A feasibility study was planned to determine the efficiency of immobilized algal cells growing in a packed bed for removing Cd from commercial effluents. To select appropriate material for an immobilized cell system, twenty-five strains of algae isolated from heavy-metal contaminated environments of known water chemistry were tested for their ability to accumulate Cd. Before accumulation experiments were initiated, ion exchange resin was employed to demonstrate that EDTA in the medium did not complex Cd to a significant degree. Svnechococcus D562 cells subcultured in Cd accumulated the most metal; little was bound to the cell wall. A continuous culture of steady- state Svnechococcus D562 cells tolerated a lower maximum concentration of metal (3.4 mg 1(^-1) Cd) than batch- cultured cells (5 mg 1(^-1) Cd), indicating that metabolic status influences the toxicity of Cd. When flasks of calcium-alginate beads were challenged with Cd, up to 60 % of the added metal was bound within 16 h; however, further incubation did not reduce the pollutant concentration. Two axenic strains which accumulated the metal to a high concentration were then immobilized and tested for their capacity to remove Cd from the circulating medium. A packed-bed reactor containing Mougeotia D536 cells proved more effective at metal removal than Svnechococcus D562, but both species grew to a lower cell density at the effluent end of the column. The medium was then aerated to overcome such growth-limiting conditions, but this treatment inhibited Cd accumulation. Column-immobilized cells reduced Cd levels more effectively than inoculated, alginate beads in stationary flasks or free cells. Energy dispersive X-ray microanalysis located Cd only in particular Svnechococcus D562 polyphosphate bodies (those with a high Ca to K ratio); peaks for Zn, Pb, Fe, Mn and Ba were also detected in algae isolated from the field. Scanning proton microanalysis provided information on the distribution of macro- and micro-elements throughout the two strains of cyanobacteria and two strains of algae selected from the Durham Culture Collection and demonstrated the presence of Cd in Klebsormidium rivulare D537.Detergent-sensitive spheroplasts of Svnechococcus D562 were produced by lysozyme and protease digestion, but were not viable for growth. To observe the extracellular mucilage of this strain by EM, lysozyme digestion proved imperative for effective ruthenium red staining to convert the material into an electron opaque material. From cultures of Svnechococcus D562 grown with or without Cd a 14 kD plasmid was isolated, which contained two Eco RI, two Bam HI and five Hind III restriction sites. A radiolabelled oligonucleotide probe based on part of the nucleotide sequence of a metallothionein from Svnechococcus FCC 6301 did not bind to a genomic and plasmid blot of Svnechococcus D562 DNA. The putative Cd-binding peptides ((yEC)(_n)G's) that were discovered only bound significant quantities of the metal when cells were exposed to 6.17 mg 1(^-1) Cd for 2 days at the end of their log-growth phase. Indigenous peptides failed to bind substantial amounts of the metal and the presence of Cd throughout growth did not influence the quantity of chelated Cd, except for Mougeotia D536. The pH of half displacement for (yEQjp's from this strain is comparable with that of other species. Reversed-phase HPLC of the peptides from Mougeotia D536 generated a thiol profile similar to that recorded for the Cd-binding peptides of Datura innoxia. The Cd-induced ultrastructural distortions that were recorded include potential Ca / P / Cd precipitates in Mougeotia D536, the loss of polyglucoside granules from Calothrix D184 together with a relaxation of its thylakoid packing and a lack of plastoglobuli in Cd-exposed Klebsormidium D537. The space between an immobilized cell and the matrix either represents shrinkage of the matrix during dehydration or mucilage which does not bind electron dense stains. Release of alkaline phosphatase into the medium by Svnechococcus D562, provided suitable material to study the inhibitory effects of Cd upon P hydrolysis. Ultrafiltration membranes proved effective as initial step towards enzyme purification and for the determination of activity under sub-optimal pH conditions. At pH 7.0, the activity of an enzyme concentrate was inhibited when 1 and 10 mg 1(^-1) Cd were added to the assay medium, but the presence of this metal in the growth medium did not reduce activity. One-dimensional SDS PAGE revealed only one protein difference between strains grown with or without Cd; a reduction in the staining intensity of a 17 kD band of Calothrix D184.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:316382 |
| Date | January 1990 |
| Creators | Jackson, Paul P. |
| Publisher | Durham University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.dur.ac.uk/6184/ |
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