Removal of cadmium from polluted water by immobilized algae

277 Pages·2014·8.76 MB·English

Durham E-Theses
Removal of cadmium from polluted water by
immobilized algae
Jackson, Paul P.
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Jackson, Paul P. (1990) Removal of cadmium from polluted water by immobilized algae, Durham theses,
Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/6184/
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Removal of cadmium from polluted water by
immobilized algae
by
Paul P. Jackson B. Sc. (Hons.), York
The copyright of this thesis rests with the author.
No quotation from it should be published without
his prior written consent and information derived
from it should be acknowledged.
A thesis submitted for the degree of Doctor of Philosophy
in the University of Durham
Department of Biological Sciences
September 1990
This thesis is entirely the result of my own work. It has not been accepted for any other degree and
is not being submitted for any other degree.
2
ABSTRACT
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. Synechococcus D562 cells
subcultured in Cd accumulated the most metal; little was bound to the cell wall. A continuous culture of steady
state Synechococcus D562 cells tolerated a lower maximum concentration of metal (3.4 mg 1-l 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 Synechococcus 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 Synechococcus 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 Synechococcus 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 radio labelled oligonucleotide probe based on part of
the nucleotide sequence of a metallothionein from Synechococcus PCC 6301 did not bind to a genomic and plasmid
blot of Synechococcus D562 DNA.
The putative Cd-binding peptides ((yEC)nG'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 (yEC)nG's from this
strain is comparable with that of other species. Reversed-phase HPLC of the pep tides 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 I PI Cd precipitates in
Mougeotia D536, the loss of poly glucoside granules from Calothrix D 184 together with a relaxation of its thylakoid
packing and a lack ofplastoglobuli 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 Synechococcus 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.
3
ABBREVIATIONS
oc
degrees Celsius
t tonne
g gramme
mg milligramme
J.lg microgramme
Mr molecular weight
kD kilo Dalton
kb kilob ase
I litre
ml millilitre
v/v volume for volume
w/v weight for volume
ppm parts per million
em centimetre
J.lm micrometre
om nanometre
yr year
day
d
h hour
min minute
s second
M molar
mM millimolar
J.1M micromolar
Cd cadmium
p phosphorus
pp polyphosphate
MT metallothionein
(yEC)nG poly(gamma-glutamylcysteinyl)glycine
PC phytochelatin
APA alkaline phosphatase activity
PME phosphomonoesterase
PDE phosphodiesterase
PAR photosynthetically active radiation
X mean
sem standard error of the mean
log logarithmic
BSA bovine serum albumin
BSO buthionine sulphoximine
DMGA dimethyl glutaric acid
EDTA ethylenediaminetetra-acetic acid (disodium salt)
HEP ES N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid
NAG N-acetyl glucosamine
NAM N-acetyl muramic acid
NTA nitrilotriacetic acid
PIPES piperazine-N ,N'-bis (2-ethane-sulphonic acid)
SDS sodium dodecyl sulphate(= sodium lauryl sulphate)
TRIS tris (hydroxymethyl) methylamine
EDXMA energy dispersive X-ray microanalysis
EM electron microscopy
FAAS flame atomic absorption spectrophotometry
HPLC high performance liquid chromatography
TEM transmission electron microscopy
SEM scanning electron microscopy
SPM scanning proton microanalysis
EC European Community
4
ACKNOWLEDGEMENTS
Research facilities in the Department of Biological Sciences were made available by Professor D. Boulter and
Northern Environmental Consultants Ltd provided the fmance for this venture. Drs B. A. Whitton and P. J. Say
supervised the project, proffering advice culled from many years of work in the field of heavy-metal accumulation.
My sincere thanks go to Mr J. W. Simon and Mr J. Gilroy for countless hours of useful discussion, providing a
whole spectrum of apparatus and readily passing on valuable experience. Notable laboratory colleagues who
maintained an effective working atmosphere, include Drs G. Davies, S. L. J. Grainger, Mr J. Hutchinson, and Dr R.
Islam. Dr G. R. W. Hawley's contribution is of particular note in the humour, encouragement and sense of
perspective provided at timely interludes throughout the period of study.
To Dr N. Harris, Mr A. Peat and Mr N. Powell, I wish to extend my appreciation of their advice on electron
microscopy, whilst Dr R. W. Banks and Mrs A. C. Richardson were always willing to indulge in extensive EDXMA
discussion without notice. Professor A. J. Brook kindly organised the submission of samples to the Oxford
Scanning Proton Microanalysis Unit, where scans of cyanobacteria and algae were performed by Dr G. W. Grime.
Productive advice on the subject of ion exchange resin was supplied by Dr D. Parker. In addition, Dr N. J.
Robinson, Mr W. Lindsay and Mr A. Gupta provided practical assistance and erudite discussion on MT genes and
Cd binding peptides.
Help with DNA manipulation was advanced by Drs G. Williams and C. Scutt. The subculture and
determination of the concentration of Cd accumulated by strains isolated from the field was executed by Mr J.
Hutchinson, whilst FAAS problem solving counsel, was readily given by Mr R. Coult and Mrs J. Dostal.
Photographs of apparatus were promptly taken by Mr P. Sidney who also parted with constructive experience on
darkroom matters.
This work would never have evolved without the continuous fmancial and moral support from my father,
mother and sister, to them I owe this investment in time and education.
