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Durham E-Theses
Physiological studies on heavy metals and blue-green
algae
Shehata, F. H. A.
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Shehata, F. H. A. (1981) Physiological studies on heavy metals and blue-green algae, Durham theses,Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/7508/
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Academic Support O�ce, Durham University, University O�ce, Old Elvet, Durham DH1 3HPe-mail: [email protected] Tel: +44 0191 334 6107
http://etheses.dur.ac.uk
PHYSIOLOGICAL STUDIES ON HEAVY METALS AND BLUE-GREEN ALGAE
by
F. H. A. SHEHATA
(B.Sc. Riyadh - Saudi Arabia)
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 f o r the degree of Doctor of Philosophy i n the Un i v e r s i t y of Durham
Department of Botany, January 1981 •-'>y.
This t h e s i s i s e n t i r e l y the r e s u l t of my own work. I t has not
been accepted f o r any other degree, and i s not being submitted
f o r any other degree.
F. H. A. SHEHATA
V
3.
TABLE OF CONTENTS
page
ABSTRACT 7
ACKNOWLEDGMENTS 8
ABBREVIATIONS 10
LIST OF TABLES H
LIST OF FIGURES 17
CHAPTER 1 INTRODUCTION 2 1
1.1 Occurrence of blue-green algae i n 21 environments r i c h i n heavy metals
1.2 Laboratory tolerance and t o x i c i t y 2 2
1.21 I s zinc e s s e n t i a l f o r growth? 2 5
1.22 E f f e c t o f zinc on n i t r o g e n f i x a t i o n 26 1.3 Adaptation 2 7
1.31 A n t i b i o t i c s 28 1.32 Morphological mutants 29 1.33 Metals 29
1.4 Factors i n f l u e n c i n g t o x i c i t y of metals 30 1.5 Accumulation of metals 34 1.6 Aims 35
CHAPTER 2 MATERIALS AND METHODS 37
2.1 Culture techniques.... 37 2.11 Cleaning of glassware 37 2.12 Culture vessels 37 2.13 S t e r i l i z a t i o n 37
2.2 Media 38 2.21 Mineral n u t r i e n t s 38 2.22 B u f f e r 42 2.23 Chelating agent 44 2.24 M a t e r i a l s used f o r t o x i c i t y t e s t s 44
2.3 Subcu l t u r i n g 47 2.31 L i q u i d 47 2.32 S o l i d 48 2.33 Incubation 48
2.4 A l g a l c u l t u r e s 49 2.41 O r i g i n s 49 2.42 I s o l a t i o n and p u r i f i c a t i o n 51
2.421 Physical 2.422 A n t i b i o t i c s 52 2.423 Tests of p u r i t y o f the c u l t u r e s . . , . — 52
4.
page
2.5 Preparation of alga f o r assay 52 2.51 Estimation o f growth 52
2.511 U n i t counts and es t i m a t i o n 52 of c e l l l e n g t h
2.512 E x t r a c t i o n s and e s t i m a t i o n 53 of pigments
2.513 Dry weight 56 2.514 T u r b i d i t y 58
2.52 Techniques used f o r comparison 58 of t o x i c i t i e s
2.6 Production of r e s i s t a n t Anacystis s t r a i n 62 2.61 NTG 62 2.62 S e r i a l subculture 63
2.7 Study o f environmental f a c t o r s 63 2.71 Factors i n f l u e n c i n g t o x i c i t y 63 2.72 Inoculum size 64 2.73 Factors i n f l u e n c i n g zinc s o l u b i l i t y 64
2.8 Experimental procedure f o r accumulation studies 65 2.81 EDTA washing 65 2.82 Acid d i g e s t i o n 71
2.9 Special techniques 71 2.91 F i l t r a t i o n 71 2.92 Atomic absorption spectrophotometry 72 2.93 Acetylene reduction assay 72
CHAPTER 3 PRODUCTION OF RESISTANT STRAINS 75 OF ANACYSTIS NIDULANS
3.1 I n t r o d u c t i o n 75 3.2 NTG 75 3.3 S e r i a l s u b c u l t u r i n g 75
3.31 Metals 75 3.32 A n t i b i o t i c s 86
3. 4 Morphology 86
CHAPTER 4 TOLERANCE AND TOXICITY STUDIES 93 ON ANACYSTIS NIDULANS STRAINS
4.1 Metals 93 4.11 Cobalt 93 4.12 N i c k e l 93 4.13 Copper 106 4.14 Zinc 106 4.15 Cadmium 106 4.16 Mercury and lead 106
4.2 A n t i b i o t i c s 130 4.3 NTG 130 4.4 Pigment composition..... 130
4.5 Zn requirement f o r growth I 3 3
5.
page CHAPTER 5 EXPERIMENTAL FACTORS AFFECTING TOXICITY %. 136
OF METALS TO ANACYSTIS NIDULANS 5.1 I n t r o d u c t i o n 136 5.2 Influence o f complementary ca t i o n s and anions 136 5.3 Infl u e n c e o f major cations 139 5.4 Influence of major anions 151 5.5 Influence of organic compounds 158 5.6 Influence of other heavy metals 166 5.7 Influence of pH 166 5.8 Infl u e n c e of inoculum size 174
CHAPTER 6 ACCUMULATION OF ZINC BY ANACYSTIS NIDULANS 1 7 7
6.1 I n t r o d u c t i o n 177 6.2 Infl u e n c e of c u l t u r e d e n s i t y on removal 179
of Zn from medium 6.3 Infl u e n c e o f environmental Zn concentrations 179
on Zn accumulation by Anacystis nidulans 6.4 Release of Zn from Anacystis by washing 188
w i t h EDTA
CHAPTER 7 STUDIES ON ZINC RESISTANCE OF STRAINS 191 ISOLATED FROM ZINC POLLUTED SITES
7.1 I n t r o d u c t i o n 191 7.2 O r i g i n s , i s o l a t i o n and c u l t u r e 191 7.3 T o x i c i t y o f Zn and Cd under standard 191
con d i t i o n s 7.4 Factors i n f l u e n c i n g t o x i c i t y 196 7.5 Nitrogen f i x a t i o n 204
CHAPTER 8 DISCUSSION 206
8.1 Production of r e s i s t a n t s t r a i n s . 206 8.2 Laboratory tolerance and t o x i c i t y 210 8.3 Environmental f a c t o r s i n f l u e n c i n g metal- 214
t o x i c i t y t o Anacystis nidulans s t r a i n s 8.31 Organic compounds i n the medium 217
8.4 Accumulation o f Zn by Anacystis nidulans. °. 218 8.5 Tolerance and adaptation shown by f i e l d m a t e r i a l . . . . 219
8.51 Nitrogen f i x a t i o n 221 8.6 Concluding remarks 221
page
SUMMARY 223
REFERENCES • 227
APPENDIX Contents l i s t A3.0 240 A4.0 2 4 1
Abstract
Mutants o f Anacystis nidulans t o l e r a n t t o high l e v e l s of Co, N i ,
Cu, Zn and Cd were obtained by repeated s u b c u l t u r i n g a t s t r o n g l y
i n h i b i t o r y l e v e l s o f metal. For instance, the l e v e l of Zn a t which
strong i n h i b i t i o n occurred was r a i s e d from 1.45 t o 16.5 mg 1 * Zn a f t e r
75 subcultures. I s o l a t e s r e s i s t a n t t o 5.0 mg 1 * Zn and 12.0 mg 1 * Zn
maintained t h e i r resistance f o r a t l e a s t 72 c e l l generations i n the
absence o f Zn, though there was subsequently an increased lag du r i n g the
f i r s t subculture back t o hi g h Zn l e v e l s . This and p l a t i n g experiments
i n d i c a t e t h a t the s t r a i n s are mutants. Assays of cross-resistance o f
each of the f i v e types of mutant were made t o the other four metals. I n
most cases changes i n cross-resistance were only s l i g h t , w i t h about
equal numbers of examples of increased and decreased r e s i s t a n c e . Examples
of marked changes were increased Co-resistance of a Cd-tolerant s t r a i n
and decreased Cd-resistance of a N i - t o l e r a n t s t r a i n . The environmental
f a c t o r s i n f l u e n c i n g t o x i c i t y were i n v e s t i g a t e d f o r Cu, Zn and Cd.
Increases i n Ca, Mn, Fe and P reduced Zn t o x i c i t y t o both w i l d - t y p e and
Zn- t o l e r a n t s t r a i n s , but the two d i f f e r e d i n t h e i r response t o pH.
E f f e c t s on morphology were evident a t high metal l e v e l s w i t h a l l s t r a i n s .
I n most cases increased l e v e l s of metal l e d t o the formation of f i l a m e n t s ,
but w i t h Cu subspherical s t r u c t u r e s sometimes made up most o f the
population. Uptake from media enriched 0.1 and 1.0 mg 1 * Zn was s i m i l a r
i n both w i l d - t y p e and Zn- t l 2 . 0 , whether judged by t o t a l Zn accumulated
or by t h a t remaining a f t e r EDTA washes.
I s o l a t e s from high Zn s i t e s were found i n general t o t o l e r a t e
considerably higher l e v e l s of Zn than l a b o r a t o r y research s t r a i n s
(presumably i s o l a t e d from environments not enriched w i t h Zn). A comparison
of the i n f l u e n c e of Zn on n i t r o g e n f i x a t i o n by a s t r a i n from low zinc s i t e
(Anabaena cylindrica) and one from a high Zn s i t e (Calothrix D184) showed
only a s l i g h t d i f f e r e n c e when Zn was f i r s t added, but a pronounced e f f e c t
a f t e r 24 h.
ACKNOWLEDGMENTS
I t i s a pleasure to acknowledge everyone who has helped me du r i n g
t h i s work. 1 am extremely g r a t e f u l t o Dr B. A. Whitton f o r h i s
supe r v i s i o n of my work and f o r h i s k i n d advice and c r i t i c i s m . I t was
h i s encouragement and i n s p i r a t i o n more than anything else t h a t enabled
me t o f u l f i l t h i s work. I wish t o thank the M i n i s t r y of Defence i n
Saudi Arabia f o r f i n a n c i a l assistance throughout the course o f the
p r o j e c t . Thanks are due t o Prince Sultan Ben-Abd-Alaziz who gave h i s
permission f o r t h a t f i n a n c i a l support. In p a r t i c u l a r I should l i k e t o
thank my parents and my wi f e f o r t h e i r help and constant encouragement
i n many ways.
Thanks are due to my colleagues i n Durham, Dr M. P o t t s , Dr C. S i n c l a i r
Dr A. Donaldson and Dr D. Livingstone who a l l gave generous help w i t h gas
chromatography f o r the acetylene r e d u c t i o n assay technique.
