K3 was Cr-sensitive, partially lysed by Cr(VI), but had the highest removal of chromate anaerobically: 92 μmol l−1 (92% of that supplied) at a specific rate of 71 nmol mg−1 protein h−1. Analysis of cell sections using transmission electron microscopy with energy dispersive X-ray analysis showed intracellular chromium in P. stutzeri but the cucurbit yellow vine disease bacterium and the Bacillus sp. precipitated chromium extracellularly. The isolates from the Cr-contaminated sites did not remove more Cr(VI), overall, than Cr-unstressed bacteria, but their tolerance to Cr(VI) is potentially useful for bioremediation, particularly since other studies have shown that the two P. stutzeri strains can bioaccumulate Cu2+.

Heavy metals removal by sand filters inoculated with metal sorbing and precipitating bacteria

Abstract Large volumes of wastewater containing metals such as Cd, Zn, Cu, Pb, Hg, Ni or Co are mainly treated by precipitation processes. However, waters treated in such ways do not always meet regulatory standards. And in many cases, ecotaxes must be paid on the heavy metals load in the discharged water. Therefore, a second polishing treatment is often necessary. In order to be economically acceptable, the technology must be cheap and adapted to the treatment of large volumes. The use of sand filters inoculated with heavy metal biosorbing and bioprecipitating bacteria fulfils these objectives. The system is based on a moving bed sand filter. A biofilm is formed on the sand grains after inoculation with heavy metal-resistant bacteria able to biosorb or to bioprecipitate heavy metals. Passage of the wastewater over these biofilms leads to the binding of the metals to the biofilm and consequently the removal of the metals from the wastewater. The metal-laden biofilm is removed from the sand grains in a sand washer created by an airlift for the continuous movement of the filter bed. The metal-loaded biomass is separated from the sand in a labyrinth on the top of the sand washer. Nutrients and a carbon source are provided continuously in the system in order to promote the regrowth of the biofilm on the sand grains. The reactor can be used for the removal of heavy metals, nitrates and some COD. The obtained biosludge contains heavy metals at concentrations of more than 10% of the dry weight. The treatment of the sludge is also taken into account.

A novel electrobiotechnology for the recovery of precious metals from spent automotive catalysts

Abstract Platinum group metals are routinely used in automotive catalysts but recycle technology lags behind demand. There is no available ‘clean technology’ and leach solutions (e.g. aqua regia) to solubilise the metals from scrap are highly aggressive. A microwave‐assisted leaching method was developed which gave 80% metals recovery, with the leach time reduced from 2 h to 15 min using 50% (aq.) diluted aqua regia to give potentially a more biocompatible leachate. Desulfovibrio desulfuricans reduces soluble platinum group metals to cell‐bound insoluble base metals (e.g. Pd(II) ? Pd(0)). For use, biofilm was immobilised on a Pd‐23% Ag solid alloy membrane which delivered H” to the cells via an electrochemical chamber at the back‐side. The biomass‐coated Pd‐Ag alloy electrode was used in a flow‐through reactor for recovery of Pd, Pt and Rh from aqua regia leachates (pH 2.5) of spent automotive catalysts with up to 90% efficiency at a flow residence time of 15 minutes. Free cells did not reduce platinum group metals from the leachates but the electrobioreactor did so using biofilm‐cells preloaded with Pd(0). Reactors lacking biomass or reactors with heat‐killed biofilm removed less platinum group metals, via electrochemically‐synthesised H’ reductant alone. The use of an active biofilm layer in a flow‐through electrobioreactor provides a simple, clean and rapid potential recycle technology.

Reduction of Cr(VI) and Bioaccumulation of Chromium by Gram Positive and Gram Negative Microorganisms not Previously Exposed to CR-Stress

Resistance to Cr(VI) is usually associated with its cellular exclusion, precluding enrichment techniques for the isolation of organisms accumulating Cr(VI) via bioreduction to insoluble Cr(III). A technique was developed to screen for potential Cr(VI) reduction in approx. 2000 isolates from a coastal environment, based on the non-specific reduction of selenite and tellurite to Se0 and Te0, and reduction of tetrazolium blue to insoluble blue formazan. The most promising strains were further screened in liquid culture, giving three, which were identified by 16S rRNA sequence analysis as Bacillus pumilus, Exiguobacterium aurantiacum and Pseudomonas synxantha, all of which reduced 100 µM Cr(VI) anaerobically, without growth. The respective removal of Cr(VI) was 90% and 80% by B. pumilus and E. aurantiacum after 48 h and 80% and by P. synxantha after 192 h. With the Gram positive strains Cr(VI) promoted loss of flagella and, in the case of B. pumilus, lysis of some cells, but Cr was deposited as an exocellular precipitate which was identified as containing Cr and P using energy dispersive X-ray microanalysis (EDAX). This prompted the testing of Citrobacter sp. N14 (subsequently re-assigned by 16S rRNA sequence analysis and biochemical studies as a strain of Serratia) which bioprecipitates metal cation phosphates via enzymatically-liberated phosphate. This strain reduced Cr(VI) at a rate comparable to that of P. synxantha but Cr(III) was not bioprecipitated where La(III) was removed as LaPO4, even though a similar amount of phosphate was produced in the presence of Cr(III). Since B. pumilus removed most of the Cr(VI), with the formation of cell-bound CrPO4 implicated, this suggests that this strain could have future bioprocess potential.

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.

Bioremediation of uranium-bearing wastewater: Biochemical and chemical factors influencing bioprocess application

A biotechnological process for the removal of heavy metals from aqueous solution utilizes enzymatically liberated phosphate ligand which precipitates with heavy metals (M) as cell-bound MHPO(4). The enzyme, a phosphatase, obeys Michaelis-Menten kinetics in resting and immobilized cells; an integrated form of the Michaelis-Menten equation was used to calculate the apparent K(m) (K(m app.)) as operating in immobilized cells in flow-through columns by a ratio method based on the use of two enzyme loadings (E(o1), E(o2)) or two input substrate concentrations (S(o1), S(o2)). The calculated K(m app.) (4.08 mM) was substituted into an equation to describe the removal of metals by immobilized cells. In operation the activity of the bioreactor was in accordance with that predicted mathematically, within 10%. The initial tests were done at neutral pH, whereas the pH of industrial wastewaters is often low; an increase in the K(m app.) at low pH was found in previous studies. Immobilized cells were challenged with acidic mine drainage wastewaters, where the limiting factors were chemical and not biochemical. Bioreactors initially lost activity in this water, but recovered to remove uranyl ion with more than 70% efficiency under steady-state conditions in the presence of competing cations and anions. Possible reasons for the bioreactor recovery are chemical crystallization factors. (c) 1997 John Wiley & Sons, Inc.