5
CONTENTS
page
ABSTRACT 2
ABBREVIATIONS 3
ACKNOWLEDGEMENTS 4
CONTENTS 5
LIST OFTA BLES 10
LIST OF FIGURES 12
1. INTRODUCTION 16
1.1 Gener.Uintroduction 16
1.11 Environmental sources 17
1.12 Industrial sources 17
1.13 Environmental pollution 18
1.14 Implications for human health 18
1.2 Metal chemistry 22
1.21 Electron distribution 22
1.22 Influence of pH 23
1.23 Redox potential 23
1.24 Metal I ligand interaction 24
1.25 Metal competition 25
1.26 Media ligands 25
1.3 Uptak:eofCd 26
1.31 Adsorption of Cd by bacterial cell-walls 26
1.32 Adsorption of Cd by algal cell-walls 27
1.33 Precipitation of Cd by algal cell-walls 29
1.34 Passive uptake 29
1.35 Cd accumulation 30
1.4 Cdremoval 32
1.41 Chemical removal 32
1.42 Algal removal 32
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1.5 Immobilized cells 32
1.51 Matrices and applications 33
1.52 Metal removal 36
1.53 Gel stabilization 38
1.54 Diffusion coefficients for immobilization matrices 39
1.6 Cd localisation 40
1.61 Energy dispersive X-ray microanalysis 40
1.62 Scanning proton microanalysis 43
1. 7 Production of spheroplasts 43
1.8 Tolerance mechanisms 44
1.81 Genomic and plasmid encoded resistance 44
1.82 Cd-binding peptides 46
1.83 Cd-binding proteins 48
1.84 Cd-binding polysaccharides 50
1.85 Polyphosphate accumulation 51
1.9 Cell disorders 51
1.91 Ultrastructural damage 51
1.92 Biochemical impairment 51
1.93 Cd I DNA interactions 52
AIMS 55
2. MATERIALS AND METHODS 56
2.1 Algal origins 56
2.2 Media and culture techniques 56
2.21 Algal growth media 56
2.22 Heavy-metal stocks 57
2.23 Bacterial test-media 57
2.24 Changes in the volume of media 65
2.25 Glassware and plastic preparation 65
2.26 Sterile technique 65
2.27 Algal subculture and experimental conditions 66
2.28 Cell harvesting 67
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2.3 General procedures 67
2.31 Mass determination 67
2.32 Cell dry weight 67
2.33 Cell density 68
2.34 pH 68
2.35 Light 68
2.36 Total protein assay 68
2.37 Flame atomic absorption spectrophotometry 69
2.38 Computing 70
2.4 Strain selection and Cd uptake 70
2.41 Isolating Cd-accumulating algae from the environment 70
2.42 Small-scale screening 70
2.43 Procedures employed to render Synechococcus D562 axenic 71
2.44 Cd complexed by the medium 75
2.45 EDTA washing 75
2.46 Dead cell uptake 76
2.47 Physiological response to dilute medium 76
2.48 Batch and continuous culture 76
2.5 Immobilized cells 80
2.51 Immobilized-cell matrices 80
2.52 Optimising calcium-alginate bead formation 81
2.53 Production of micro beads 81
2.54 Fluidised-bed reactor 81
2.55 Toxicity of sodium alginate 81
2.56 Influence of aeration 83
2.57 Free versus immobilized cells 83
2.6 Microscopy 88
2.61 Light microscopy 88
2.62 Energy dispersive X-ray microanalysis 88
2.63 Determination of machine efficiency-factors 89
2.64 Scanning proton microanalysis 89
2.7 Tolerance mechanisms 90
2.71 Production of Synechococcus D562 spheroplasts 90
2.72 Plasmid extraction 90
2.73 Genomic extraction 91
2.74 DNA restriction 91
2.75 Southern blotting 92
2.76 Gel permeation HPLC of Cd-binding peptides 92
2.77 Reversed-phase HPLC of Cd-binding peptides 93
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2.8 Ultrastructural, enzyme and protein damage 94
2.81 Electron microscopy 94
2.82 Grid coating 95
2.83 Electron microscopy of immobilized cells 95
2.84 Alkaline phosphatase activity 96
2.85 SDS polyacrylamide gel electrophoresis 97
RESULTS 101
3. Strain selection 101
3.1 Introduction 101
3 .2 Culture collection strains 101
3.3 Strains recently isolated from the field 102
4. Accumulation of Cd 105
4.1 Introduction 105
4.2 Concentration of divalent Cd in the medium 105
4.3 Small-scale screening 107
4.4 Physiological response to dilute medium 108
4.5 Cd adsorbed by the cell wall 108
4.6 Batch and continuous culture 112
5. Immobilized cells 115
5.1 Introduction 115
5.2 Accumulation of Cd by the matrix 115
5.3 Accumulation of Cd by immobilized cells 115
6. Microscopy and Cd localisation 126
6.1 Introduction 126
6.2 Light microscopy 126
6.3 Lysozyme hydrolysis and ruthenium red staining 126
6.4 Immobilized cells 127
6.5 Energy dispersive X-ray microanalysis 136
6.6 Scanning proton microanalysis 168
7. Tolerance mechanisms 179
7.1 Introduction 179
7.2 Production of Synechococcus D562 spheroplasts 179
7.3 DNA extraction and restriction 183
7.4 Cd-binding peptides 186