Dr D. Livingstone also gave advice and h e l p f u l discussion on c h l o r o p h y l l a
e x t r a c t i o n . Dr P. J. Say, Dr J. P. C. Harding, Dr N. T. H. Holmes,
Mr B. M. Diaz, Mr J, D. Wehr, Mr G. Patterson, Mr C. Rajendran,
Mr I . G. Burrows, Mr Abdullah Al-Mousawi and Mr Abdul B. H. Z o l a l y gave
many i n t e r e s t i n g discussions on heavy metals. Special thanks are due
to Mr J. W. Simon f o r h i s h e l p f u l assistance i n s o l v i n g many t e c h n i c a l
problems and f o r valuable advice. I am g r a t e f u l t o Dr A. Yarwood and
Mr G. H. Banbury f o r a l l o w i n g me t o attend t h e i r l e c t u r e s . Mr T. B r e t t and
Mr B. Coult advised me on the use of the atomic absorption spectrophotomete
I am also g r a t e f u l t o Dr N. H a r r i s , Mr J. G i l r o y , Mr G. Bainbridge,
Mrs G. A. Walker, Mr A. Jamieson, Mr P. Masters, Mr J. H. W h i t t l e f o r
various types of t e c h n i c a l advice; t o the s t a f f of Durham U n i v e r s i t y
Science L i b r a r y f o r t r a c i n g my obscure references and f o r p r o v i d i n g a
favourable working environment d u r i n g w r i t i n g up the work.
I wish t o thank Prof. D. Boulter f o r the p r o v i s i o n of research
f a c i l i t i e s i n the Botany Department. F i n a l l y very many thanks are
o f f e r e d t o Mrs H. Winn who took on very e f f i c i e n t l y the arduous task
of t y p i n g t h i s t h e s i s .
10.
ABBREVIATIONS
C degrees Celcius d day EDTA eth y l e n e d i a B i n e t e t r a - a c e t i c acid (disodium s a l t ) g gramme h hour 1 l i t r e M molar mg milligramme min minute ml m i l l i l i t r e )j.g microgramme um micrometer n number of measurements nm nanometer s.d. standard d e v i a t i o n u.v. u l t r a - v i o l e t x mean value f i l t r a b l e capable of passing through f i l t e r n o n - f i l t r a b l e incapable of passing through f i l t e r NTG N-methyl-N'-nitro-N-nitrosoguanidine T.I.C. Tolerance Index Concentration (seep. 62) HEPES N-2-hydroxyethylpiperazine-N 1-2-ethanesulphonic a c i d P-A.R. Pho t o s y n t h e t i c ' a c t i v e r a d i a t i o n between 400 - 700 nm lux Photometric measurements of il l u m i n a n c e
(one lumen m ^) Zn-t5.0 Anacystis nidulans t o l e r a n t t o 5.0 mg 1 * Zn Zn-tl2.0 " " " "12.0 mg l " " 1 Zn Co-tl.8 " " " "1.8 mg l " 1 Co
-1 N i - t l . 0 " " " " 1.0 mg 1 Ni Cu-t0.5 " " " "0.5 mg 1 _ 1 Cu Cd-t2.0 " " " "2.0 mg l " 1 Cd
11.
LIST OF TABLES
Table Page
2.1 Composition of media (mg 1 1 of s a l t s ) . 39
2.2 Composition of media (mg 1 " of elements) 40
2.3 Amount of zinc as i m p u r i t i e s i n the stock (mg 1 ^) 41
2.4 Levels of EDTA and HEPES used f o r various experiments. ... 43
2.5 Influence of EDTA on Zn s o l u b i l i t y i n ACM 45
medium (pH 7.0; + HEPES: Table 2.2).
2.6 Factors i n f l u e n c i n g Zn t o x i c i t y 46
2.7 Sources of c u l t u r e s 50
2.8 Comparison between methods used f o r phycocyanin 55
e x t r a c t i o n from Anacystis nidulans.
2.9 E f f e c t of inc u b a t i o n medium and time on the release 57
and subsequent degradation of phycocyanin from
Anacystis nidulans.
2.10a Influence of Mn as MnCl^ on t u r b i d i t y o f ACM medium 59
2.10b Influence of Ca as CaCl on t u r b i d i t y of ACM medium 59
2.11 Influence of pH on Zn s o l u b i l i t y i n ACM medium 66
(10 mg I " 1 EDTA + HEPES).
2.12 Influence of Ca on Zn s o l u b i l i t y i n ACM medium 67
(pH 7.0 + HEPES + 0.5 mg 1 _ 1 EDTA).
2.13 Influence of PO -P on Zn s o l u b i l i t y i n ACM medium 68 4
-1
(pH 7.0 + HEPES + 1 0 mg 1 EDTA).
2.14 Inf l u e n c e of aut o c l a v i n g and non-autoclaving on 69
Zn s o l u b i l i t y i n ACM medium (pH 7.0 + HEPES + 10 mg 1 _ 1 EDTA).
2.15 In f l u e n c e of EDTA (pH 5.4) on removal of Zn from 70
Anacystis nidulans.
12.
Table page
3.1 Resistance obtained (so f a r ) on repeated subculture 76
of Anacystis nidulans t o p r o g r e s s i v e l y higher metal
concentrations.
3.2 Tolerance of mutants used f o r experiments t o the 80
metals used i n t h e i r i s o l a t i o n .
3.3 Influence of p r i o r growth c o n d i t i o n and Zn concen- 85
t r a t i o n s on growth, lag shown by Zn-t5.0 and Zn-tl2.0.
4.1 Influence of growth stage on colony-forming a b i l i t y of . .. 126
wil d - t y p e Anacystis on 1% agar p l a t e s .
4.2 Influence of Zn on growth of w i l d - t y p e Anacystis nidulans 127
on 1% agar p l a t e s .
4.3 Changes (greater than a f a c t o r of two) i n resistance .... 129
t o other metals of m e t a l - t o l e r a n t s t r a i n s of Table 8.1.
4.4 Comparison of response of w i l d - t y p e and Z n - t o l e r a n t ..... 131
s t r a i n s t o p e n i c i l l i n , p o l y m i x i n and streptomycin.
4.5 Comparison of c h l a content per u n i t length of alga 131
i n Zn t r e a t e d c u l t u r e s .
5.1 Influence of environmental f a c t o r s on t o x i c i t y of 137
Cu, Zn and Cd t o various s t r a i n s of Anacystis nidulans
Results based on t u r b i d i m e t r i c measurements.
5.2 Key environmental f a c t o r s changing t o x i c i t y o f metals .... 138
t e s t e d , based on e n t r i e s i n Table 5.1 d i f f e r i n g by
at l e a s t two scores.
5.3 Inf l u e n c e o f Na on Zn t o x i c i t y t o w i l d - t y p e Anacystis.. 140
5.4 Influence of CI on Zn t o x i c i t y t o w i l d - t y p e Anacystis. 140
5.5 Inf l u e n c e of SO -S on Cd t o x i c i t y t o w i l d - t y p e Anacystis 141
13.
Table page
5.6 Influence of Mg on Zn t o x i c i t y t o wi l d - t y p e Anacystisi ... 141 (high Mg le e l s ) .
5.7 Influence of Mg on Zn t o x i c i t y t o Zn-t5.0 Anacystis 142
5.8 Influence of Mg on Zn t o x i c i t y t o Zn-tl2.0 Anacystis 142
5.9 Influence of Mg on Zn t o x i c i t y t o wi l d - t y p e Anacystis; ... 143
(low Mg l e v e l s ) .
5.10 Influence of Mg on Cu t o x i c i t y t o wi l d - t y p e Anacystis 143
5.11 Influence of Mg on Cu t o x i c i t y to Cu-t0.5 Anacystis 145
5.12 Influence of Mg on Cd t o x i c i t y t o w i l d - t y p e Anacystis 145
5.13 Influence of Ca on Cu t o x i c i t y t o wi l d - t y p e Anacystis 148
5.14 Influence of Ca on Cu t o x i c i t y t o Cu-t0.5 Anacystis 148
5.15 Influence of Mn on Zn t o x i c i t y t o wild- t y p e Anacystis 149
5.16 Influence of Mn on Cd t o x i c i t y t o wi l d - t y p e Anacystis 149
5.17 Influence of Fe on Zn t o x i c i t y t o w i l d - t y p e Anacystis 152
5.18 Inf l u e n c e of Fe on Cd t o x i c i t y t o wi l d - t y p e Anacystis 152
5.19a Influence of PO -P on Zn t o x i c i t y t o w i l d - t y p e 155
Anacystis; starved inoculum.
5.19b Influence of PO -P on Zn t o x i c i t y t o w i l d - t y p e 155
Anacystis; r i c h inoculum.
5.20 Influence of PO -P on Zn t o x i c i t y t o w i l d - t y p e 156
Anacystis; (PO^-P was introduced a f t e r a l g a l
i n o c u l a t i o n and contact w i t h Zn f o r 10 min.).
5.21 Influence of PO -P on Cu t o x i c i t y t o w i l d - t y p e 156
Anacystis.
5.22 Inf l u e n c e of PO -P on Cu t o x i c i t y t o Cu-t0.5 157 4
Anacystis.
5.23a Inf l u e n c e of P04~P (as /^-glycerophosphate) on Zn 159
t o x i c i t y t o w i l d - t y p e Anacystis; u n i t s ml * was used
as a growth c r i t e r i o n .
14.
Table page
5.23b Influence of PO -P (as >§ -glycerophosphate) on Zn 160
t o x i c i t y t o w i l d - t y p e Anacystis; c h l a was used as
a growth c r i t e r i o n .
5.24a Influence of PO -P ( <\ -D glucose-l-phosphate) on 161
Zn t o x i c i t y t o w i l d - t y p e Anacystis; u n i t s ml * were
used as a growth c r i t e r i o n .
5.24b Influence of PO -P ( <K -D glucose-l-phosphate) on 162
Zn t o x i c i t y t o wi l d - t y p e Anacystis; c h l a was used
as a growth c r i t e r i o n .
5.25 Inf l u e n c e of NO -N on Cd t o x i c i t y t o wi l d - t y p e Anacystis.. 163
5.26 Influence of HEPES on the growth of w i l d - t y p e Anacystisy.. 167
at two l e v e l s o f Zn.
5.27 Influence of Ni on Zn t o x i c i t y t o w i l d - t y p e Anacystis 168
5.28 Influence of Cu on Zn t o x i c i t y t o w i l d - t y p e Anacystis 168
5.29 Influence of Hg on Zn t o x i c i t y t o w i l d - t y p e Anacystis 169
5.30 Influence of Pb on Zn t o x i c i t y to w i l d - t y p e Anacystis 169
5.31 In f l u e n c e of Ni on Cd t o x i c i t y t o w i l d - t y p e Anacystis 170
5.32 Inf l u e n c e of Cu on Cd t o x i c i t y t o w i l d - t y p e Anacystis..... 170
5.33 Inf l u e n c e of Hg on Cd t o x i c i t y t o w i l d - t y p e Anacystis 171
5.34 Influence of Pb on Cd t o x i c i t y t o w i l d - t y p e Anacystis 171
6.1 I n f l u e n c e of c u l t u r e d e n s i t y on removal of Zn from 180
c u l t u r e medium over a sho r t p e r i o d of time.
6.2 Accumulation of Zn during batch c u l t u r e growth of 181
w i l d - t y p e Anacystis. Results show values estimated when
c o n t r o l s (Nuclepore f i l t e r through which 0.1 mg 1 * Zn
had been passed) are subtracted from the measured values.
Accumulation of Zn during batch c u l t u r e growth of
wi l d - t y p e Anacystis. Results show values estimated
when c o n t r o l s (Nuclepore f i l t e r through which -1
1.0 mg 1 Zn had been passed) are subtracted from
measured values.
Accumulation of Zn during batch c u l t u r e growth of
Zn-tl2.0 Anacystis s t r a i n . Results show values
estimated when c o n t r o l s (Nuclepore f i l t e r through
which 0.1 mg 1 * Zn had been passed) are subtracted
from measured values.
Accumulation of Zn during batch c u l t u r e growth of ....
Zn-tl2.0 Anacystis s t r a i n . Results show values
estimated when c o n t r o l s (Nuclepore f i l t e r through
which 1.0 mg 1 * Zn had been passed) are subtracted
from measured values.
Accumulation of Zn du r i n g batch c u l t u r e s growth o f . . .
Zn-tl2.0 Anacystis s t r a i n . Results show values
estimated when c o n t r o l s (Nuclepore f i l t e r through
which 10.0 mg 1 * Zn had been passed) are subtracted
from measured values.
Comparison o f tolerance t o zinc o f organisms
i s o l a t e d from high zinc s i t e s w i t h tolerance
s t r a i n s taken from c u l t u r e c o l l e c t i o n and almost
c e r t a i n l y i s o l a t e d i n i t i a l l y from environment w i t h
low z i n c l e v e l s .
I n fluence o f Ca on Zn t o x i c i t y t o Phormidium autumnal
D476 as measured by c h l a.
16.
Table page
7.3 Inf l u e n c e of Ca on Cd t o x i c i t y to Phormidium autumnale ... 198 D476 as measured by c h l a.
7.4 Influence of PO -P on Zn t o x i c i t y t o Phormidium autumnale 199 4
D476 as measured by c h l a.
7.5 Inf l u e n c e of PO -P on Cd t o x i c i t y t o Phormidium autumnale 200 4
D476 as measured by c h l a.
7.6 Influence of pH on Zn t o x i c i t y t o Phormidium autumnale.... 201
D476 as measured by c h l a.
7.7 Inf l u e n c e of pH on Cd t o x i c i t y t o Phormidium autumnale 202
D476 as measured by c h l a.
7.8 Influence of Zn on Cd t o x i c i t y t o Phormidium autumnale ... 203
D476 as measured by c h l a.
7.9 Inf l u e n c e of Zn on acetylene r e d u c t i o n ( n i t r o g e n 205
f i x a t i o n ) by Z n - r e s i s t a n t and Zn-sensitive
heterocystous organisms.
8.1 Cross-resistance of s t r a i n s t o l e r a n t t o one 207
p a r t i c u l a r metal t o the other four metals. S t r a i n s
are named according t o s t r o n g l y i n h i b i t o r y l e v e l
of metal a t the time of i s o l a t i o n . 8.2 Tolerance t o Cu and Zn of soecies studied 211
by Rana and Kumar (1974).
8.3 Summaries of l i t e r a t u r e on Zn requirement by 213
various organisms.
17.
LIST CF FIGURES page
F i g u r e
2.1 R e l a t i o n s h i p between p o p u l a t i o n d e n s i t y and t u r b i d i t y 60
d u r i n g g r o w t h o f w i l d - t y p e and Z n - t o l e r a n t s t r a i n s o f
Anacystis nidulans.
3.1 I n c r e a s e i n r e s i s t a n c e t o Zn on r e p e a t e d s u b c u l t u r i n g 78
a t p r o g r e s s i v e l y h i g h e r Zn c o n c e n t r a t i o n .
3.2 I n f l u e n c e o f Co on g r o w t h o f C o - t l . 8 81
3.3 I n f l u e n c e o f N i on g r o w t h o f N i - t l . 0 81
3.4 I n f l u e n c e o f Cu on g r o w t h o f Cu-t0.5 82
3.5 I n f l u e n c e o f Zn on g r o w t h o f Zn-t5.0 82
3.6 I n f l u e n c e o f Zn on g r o w t h o f Z n - t l 2 . 0 83
3.7 I n f l u e n c e o f Cd on g r o w t h o f Cd-t2.0 83
3.8 I n f l u e n c e o f Zn on g r o w t h o f Z n - t 5 . 0 , grown f o r 72 c e l l ... 84
g e n e r a t i o n s i n b a s a l medium.
3.9 I n f l u e n c e o f Zn on g r o w t h o f Z n - t l 2 . 0 grown f o r 96 c e l l ... 84
g e n e r a t i o n s i n b a s a l medium.
3.10 Diagrams showing r e p r e s e n t a t i v e forms o f Anacystis 88
u n i t s i n b a t c h c u l t u r e .
3.11 I n f l u e n c e o f Zn and t i m e o f h a r v e s t i n g i n b a t c h ••• 89
c u l t u r e on l e n g t h o f Anacystis.
3.12 I n f l u e n c e o f Zn and t i m e o f h a r v e s t i n g i n b a t c h 90
c u l t u r e on l e n g t h o f Z n - t 5 . 0 ; i n o c u l u m f r o m
b a s a l medium.
3.13 I n f l u e n c e o f Zn and t i m e o f h a r v e s t i n g i n b a t c h »... 91
c u l t u r e on l e n g t h o f Z n - t 5 . 0 ^ i n o c u l u m f r o m
s t r o n g l y i n h i b i t o r y l e v e l o f Zn.
18.
F i g u r e page
4.1 I n f l u e n c e o f Co on g r o w t h o f w i l d - t y p e Anacystis 94
4.2 I n f l u e n c e o f Co on g r o w t h o f N i - t l . O . 95
4.3 I n f l u e n c e o f Co on g r o w t h o f Cu-t0.5. 96
4.4 I n f l u e n c e o f Co on g r o w t h o f Zn-t5.0 . = .. 97
4.5 I n f l u e n c e o f Co on g r o w t h o f Z n - t l 2 . 0 98
4.6 I n f l u e n c e o f Co on g r o w t h o f Cd-t2.0 99
4.7 I n f l u e n c e o f N i on g r o w t h o f w i l d - t y p e 100
4.8 I n f l u e n c e o f N i on g r o w t h o f C o - t l . 8 101
4.9 I n f l u e n c e o f N i on g r o w t h o f Cu-t0.5. 102
4.10 I n f l u e n c e o f N i on g r o w t h o f Zn-t5.0 103
4.11 I n f l u e n c e o f N i on g r o w t h o f Z n - t l 2 . 0 104
4.12 I n f l u e n c e o f N i on g r o w t h o f Cd-t0.2. 105
4.13 I n f l u e n c e o f Cu on g r o w t h o f w i l d - t y p e Anacystis 107
4.14 I n f l u e n c e o f Cu on g r o w t h o f C o - t l . 8 108
4.15 I n f l u e n c e o f Cu on g r o w t h o f N i - t l . O 109
4.16 I n f l u e n c e o f Cu on g r o w t h o f Zn-t5.0 110
4.17 I n f l u e n c e o f Cu on g r o w t h o f Z n - t l 2 . 0 . H I
4.18 I n f l u e n c e o f Cu on g r o w t h o f Cd-t2.0 112
4.19 I n f l u e n c e o f Zn on g r o w t h o f w i l d - t y p e Anacystis. H 3
4.20 I n f l u e n c e o f Zn on g r o w t h o f C o - t l . 8 . H 4
4.21 I n f l u e n c e o f Zn on g r o w t h o f N i - t l . O . 1 1 5
4.22 I n f l u e n c e o f Zn on g r o w t h o f Cu-t0.5. H 6
4.23 I n f l u e n c e o f Zn on g r o w t h o f Cd-t2.0. 1 1 7
4.24 I n f l u e n c e o f Cd on g r o w t h o f w i l d - t y p e Anacystis
4.25 I n f l u e n c e o f Cd on g r o w t h o f C o - t l . 8 . 1 1 9
4.26 I n f l u e n c e o f Cd on g r o w t h o f N i - t l . O . 1 2 0
1 2 1
4.27 I n f l u e n c e o f Cd on g r o w t h o f Cu-t0.5. • • *.
4.28 I n f l u e n c e o f Cd on g r o w t h o f Z n - t 5 . 0 . 1 2 2
4.29 I n f l u e n c e o f Cd on g r o w t h o f Z n - t l 2 . 0 . 1 2 3
19.
F i g u r e page
4.3 0 I n f l u e n c e o f Hg on g r o w t h o f w i l d - t y p e Anacystis. 124
4.31 I n f l u e n c e o f Pb on g r o w t h o f w i l d - t y p e Anacystis. 124
4.32 I n f l u e n c e o f Zn on g r o w t h o f Z n - t l 2 . 0 i n s t a n d i n g 128
c u l t u r e .
4.33 I n f l u e n c e o f NTG on g r o w t h o f w i l d - t y p e Anacystis; 132
p o p u l a t i o n d e n s i t y and c h l a were used as c r i t e r i a
o f g r o w t h .
4.34 Pigment changes d u r i n g g r o w t h o f w i l d - t y p e Anacystis 134
a t d i f f e r e n t Cd c o n c e n t r a t i o n s i n b a t c h c u l t u r e .
4.35 I n f l u e n c e o f minimum and maximum c o n c e n t r a t i o n s o f 135
Zn f o r t h e optimum g r o w t h o f w i l d - t y p e Anacystis.
5.1 I n f l u e n c e o f Ca on Zn t o x i c i t y t o w i l d - t y p e Anacystis. •••• 146
5.2 I n f l u e n c e o f Ca on Zn t o x i c i t y t o Zn-t5.0 Anacystis. •••• 146
5.3 I n f l u e n c e o f Ca on Zn t o x i c i t y t o Z n - t l 2 . 0 Anacystis. .... 147
5.4 I n f l u e n c e o f Ca on Cd t o x i c i t y t o w i l d - t y p e Anacystis. .... 147
5.5 I n f l u e n c e o f Mn on Zn t o x i c i t y t o Zn-t5.0 Anacystis. 1^0
5.6 I n f l u e n c e o f Mn on Zn t o x i c i t y t o Z n - t l 2 . 0 Anacystis. 150
5.7 I n f l u e n c e o f PO^-P on Zn t o x i c i t y t o w i l d - t y p e Anacystis... 153
5.8 I n f l u e n c e o f PO^-P on Zn t o x i c i t y t o Zn-t5.0 Anacystis. ... 153
5.9 I n f l u e n c e o f P0 4~P on Zn t o x i c i t y t o Z n - t l 2 . 0 Anacystis. . .. 154
5.10 I n f l u e n c e o f PO^-P on Cd t o x i c i t y t o w i l d - t y p e Anacystis. .. 154
5.11 I n f l u e n c e o f EDTA on Zn t o x i c i t y t o w i l d - t y p e Anacystis.-.. 164
5.12 I n f l u e n c e o f EDTA on Zn t o x i c i t y t o Zn-t5.0 Anacystis 164
5.13 I n f l u e n c e o f EDTA on Zn t o x i c i t y t o Z n - t l 2 . 0 Anacystis. .... 165
5.14 I n f l u e n c e o f EDTA on cd t o x i c i t y t o w i l d - t y p e Anacystis. 165
5.15 I n f l u e n c e o f Zn on Cd t o x i c i t y t o w i l d - t y p e Anacystis 172
5.16 I n f l u e n c e o f Zn on Cd t o x i c i t y t o Zn-t5,0 Anacystis 172
5.17 I n f l u e n c e o f pH on Zn t o x i c i t y t o w i l d ^ t y p e Anacystis 173
20.
F i g u r e page
5.18 I n f l u e n c e o f pH on Zn t o x i c i t y t o Zn-t5.0 Anacystis 173
5.19 I n f l u e n c e o f pH on Zn t o x i c i t y t o Z n - t l 2 . 0 Anacystis 175
5.20 I n f l u e n c e o f pH on Cd t o x i c i t y t o w i l d - t y p e Anacystis 175
5.21 I n f l u e n c e o f i n o c u l u m s i z e on Zn t o x i c i t y t o w i l d - t y p e . . . . 176
Anacystis ; c h l a used as g r o w t h c r i t e r i o n .
1 7 R
6.1 I n f l u e n c e o f Zn on g r o w t h o f w i l d - t y p e Anacystisf
d r y w e i g h t was used as a g r o w t h c r i t e r i o n .
6.2 I n f l u e n c e o f Zn on g r o w t h o f Z n - t l 2 . 0 Anacystis,: 178
d r y w e i g h t was used as a g r o w t h c r i t e r i o n .
6.3 R e l a t i o n s h i p between l o g a r i t h m o f Zn c o m p o s i t i o n o f 187
w i l d - t y p e A mcystis and d r y w e i g h t o f a l g a d u r i n g
g r o w t h of b a t c h c u l t u r e .
6.4 R e l a t i o n s h i p between l o g a r i t h m o f Zn c o m p o s i t i o n o f 187
Z n - t l 2 . 0 Anacystis and d r y w e i g h t o f a l g a d u r i n g
g r o w t h o f b a t c h c u l t u r e .
6.5 R e l a t i o n s h i p between l o g a r i t h m o f Zn c o m p o s i t i o n o f 190
w i l d - t y p e Anacystis and d r y w e i g h t o f a l g a d u r i n g
g r o w t h o f b a t c h c u l t u r e .
6.6 R e l a t i o n s h i p between l o g a r i t h m o f Zn c o m p o s i t i o n o f 190
Z n - t l 2 . 0 Anacystis and d r y w e i g h t o f a l g a d u r i n g
g r o w t h o f b a t c h c u l t u r e .
7.1 I n f l u e n c e o f Zn on g r o w t h o f Calothrix D184, i n t h e ••• 194
presence and absence o f combined n i t r o g e n .
7.2 I n f l u e n c e o f Zn on g r o w t h o f Phormidium autumnale D475.... 195
7.3 I n f l u e n c e o f Cd on g r o w t h o f Phormidium autumnale D475. ... 195
21 .
CHAPTER 1 INTRODUCTION
1.1 Occurrence o f b l u e - g r e e n algae i n e n v i r o n m e n t r i c h i n
heavy m e t a l s
Blue-green algae a r e widespread and sometimes abundant i n many
d i f f e r e n t t y p e s o f e n v i r o n m e n t (Fogg 1973). They are a l s o p r e s e n t i n
many h a b i t a t s which are p o l l u t e d w i t h o r g a n i c wastes (Palmer 1969) or
heavy m e t a l s ( W h i t t o n 1980) . Examples a re widespread i n Europe, U.S.A.
and elsewhere o f r i v e r s and streams which are c o n t a m i n a t e d w i t h zinc-
as a r e s u l t o f m i n i n g a c t i v i t i e s ( W h i t t o n 1980) . For i n s t a n c e streams
i n n o r t h Wales d r a i n i n g o l d lead mine t i p s and c a r r y i n g l e v e l s o f z i n c
g r e a t e r than about 1.5 mg 1 ^ Zn were r e o o r t e d by W h i t t o n
(1970b) t o have a v e r y reduced f l o r a . A t one s i l t e d s i t e t h e r e were
o n l y t h r e e s p e c i e s o f a l g a e , among them the b l u e - g r e e n a l g a Lyngbya sp.
Gale e t al. (1973) r e p o r t e d t h a t i n some p o l l u t e d streams i n t h e New
Lead B e l t r e g i o n o f s o u t h e a s t e r n M i s s o u r i , a l g a l g r o w t h d e v e l o p e d t o
such an e x t e n t as t o cause problems. Besides Oscillatoria t h e
f o l l o w i n g were fo u n d t o c o n t r i b u t e t o these p r o b l e m s : Cladophora,
Mougeotia, Zygnema, Spirogyra. W h i t t o n (1980) r e p o r t e d f r o m the same
area t h a t i n June 1977 among f l o e s i n h e a v i l y c o n t a m i n a t e d streams
below t h e s m e l t e r a t G l o v e r , phormidium and she a t h e d , v e r y narrow forms
o f O s c i l l a t o r i a c e ; e were i m p o r t a n t . Oscillatoria was a l s o found by
T r o l l o p e and Evans (1976) i n f r e s h w a t e r (1.96 mg 1 * Zn) nea r a z i n c
s m e l t i n g waste i n t h e lower Swansea v a l l e y , Wales. I n some seepages
and s p r i n g s w i t h v e r y h i g h z i n c l e v e l s a t E l v i n s T a i l i n g s , O ld Lead
B e l t , M i s s o u r i , an e s p e c i a l l y c h a r a c t e r i s t i c community o c c u r s i n
seepages below o l d t i p s w i t h pH v a l u e s w i t h i n o r near t h e 6.5 t o 7.0
ran g e s . The d o m i n a n t s , Plectonema and t h e protonema o f Dicranella varia,
22.
were the same, and a s s o c i a t e d s p e c i e s q u i t e s i m i l a r t o t h o s e a t o t h e r s i t e s
w i t h e l e v a t e d z i n c b o t h i n Europe and t h e U.S.A. ( W h i t t o n 1980). Phormidium
autumnale has been r e c o r d e d from t h e Riou M o r t , France w i t h 15.7
mg 1 * Zn a t pH 6.7. I n a stream o f t h e N o r t h e r n Pennine O r e f i e l d
showing a g r a d i e n t o f z i n c (30 t o 1.5 rag 1 ' f i l t r a b l e Zn) Plectonema
gracillimum was r e c o r d e d f o r over two y e a r s among t h e a l g a l s p e c i e s
near t h e t o p o f t h i s g r a d i e n t . While n o n - h e t e r o c y s t o u s b l u e - g r e e n
a l g a e are widespread i n streams w i t h h i g h Zn l e v e l s , t h e o n l y p u b l i s h e d
r e c o r d f o r a h e t e r o c y s t o u s one from such an e n v i r o n m e n t i n v o l v e s
Nodularia spumigena (Gopal et al. 1975); t h i s c o v e r s m o i s t s o i l near
t h e e f f l u e n t o f a z i n c s m e l t e r a t D e b a r i , I n d i a . Waste w a t e r s f r o m
t h e s m e l t e r were shown t o be t o x i c t o o t h e r h e t e r o c y s t o u s b l u e - g r e e n
a l g a e h e l d i n l a b o r a t o r y c u l t u r e (Tolypothrix tenuis, Calothrix
brevissima, Anabaena doliolum, Fischerella muscicola). The non-
h e t e r o c y s t o u s b l u e - g r e e n a l g a Plectonema boryanum t a k e n f r o m the same
s i t e was e x t r e m e l y t o l e r a n t t o Zn (Rana e t al. 1971). Anabaena, Nostoc
and Scytonema a r e abundant on h i g h copper s o i l s i n Zimbabwe ( W i l d 1968).
1.2 L a b o r a t o r y t o l e r a n c e and t o x i c i t y
I t has l o n g been r e c o g n i s e d t h a t h i g h c o n c e n t r a t i o n s o f heavy
m e t a l s such as Zn, Cu and Cd may pro v e t o x i c t o a q u a t i c p l a n t s and
a n i m a l s . A l a r g e body o f l i t e r a t u r e e x i s t s c o n c e r n i n g t h e t o x i c i t y o f
v a r i o u s m e t a l s t o v a r i o u s organisms i n c l u d i n g a l g a e ( W h i t t o n and Say
1975). The f o l l o w i n g b r i e f r e v i e w c o n c e n t r a t e s on t h e t o l e r a n c e and
t o x i c i t y o f m e t a l s t o a l g a e , p a r t i c u l a r l y b l u e - g r e e n , an a s p e c t t h a t
has r e c e i v e d l e s s a t t e n t i o n t h a n f o r example, t h e t o x i c i t y o f heavy
m e t a l s t o f i s h .
Many l a b o r a t o r y s t u d i e s have been c a r r i e d o u t on t h e t o x i c i t y o f
d i f f e r e n t heavy m e t a l s t o d i f f e r e n t s p e c i e s o f a l g a e . A l a r g e number
o f t h e s e s t u d i e s have been concerned w i t h t h e p o s s i b l e uses o f a l g a e
as m o n i t o r s o f p o l l u t i o n , o r w i t h t h e t o x i c e f f e c t s o f m e t a l -
c o n t a i n i n g a l g i c i d e s ( W h i t t o n and Say 1975) .
S e v e r a l w o r k e r s have used s e l e c t e d s p e c i e s o f algae t o assess
t h e i n f l u e n c e o f one o r more m e t a l s under c o n t r o l l e d c o n d i t i o n s i n
the l a b o r a t o r y . The s p e c i e s used, which a re f r e q u e n t l y o b t a i n e d from
c u l t u r e c o l l e c t i o n s , are t h e r e f o r e b e i n g used as b i o a s s a y o r g a n i s m s .
For example B a r t l e t t et al. (1974) found t h a t a l g i c i d a l c o n c e n t r a t i o n s
o f Cu, Zn and Cd f o r Selenastrum capricornutum were 0.3 mg 1 -1 -1
0.7 mg 1 and 0.65 mg 1 r e s p e c t i v e l y . R a c h l i n and F a r r a n (1974)
d e m o n s t r a t e d t h a t a c o n c e n t r a t i o n c f 2 - 4 mg 1 ' Zn b r o u g h t about 50%
r e d u c t i o n i n the g r o w t h r a t e o f Chlorella vulgaris w i t h i n 96 h o u r s .
I n a r a t h e r s i m i l a r s t u d y , Malanchuk and G r u e n d l i n g (1973) e s t i m a t e d 14
t h e ^D^Q (median e f f e c t i v e dose c a u s i n g 50% r e d u c t i o n o f CO^ f i x a t i o n )
o f Pb f o r f o u r s p e c i e s o f a l g a e . T h i s c o n c e n t r a t i o n l a y w i t h i n t h e
range 15 - 18 mg 1 ^ Pb f o r s p e c i e s o f Anabaena, Chlamydomonas and
Navicula, b u t was o n l y 5 mg 1 * f o r Cosmarium sp.
Only a few o t h e r w o r k e r s have a t t e m p t e d t o compare t h e s e n s i t i v i t y
o f d i f f e r e n t groups o f a l g a e t o heavy m e t a l s . W h i t t o n (1970b) c a r r i e d
o u t a l a b o r a t o r y s t u d y o f t h e t o x i c i t y o f Cu, Zn and Pb t o 37 s p e c i e s
o f green a l g a e . Microspora and Ulothrix tended t o be r e s i s t a n t t o a l l
t h r e e m e t a l s , w h i l s t Oedogonium spp. tended t o be r e l a t i v e l y s e n s i t i v e .
Zygnemales were on t h e whole i n t e r m e d i a t e i n t h e i r r e s i s t a n c e t o Cu
and Pb, b u t showed a wide range o f r e s i s t a n c e t o Pb. On t h e b a s i s o f
t h i s s t u d y , he c o n c l u d e d t h e t o l e r a n c e o f a l g a e t o one heavy m e t a l o r
a n o t h e r v a r i e s f r o m s p e c i e s t o s p e c i e s . Such v a r i a t i o n i s sometimes
e v i d e n t among algae b e l o n g i n g t o the same c l a s s and even i n t h e .same
f a m i l y . T h i s c o n f i r m e d t h e r e s u l t s o f V e l i c h k o (1968) who showed t h a t
0.05 mg 1 1 Zn had p r a c t i c a l l y no e f f e c t on r e d u c i n g t h e c e l l -numbers
o f Microcystis aeruginosa, w h i l e 0.1 and 0.2 mq 1 Zn reduced t h e c e l l
numbers by 2.3x and 25x r e s p e c t i v e l y , w h i l e a t t h e same t i m e r e s p i r a t i o n
and p h o t o s y n t h e s i s were i n h i b i t e d c o m p l e t e l y . On t h e o t h e r hand, 0.2 mg 1
had a p p r o x i m a t e l y the same ^ f r V c i r on M. tmlver&a as 0.5 ma 1 ' Zn
d i d on M. aeruginosa. A c l e a r a l g i c i d a l e f f e c t on the fo r m e r was
p r e s e n t o n l y a t 0.5 rng 1 1 Zn and 1.0 mg .1 1 Zn i n h i b i t s p h o t o s y n t h e s i s and
r e s p i r a t i o n c o m p l e t e l y . Rana and Kumar (1974b)used b i o a s s a y t e c h n i q u e s
t o e v a l u a t e t h e t o x i c i t y o f a Z n - c o n t a i n i n g e f f l u e n t ( f r o m a s m e l t e r )
t o t e n s p e c i e s o f a l g a e . The r e s u l t s suggested t h a t Chlorella vulgaris,
Scenedesmus sp. and Plectonema boryanum were r e l a t i v e l y t o l e r a n t t o
Zn, w h i l s t Anacystis nidulans, Oscillatoria sp. and Nodularia spumigena
were r e l a t i v e l y s e n s i t i v e , O v e r n e l l (1976) used t h e p r o d u c t i o n o f
oxygen t o measure the i n h i b i t o r y e f f e c t s o f heavy m e t a l s on p h o t o
s y n t h e s i s i n seven s p e c i e s o f marine a l g a e . Of t h e s e , Phaeodactylum
tricornutum and Dunallella tertiolecta were fo u n d t o be most s e n s i t i v e
t o Zn,Cu, Cd and Hg.
I t i s n o t t h e purpose o f t h e p r e s e n t s e c t i o n t o c o n s i d e r t h e
p r e s e n t knowledge o f t h e b i o c h e m i c a l e f f e c t s o f t o x i c o r n o n - t o x i c
c o n c e n t r a t i o n s o f heavy m e t a l s . Say (1977) has r e v i e w e d such a s p e c t s
f o r Zn. Say a l s o r e v i e w e d t h e methods employed by d i f f e r e n t workers
t o assess the t o x i c i t y o f m e t a l s , and n o t e d t h a t i n s e v e r a l cases t h e
e f f e c t s o f m e t a l have been q u a n t i f i e d by t h e degree o f i n h i b i t i o n o f a
p a r t i c u l a r m e t a b o l i c p r o c e s s . For a l g a e , such p r o c e s s e s i n c l u d e the
p r o d u c t i o n o f c h l o r o p h y l l a (Hargreaves and W h i t t o n 1 9 7 6 ) , t h e r a t e o f
r e s p i r a t i o n ( V e l i c h k o 1968), t h e r a t e o f a c e t y l e n e r e d u c t i o n o r
n i t r o g e n a s e a c t i v i t y (Home and Goldman 1974; H e n r i k s s o n and D a s i l v a
1978; D e l m o t t e 1980) and s p e c i f i c p h o t o s y n t h e t i c r e a c t i o n s ( O v e r n e l l
1975; Da F i l l i p i s and P a l l a g h y 1976a ; Delmotte 1980).
I n a d d i t i o n t o b i o c h e m i c a l c o n s i d e r a t i o n s , s e v e r a l w o r k e r s
have made o b s e r v a t i o n s on t h e e f f e c t o f l e t h a l o r s u b - l e t h a l concen
t r a t i o n s o f heavy m e t a l s on t h e g r o w t h r a t e o r morphology o f d i f f e r e n t
s p e c i e s o f a l g a e . Thus B a r t l e t t e t al. (1974) f o u n d t h a t t h e most
n o t i c e a b l e e f f e c t o f s u b - l e t h a l c o n c e n t r a t i o n s o f Zn, Cu, Cd on
Selenastrum capricornutum was an e x t e n s i o n o f t h e l a g phase of growth
i n c u l t u r e . S i m i l a r l y Rosko and R a c h l i n (1977) f o u n d t h a t concen
t r a t i o n s o f 0.32 mg l " 1 Cu, 2.4 mg l " 1 Hg, 0.32 mg l " 1 Cd, 7.5 mg l " 1
Zn and 32.0 mg 1 1 Pb extended t h e l a g phase f o r Chlorella vulgaris
t o 28, 17, 1 1 , 7 and 3 days r e s p e c t i v e l y . I n a d d i t i o n they observed
t h a t t h e s e c o n c e n t r a t i o n s l e d t o decreases i n t h e mean s i z e of the
c o n t r o l c e l l s by about 84% a f t e r 33 days. Some s t u d i e s have a l s o
d e m o n s t r a t e d t h e adverse e f f e c t s o f m e t a l t o x i c a n t s on the c e l l
d i v i s i o n and morphology o f a l g a e . For i n s t a n c e Say e t al. (1977)
o b s e r v e d an i n c r e a s e d f r e q u e n c y o f g e n i c u l a t i o n i n f i l a m e n t s o f some p o p u l a t i o n s
o f Hormidium rivulare. R e c e n t l y Cu and Zn have been found t o i n c r e a s e
t h e c h a i n l e n g t h o f t h e mar i n e d i a t o m Thalassiosira aestivalis, and
to i n h i b i t t h e normal s e p a r a t i o n o f c e l l s (Thomas e t al. 1980).
1.21 I s z i n c e s s e n t i a l f o r growth?
A Zn r e q u i r e m e n t f o r no r m a l p l a n t g r o w t h was f i r s t recognised i n
t h e l a t e n i n t e e n t h c e n t u r y , b u t acceptance o f t h i s e l e m e n t as an
e s s e n t i a l p l a n t n u t r i e n t d i d n o t oc c u r u n t i l t h e e a r l y 1930's.
R e l a t i v e l y few s t u d i e s have been conducted to d e t e r m i n e the requirement
o f z i n c t o p u r e c u l t u r e s of a l g a e . The reason has been due l a r g e l y t o
d i f f i c u l t y i n d e v e l o p i n g s a t i s f a c t o r y s y n t h e t i c growth media low enough
i n Zn t o d e m o n s t r a t e i t s r e q u i r e m e n t (Coleman e t al. 1971). The amount
o f Zn r e q u i r e d by most organisms i s so s m a l l t h a t i t i s f r e q u e n t l y
s u p p l i e d i n c o n s i d e r a b l e p a r t by the i n c i d e n t a l i m p u r i t i e s i n c l u d e d i n
t h e c u l t u r e medium.
112 y e a r s have passed s i n c e R a u l i n (.1869) d i s c o v e r e d the r o l e o f
Zn as a n u t r i e n t f o r t h e fungus Aspergillus niger. The a s s o c i a t i o n o f
m e t a l s w i t h p r o t e i n s has been r e c o g n i s e d and t h e i r e s s e n t i a l f u n c t i o n
i n t h e a c t i o n o f many enzymes has been r e p e a t e d l y d e m o n s t r a t e d ( V a l l e e
1962). However no such c r i t i c a l r e s e a r c h has been r e p o r t e d f o r Zn i n
a l g a e . The e a r l i e s t d e m o n s t r a t i o n o f a r e q u i r e m e n t i n a l g a e was
p r o b a b l y f o r t h e green a l g a Stichococcus bacillaris ( E i l e r s 1926)„ He
gave no d e t a i l s o f the e x p e r i m e n t o r c r i t i c a l l e v e l o f Zn r e q u i r e d .
Arnon (1958) s t a t e d i n a r e v i e w a r t i c l e t h a t Anabaena cylindrica r e q u i r e s
a 'micro' l e v e l o f z i n c i n the medium f o r g r o w t h ; t h e r e s e a r c h was c a r r i e d
o u t i n h i s own l a b o r a t o r y , b u t no e x p e r i m e n t a l d e t a i l s were g i v e n . Coleman
e t a l . (1971) found t h a t t h e optimum g r o w t h ( d r y w e i g h t ) o f Pediastrum
tetras and Euglena viridis o c c u r r e d a t 4.2 mg 1 * Zn, w h i l e t h a t o f
Chlorella vulgaris o c c u r r e d a t 18.03 mg 1 * Zn. Rana and Kumar (1974b)
observed t h a t i n c r e a s e s i n Zn i n t h e medium i n i t i a l l y i n c r e a s e d t h e g r o w t h
o f Plectonema boryanum, b u t as t h e c o n c e n t r a t i o n exceeded 0.5 mg 1 * g r o w t h
was r e t a r d e d .
1.22 E f f e c t o f z i n c on n i t r o g e n f i x a t i o n
L i t t l e seems t o be known about t h e e f f e c t s o f m e t a l s on n i t r o g e n
f i x a t i o n by b l u e - g r e e n a l g a e . O b s e r v a t i o n a t many f i e l d s i t e s p o l l u t e d
by Zn have shown t h a t most, i f n o t a l l , b l u e - g r e e n a l g a e a re non-
h e t e r o c y s t o u s t y p e s such as Phormidium, Oscillatoria, Lyngbya and
Plectonema. The o n l y h e t e r o c y s t o u s b l u e - g r e e n a l g a f o u n d a t a Zn
s m e l t e r s i t e was Nodularia spumigena (Gopal e t al. 1975). A c e t y l e n e
r e d u c t i o n assays were c a r r i e d o u t by W h i t t o n (1980) b o t h i n t h e
l a b o r a t o r y and f i e l d , on a Plectonema mat c o l l e c t e d f r o m t h e Harz.
A l t h o u g h a s t r a i n o f Plectonema was used f o r t h e f i r s t d e m o n s t r a t i o n o f
n i t r o g e n f i x a t i o n by a n o n - h e t e r o c y s t o u s f i l a m e n t o u s b l u e - g r e e n a l g a e
( S t e w a r t arid Lex 1970) t h e r e was no i n d i c a t i o n o f n i t r o g e n a s e a c t i v i t y
27 .
i n t h e Harz m a t e r i a l . The a d d i t i o n o f Cu a t 0.005 - 1 mg 1 *
depressed n i t r o g e n f i x a t i o n by Aphanizomenon and Anabaena i n C l e a r
Lake, C a l i f o r n i a (Home and Goldman 1974; E l d e r and Home 1978).
However 0.002 mg 1 * Cu s t i m u l a t e d n i t r o g e n f i x a t i o n . I n h i b i t i o n o f
n i t r o g e n f i x a t i o n m i g h t be a secondary e f f e c t , caused by a decrease
i n t h e energy s u p p l y t o h e t e r o c y s t s , w i t h subsequent breakdown i n
the removal o f oxygen from h e t e r o c y s t s and i n a c t i v a t i o n o f n i t r o g e n a s e .
A r e c e n t d e t a i l e d l a b o r a t o r y s t u d y was c a r r i e d o u t by H e n r i k s s o n
and D a s i l v a (1978), who found t h a t 0.005 - 0.025 mg l " 1 Zn s t i m u l a t e d
n i t r o g e n f i x a t i o n by Nostoc muscorum. Nostoc so., Chlorogloea tritschii and
Westiellopsis sp. At c o n c e n t r a t i o n s above t h i s , however, t h e r e was
i n h i b i t i o n . T h i s i n h i b i t i o n may be due t o t h e p a r t i c u l a r s e n s i t i v i t y
t o t h e m e t a l o f t h e n i t r o g e n a s e enzyme o r some o t h e r f e a t u r e o f t h e
n i t r o g e n f i x e r s .
1.3 A d a p t a t i o n
An a l t e r e d response t o a heavy m e t a l may oc c u r t h r o u g h m o d i f i c a
t i o n o f t h e c e l l s t h e m s e l v e s , e i t h e r p h e n o t y p i c o r g e n o t y p i c . Where
m u t a t i o n and s e l e c t i o n i s i n v o l v e d i t i s assumed t h a t mutant c e l l s
( i . e . mutants w i t h a g e n e t i c a l l y d e t e r m i n e d i n c r e a s e i n m e t a l r e s i s
t a n ce) are always p r e s e n t i n the c e l l p o p u l a t i o n b u t i n e x t r e m e l y low
numbers. Upon a d d i t i o n o f t h e a p p r o p r i a t e m e t a l t h e m a j o r i t y o f c e l l s
are k i l l e d o r i n h i b i t e d w h i l e t h e mutant c e l l s grow o u t , u l t i m a t e l y
becoming t h e p r e d o m i n a n t c e l l t y p e . I n t h i s k i n d o f v a r i a t i o n t h e
m o d i f i c a t i o n i s permanent i . e . r e s i s t a n c e i s m a i n t a i n e d even when c e l l s
are passed t h r o u g h non-metal c o n t a i n i n g media. I n most r e p o r t e d cases
o f t h e development o f heavy m e t a l r e s i s t a n c e i t i s d i f f i c u l t t o d e c i d e
whether p h e n o t y p i c a d a p t a t i o n o r m u t a t i o n i s i n v o l v e d . T h i s i s
p a r t i c u l a r l y so where r e s i s t a n t s t r a i n s have been o b t a i n e d by
s u c c e s s i v e t r a n s f e r i n t o media c o n t a i n i n g i n c r e a s i n g c o n c e n t r a t i o n s
o f t h e m e t a l . Golomzik and Ivanov (1964) used t h i s method t o o b t a i n
c u l t u r e s o f Thiobacillus ferrooxidans which o x i d i z e d f e r r o u s i r o n i n
the presence of v e r y h i g h c o n c e n t r a t i o n s o f f e r r o u s i r o n and copper
more r a p i d l y t h a n c e l l s o f t h e o r i g i n a l p o p u l a t i o n . S i m i l a r l y s t r a i n s
o f Proteus have been o b t a i n e d by s u c c e s s i v e t r a n s f e r which a re
r e s i s t a n t t o 150 mg 1 ^ c o b a l t , a c o n c e n t r a t i o n which c o m p l e t e l y
i n h i b i t s t h e p a r e n t s t r a i n s . I t has a l r e a d y been r e v i e w e d by Ashida
(1965) t h a t , b a c t e r i a , f u n g i and y e a s t are capable o f f o r m i n g
p o p u l a t i o n s which a re more t o l e r a n t t o heavy m e t a l s a f t e r exposure
f o r many passages t h r o u g h media w i t h m e t a l c o n c e n t r a t e s which p a r t i a l l y
i n h i b i t g r o w t h o f i n o c u l a n t s .
B l u e - g r e e n algae are c o m p a r a t i v e l y new t o g e n e t i c a l r e s e a r c h .
U n t i l about a decade and a h a l f ago n o t h i n g was known about t h e i r
m u t a g e n i c i t y , r e c o m b i n a t i o n o r g e n e t i c systems. A few y e a r s l a t e r ,
r e s e a r c h has n o t o n l y e s t a b l i s h e d t h e e x i s t e n c e o f g e n e t i c recombin
a t i o n i n some cyanophytes b u t a l s o shown t h a t these p r o k a r y o t e s are
no d i f f e r e n t f r o m o t h e r l i v i n g organisms i n t h e i r response t o
m u t a t i o n a l s t i m u l i (Ladha and Kumar 1978) .
1.31 A n t i b i o t i c s
A l t h o u g h a n t i b i o t i c and d r u g r e s i s t a n t mutants have been r e p o r t e d
i n b l u e - g r e e n a l g a e , e s p e c i a l l y Anacystis nidulans, Kumar (1964) and
Singh and Sinha (1965) s e l e c t e d s t r a i n s by s u c c e s s i v e s u b c u l t u r e s i n
l i q u i d media c o n t a i n i n g i n c r e a s i n g c o n c e n t r a t i o n s o f t h e a n t i b i o t i c s
p e n i c i l l i n and s t r e p t o m y c i n . These a n t i b i o t i c - r e s i s t a n t t r a i t s were
f u l l y s t a b l e even a f t e r s e v e r a l s u b c u l t u r e s i n a n t i b i o t i c - f r e e medium
(Kumar 1964; Gupta and Kumar 1970). W i t h t h e advent o f p l a t i n g
t e c h n i q u e s (Van Baalen 1.965), s u c c e s s f u l c l o n a l i s o l a t i o n has been
made b o t h a f t e r spontaneous s c r e e n i n g and a f t e r exposure t o mutagens.
However Ladha and Kumar (1978) p o i n t e d o u t t h a t the e f f i c a c y o f
c l o n i n g on agar needs t o be c o n s i d e r e d c a r e f u l l y ; i t may n o t be f u l l y
j u s t i f i e d t o r e g a r d a l l c o l o n i e s d e v e l o p i n g on an agar p l a t e as
s t r i c t l y c l o n a l , f o r t h e chances are t h a t a t l e a s t some c o l o n i e s would
have dev e l o p e d from two o r more c e l l s , r a t h e r t h a n s i n g l e c e l l s .
1.32 M o r p h o l o g i c a l mutants
Many w o r k e r s have i s o l a t e d f i l a m e n t o u s mutants o f t h e u n i c e l l u l a r
Anacystis nidulans (see Ladha and Kumar 1978)„ These mutants a r e o f
two b a s i c t y p e s : ( i ) f i l a m e n t s h a v i n g c r o s s - w a l l s and ( i i ) f i l a m e n t s
w i t h o u t c r o s s - w a l l s . Both t y p e s can be s t a b l e w i t h o u t h a v i n g a l t e r e d
g r o w t h r a t e ; however t h e y show changes i n c o l o n y morphology. I n t h e
second c l a s s o f m u t a n t s , f i l a m e n t s may be q u i t e l o n g , up t o 100 t i m e s
l o n g e r t h a n n o r m a l , w i t h o u t e x h i b i t i n g any d i s c o n t i n u i t y i n t h e
p h o t o s y n t h e t i c l a m e l l a e t h r o u g h o u t t h e l e n g t h o f t h e f i l a m e n t
(Kunisawa and Cohen-Bazire 1970).
1.33 M e t a l s
A r e l a t i v e l y l a r g e number o f mutant s t r a i n s o f b l u e - g r e e n a l g a e
have been i s o l a t e d , b u t a l m o s t a l l t h o s e r e l a t i n g t o m i n e r a l n u t r i t i o n
o r t o x i c i t y i n v o l v e n i t r o g e n a s s i m i l a t i o n pathways (Ladha and Kumar
1978). A r e c e n t s t u d y by Singh e t al. (1978) d e a l s w i t h t h e heavy
m e t a l s W and Cr. Spontaneous mutants o f Nostoc wuscorum grown i n
t h e presence o f these elements n o t o n l y t o l e r a t e d them b u t r e q u i r e d
them f o r g r o w t h w i t h o r NO^ as the n i t r o g e n s o u r c e , I n c o n t r a s t ,
a t t e m p t s by Sarma (1979) t o produce spontaneous mutants o f Anacystis
nidulans r e s i s t a n t t o Cu p r o v e d u n s u c c e s s f u l .
30.
1.4 F a c t o r s i n f l u e n c i n g t h e t o x i c i t y o f m e t a l s
The response o f a p a r t i c u l a r o r g a n i s m t o a p a r t i c u l a r m e t a l may
be a l t e r e d i n a number o f ways. These i n c l u d e m o d i f i c a t i o n o f t h e
en v i r o n m e n t o f t h e organism o r o f t h e or g a n i s m i t s e l f by a d a p t a t i o n
o r m u t a t i o n ( S a d l e r and T r u d i n g e r 1967). O v e r n e l l (1976) n o t e d t h a t
the a d d i t i o n o f EDTA i s u s u a l l y n e c e s s a r y t o p r e v e n t t h e p r e c i p i t a t i o n
o f t r a c e n u t r i e n t s such as i r o n , b u t i t may swamp the e f f e c t s o f s m a l l
q u a n t i t i e s o f added heavy m e t a l s . S t u d i e s o f t h e e f f e c t o f EDTA on t h e
Zn t o x i c i t y t o b l u e - g r e e n algae are r a r e b u t Stokes and H u t c h i n s o n
(1976) q u o t e d f r o m a s t u d y by H a l l (1974) t h a t EDTA i n c r e a s e s
Fe a v a i l a b i l i t y , w h i l e d e c r e a s i n g Zn t o x i c i t y t o Microcystis. Say e t al.
(1977) f o u n d t h a t r a i s i n g t he l e v e l o f EDTA from 5 - 20 mg 1 _ 1 had a
marked e f f e c t , i n c r e a s i n g t h e r e s i s t a n c e to Zn of Hormidium rivulare«
There i s a l s o i n f o r m a t i o n on t h e e f f e c t o f EDTA i n r e d u c i n g Cu t o x i c i t y ,
as a r e s u l t o f t h e f a c t t h a t Cu i s w i d e l y used f o r c o n t r o l l i n g a l g a l
blooms. Fogg and Westlake (1955) found e x t r a c e l l u l a r " p o l y p e p t i d e "
reduced Cu t o x i c i t y t o Anabaena cylindrica. S t u d i e s w i t h p h y s i o l o g i c a l l y
d i s t i n c t p o p u l a t i o n s o f Aphani zomenon (Home and Goldman 1974) have -1
shown t h a t t h e t o x i c e f f e c t s o f up t o 70 uq 1 Cu were removed i f EDTA
i s mixed w i t h Cu a few m i n u t e s b e f o r e a d d i t i o n t o t h e l a k e w a t e r , b u t
n o t when EDTA i s added s i m u l t a n e o u s l y . F e u i l l a d e and F e u i l l a d e (1977)
r e p o r t e d t h a t EDTA reduced t h e l e t h a l a c t i o n o f Cu t o Oscillatoria
rubescens. Among t h e f a c t o r s which have been mentioned as r e d u c i n g
Zn t o x i c i t y , t h e one q u o t e d t h e most o f t e n i s t h e hardness o f t h e w a t e r .
For i n s t a n c e , i t seems w i d e l y a c c e p t e d t h a t Zn i s al m o s t always l e s s
t o x i c t o f i s h i n h a r d t h a n i n s o f t w a t e r s (Mount 1966). L a b o r a t o r y
s t u d i e s o f b a c t e r i a (Abelson and Aldous 1950) i n d i c a t e d t h a t t o x i c
31 .
e f f e c t s o f N i , Co, Cd, Zn and Mn t o Escherichia coli were decreased
w i t h a h i g h Mg c o n t e n t i n t h e medium. Haavik (1976) d e m o n s t r a t e d
t h a t amounts o f Mn, Fe, Co, Ni and Cu i n h i b i t o r y t o Bacillus
licheniformis c o u l d be a n t a g o n i s e d by t h e a d d i t i o n o f Mg (1 g 1 ')
i n t h e medium, a l t h o u g h t o x i c c o n c e n t r a t i o n s o f Zn and Cd were
reduced l e s s e f f e c t i v e l y . However Break e t al. (1976) n o t e d t h a t Zn
t o x i c i t y t o f o u r s p e c i e s o f a l g a e Phaeodactylum tricornutum,
Skeletonema costatum, Thalissiosira pseudonana, Amphidinium carter!
was reduced by e l e v a t e d c o n c e n t r a t i o n s o f Mg. They suggested t h a t
t h i s m i g h t i n d i c a t e a common r o u t e f o r d i v a l e n t m e t a l i o n s e n t e r i n g
a l g a l c e l l s . I n an i n v e s t i g a t i o n o f the green a l g a Hormidium,
Say and W h i t t o n (1977) f o u n d t h a t b o t h Mg and Ca reduced t h e t o x i c i t y
o f Zn. T h i s e f f e c t was marked w i t h two Z n - t o l e r a n t p o p u l a t i o n s o f
Hormidium rivulare; t h e e f f e c t o f Mg was g r e a t e r than t h a t o f Ca a t
lo w e r c o n c e n t r a t i o n s b u t t h e i n f l u e n c e o f Ca i n c r e a s e d o v e r a much
g r e a t e r range o f c o n c e n t r a t i o n s . The i n f l u e n c e o f Mg was r e l a t i v e l y
s m a l l t o t h e Z n - s e n s i t i v e p o p u l a t i o n i n agreement w i t h t h e
o b s e r v a t i o n s o f H a r d i n g and W h i t t o n (1976) on Stigeoclonum tenue.
I n c o n t r a s t Gachter (1976) found t h a t t h e c o n c e n t r a t i o n o f Ca d i d n o t
appear t o a f f e c t t h e t o x i c i t y o f Zn, Pb, Hg o r Cu t o n a t u r a l p o p u l a t i o n s
o f p h y t o p l a n k t o n . Among al g a e t h e f u r t h e r f a c t o r s which seem t o have
no c l e a r antagonism t o Zn t o x i c i t y are Na and CI (Hormidium rivulare:
Say and W h i t t o n 1977; Stigeoclonum tenue: H a r d i n g and W h i t t o n 1977).
The s i t u a t i o n i s f u r t h e r c o m p l i c a t e d by t h e f a c t t h a t a ntagonism can
occur n o t o n l y between heavy m e t a l s and e s s e n t i a l elements as p r e v i o u s l y
d i s c u s s e d , b u t a l s o among heavy m e t a l s t h e m s e l v e s . For i n s t a n c e , i n
t h e case o f f i s h , b o t h f i e l d and l a b o r a t o r y s t u d i e s have shown t h a t
t h e c o n c e n t r a t i o n s o f o t h e r m e t a l s may i n f l u e n c e t h e t o x i c i t y o f any
p a r t i c u l a r m e t a l . Examples are known o f a n t a g o n i s t i c and s y n e r g i s t i c
i n t e r a c t i o n s (Jones 1964) . I n l a b o r a t o r y s t u d i e s o f Anabaena
inaequalis, S t r a t t o n and Corke (1979) found t h a t t h e response towards
c o m b i n a t i o n s o f Hg I I and Cd o r N i and Cd r e s u l t i n g i n e i t h e r
s y n e r g i s m o r antagonism towards g r o w t h , depended upon the a c t u a l m e t a l
c o m b i n a t i o n s used. When t h e p a i r s o f m e t a l s Hg I I and Cd or N i and Cd
were used a t a s u b l e t h a l c o n c e n t r a t i o n and i n c o r p o r a t e d s i m u l t a n e o u s l y ,
t h e y i n t e r a c t e d s y n e r g i s t i c a l l y and a n t a g o n i s t i c a l l y i n t h e i r e f f e c t
on g r o w t h r a t e , r e s p e c t i v e l y . Cd has a l s o been fo u n d t o reduce t h e
i n h i b i t i o n o f g r o w t h o f Selenastrum capricornutum by Cu ( B a r t l e t t et al.
1974). Say and W h i t t o n (1977) found t h a t t h e t o x i c e f f e c t s o f Cd and
Zn a r e s y n e r g i s t i c t o w a r d t h e g r o w t h o f Hormidium rivulare, t h u s
r e s e m b l i n g t h e response f o u n d by H u t c h i n s o n and Czyrska (1972) f o r
Lemna valdiviana. Any l e v e l o f Cd above 0.01 mg 1 * must be s u s p e c t e d
o f p r o d u c i n g a s i g n i f i c a n t i n c r e a s e i n the t o x i c i t y t o Hormidium o f
any Zn p r e s e n t . Cu and N i i n t e r a c t s y n e r g i s t i c a l l y t o w a r d s t h e g r o w t h
o f some green a l g a e , w h i l e Se a n t a g o n i z e Cd t o x i c i t y ( H u t c h i n s o n 1973).
I n a d e t a i l e d l a b o r a t o r y s t u d y , Nakano e t al. (1978) f o u n d
a n t a g o n i s t i c a c t i o n between Zn and Cd t o Euglena gracilis. I n t h e
presence o f 20 mg 1 ^ Cd, t h e g e n e r a t i o n t i m e i n Z n - f r e e medium o f
57 h was reduced t o 27 h a f t e r a d d i t i o n o f 2 mg 1 * Zn. T h i s i s i n
agreement w i t h Falchuk e t al. (1975) who found Zn redu c e d Cd t o x i c i t y
t o Euglena gracilis; i t a l s o agrees w i t h r e s u l t s by Pakalne et al.
(1970) and U p i t i s e t al. ( 1 9 7 3 ) , who fou n d t h a t Zn p r o t e c t s Chlorella
a g a i n s t Cd t o x i c i t y .
S t u d i e s o f v a s c u l a r p l a n t s show t h a t h i g h c o n c e n t r a t i o n s o f s o i l
p hosphate a n t a g o n i z e t h e t o x i c i t y o f Zn. D i r e c t e x p e r i m e n t a l e v i d e n c e
f o r t h i s has been obtained f o r Thlaspi al pestre ssp. calaminare
(Ernst 1974). Rana and Kumar (1974 a)studied the i n f l u e n c e of phosphate
and n i t r a t e on Zn t o x i c i t y t o the blue-green alga Plectonema boryanum
as w e l l as the green alga Chlorella vulgaris. R e l a t i v e l y high concen-
g r a t i o n of phosphate, but not n i t r a t e , improved the growth of both
algae and pr o t e c t e d them against Zn t o x i c i t y . These r e s u l t s are i n
apparent c o n t r a s t t o those of Green et al. (1975), who reported t h a t
Zn t o x i c i t y t o the green alga Selenastrum capricornutum was not a f f e c t e d
s i g n i f i c a n t l y by low PO -P i n range of 0.047 mg 1 ' to 0.93 mg 1
Phosphate also reduced Zn t o x i c i t y t o green algae, Hormidium rivulare
(Say et al. 1976; Say and Whitton 1977) and Stigeoclonum tenue (Harding
1978). Sulphate had no detectable i n f l u e n c e i n reducing Zn t o x i c i t y t o
Hormidium rivulare (Say and Whitton 1977) .
Hydrogen ion concentrations may play a determinant r o l e i n a f f e c t
i n g Zn t o x i c i t y t o the algae. Most of the experimental studies reported
i n the l i t e r a t u r e on the i n f l u e n c e of pH on Zn t o x i c i t y have so f a r
been concentrated on green algae. For instance, Harding and Whitton
(1977) reported an obvious decrease i n Zn t o x i c i t y t o a Z n - t o l e r a n t
p o p u l a t i o n of Stigeoclonum tenue w i t h a r i s e i n pH from 6.1 t o 7.6,
but a s i m i l a r response was scarcely detectable w i t h a Zn-sensitive
p o p u l a t i o n . The i n f l u e n c e of pH on Hormidium rivulare c o n t r a s t s w i t h
t h a t on Stigeoclonum. With both the Zn-sensitive and Zn - t o l e r a n t
populations of Hormidium rivulare i s o l a t e d from a reach w i t h a mean
f i e l d pH of 4.4 and 6.8, r e s p e c t i v e l y the t o x i c i t y of Zn decreased w i t h
a f a l l i n pH between 8 - 3 (Say and Whitton 1977) , agreeing w i t h the
observations of Hargreaves and Whitton (1977) who showed t h a t the
t o x i c i t y of Zn t o Hormidium rivulare i s o l a t e d from a stream a t pH 3.1,
increased over the range of pH 3.5 - pH 7.0. The t o x i c i t y of Cu was
found t o be pH-dependent; pH may also play a r o l e i n reducing Cd
t o x i c i t y . For instance, Hart and Scaife (1977) found t h a t Cd i n h i b i t e d
the growth of the green alga Chlorella pyrenoidosa, the extent of
i n h i b i t i o n being more pronounced at pH 7.0 than at pH 8.0.
1 .5 Accumulation of metals
Freshwater algae exposed t o Zn concentrations above "normal"
background l e v e l s have the a b i l i t y and tendency t o accumulate Zn, as
shown ex p e r i m e n t a l l y (Coleman et al. 1971) and i n the f i e l d ( T r ollope
and Evans 1976). I t i s c l e a r t h a t freshwater algae can be very
s i g n i f i c a n t environmental f a c t o r s i n the uptake and movement of Zn i n
freshwater systems. I n water near Zn smelting wastes i n the Lower
Swansea V a l l e y , w i t h 1.96 mg 1 1 Zn, a bloom of Oscillatoria accumulated -1
1.88 mg Z n g dry w e i g h t (Trollope and Evans 1976). Studies i n S t r o t h e r
Creek, M i s s o u r i , w i t h 0.041 mg 1 1 Zn (not f i l t e r e d ) , 0.026 mg 1 1 Zn
( f i l t e r e d ) , showed t h a t two Oscillatoria samples found by Jennett et al.
(1980) concentrated 3260 ug Zn g * dry weight and 4030 u.g Zn g * dry
weight, r e s p e c t i v e l y . The authors reported a lower concentration f a c t o r
f o r Cd by blue-green algae; three young c u l t u r e s of Nostoc muscorum,
Nostoc sp. and Schizothrix calcicola d i d not remove Cd s i g n i f i c a n t l y a t
any pH, while only one green alga Mougeotia showed such negative r e s u l t s .
They suggested that, green algae appear t o be much more e f f i c i e n t a t
accumulating Cd than the blue-greens.
Laboratory studies on Zn uptake and accumulation have been made on
vascular p l a n t s (Adams e t al. 1973; Mathys 1980), bryophytes (Pi c k e r i n g
and Puia 1969) and green algae (Coleman et al. 1971; Wixson and Gale
1975; Whitton and Say 1975) . Few accounts f o r blue-green algae have been
reported. S p a r l i n g (1968) found i n l a b o r a t o r y studies t h a t Gloeocapsa sp.
Nostoc muscorum, Anacystis nidulans and Merismopedia sp. a l l took up a
s i g n i f i c a n t amount of Zn, Cu, Cd and Ni from s o l u t i o n , enough t o
i n f l u e n c e the metal balance i n n a t u r a l waters. I n a study of metal
uptake by Anacystis nidulans, K a t a g i r i (1975) found t h a t a l o g a r i t h m
i c a l l y grown c u l t u r e exposed t o 0.5 mg 1 * Cd f o r 24 h accumulated
1.5 mg 1 * Cd. However Cd uptake was pH dependent, more Cd being
accumulated a t pH 7.0 than at pH 8.0. Jones et al. (1978) found a
considerable v a r i a t i o n i n Zn and Cd accumulation between s t r a i n s o f
blue-green algae, a f t e r they had been grown i n medium c o n t a i n i n g
74 ug 1 1 Zn and 20 ug 1 1 Cd, as shown below:
Mg g zn Mg g cd
Anabaena cylindrica 93 1.5
Anabaena variablis 48 2.3
Anacystis nidulans 81 2.3
Chlorogloea fritschii 109 2.9
Nostoc muscorum 479 9.1
1.6 Aims
Blue-green algae are o f t e n the dominant organisms a t moist s i t e s
combining high l e v e l s of Zn w i t h high pH. Laboratory studies have
confirmed t h a t the s t r a i n s present are h i g h l y r e s i s t a n t t o Zn. The
question arises as t o how easy i t i s f o r s t r a i n s l a c k i n g r e s i s t a n c e t o
develop i t . ? A r e l a t i v e l y large number of mutant s t r a i n s of blue-green
algae have been i s o l a t e d , but so f a r not ones r e s i s t a n t t o heavy metals
(1.33). On the other hand there are several r e p o r t s i n the l i t e r a t u r e
showing a large number of Anacystis nidulans mutants r e s i s t a n t t o drugs.
As a r e s u l t of reviewing the above l i t e r a t u r e , i t was planned t o
attempt t o i s o l a t e s t r a i n s of A. nidulans r e s i s t a n t t o Co, N i , .
Cu, Zn and Cd and make an account of the p r o p e r t i e s of these s t r a i n s .
I t seemed l o g i c a l p r i o r t o the s t a r t of these experiments t o i n v e s t i g a t e
the e f f e c t of these metals on the growth and morphology of t h i s s t r a i n .
Many reports in the l i t e r a t u r e have shown that the behaviour of metals
may be influenced by other factors (section 1.4) so experiments were
also planned to determine the eff e c t s of such factors on metal t o x i c i t y
to A. nidulans. Experiments were also planned to determine the extent
to which A. nidulans can accumulate Zn.
37.
CHAPTER 2
MATERIALS AND METHODS
2.1 Culture techniques
2.11 Cleaning of glassware
Glassware was washed w i t h d i s t i l l e d water, soaked f o r at l e a s t
24 h i n 10% Analar HC1 ( e a r l i e r experiments) or 2% Analar ( l a t e r
experiments), r i n s e d i n d i s t i l l e d water, and d r i e d a t 105°C.
2.12 Culture vessels
The c u l t u r e vessels f o r maintenance of stock c u l t u r e s or preparing
inocula i n l i q u i d medium were 100 ml and 250 ml Pyrex c o n i c a l f l a s k s .
For t o x i c i t y experiments, 50 ml b o i l i n g tubes were used. P l a s t i c
disposable s t e r i l i s e d p e t r i dishes were used f o r s o l i d media.
Good q u a l i t y c o t t o n wool was used f o r plugging the c o n i c a l f l a s k s .
Morton closures (Bellco s t a i n l e s s s t e e l ) or Axa closures (Axa Ltd)
were used f o r r o u t i n e t o x i c i t y t e s t s f o r which many tubes were r e q u i r e d .
2.13 S t e r i l i z a t i o n
A l l f l a s k s and tubes c o n t a i n i n g medium were s t e r i l i z e d by auto-o -2 -2 c l a v i n g a t 121 C (10 KNm j 15 l b i n ) f o r 15 min. The medium was
allowed t o stand overnight before i n o c u l a t i o n t o allow r e q u i l i b r a t i o n
w i t h the atmosphere; i f the medium was t o be stored longer than t h i s
i t was kept i n the dark i n order t o p r o t e c t EDTA-metal chelates against
p h o t o - d e t e r i o r a t i o n .
In media w i t h high phosphate, calcium or z i n c , these were auto-
claved separately and added a s e p t i c a l l y t o each tube or f l a s k , t o avoid
p r e c i p i t a t i o n . I n media cont a i n i n g c e r t a i n organic compounds or
a n t i b i o t i c s which might be heat s e n s i t i v e the f i l t e r procedure i n
2.91 ( i i i ) was used.
2.2 Media
2.21 Mineral n u t r i e n t s
Tables 2.1 (p. 3',' ) and 2.2 (p. 40 ) show the composition of the
media and the s a l t s used i n t h e i r p r e p a r a t i o n . The percentages of Zn
present i n these s a l t s as i m p u r i t i e s are given i n Table 2.3 (p. 4-| ) .
A l l s a l t s were prepared i n high concentrations, from Analar products
i n deionized d i s t i l l e d water and stored a t 4°C i n the dark. I n
studies of Zn t o x i c i t y t o Anacystis nidulans a medium (ACM) was used
derived from medium *C of Kratz and Myers (1955). The m o d i f i c a t i o n
was performed as f o l l o w s : the l e v e l of phosphate was reduced by a -1
f a c t o r of 100 (to 1.78 mg 1 P); zn was omitted from the microelement -1
stock leaving about 0.04 mg 1 Zn derived as contaminants. Analysis
of stock s o l u t i o n show t h a t the amount of Zn as i m p u r i t i e s of chemical
i s 0.024 mg 1 The r e s t o f Zn may perhaps come from glassware, and
deionized water; the l e v e l o f c h e l a t i n g agent was r e l a t i v e l y low, w i t h
0.5 mg 1 1 ( = 0.0017 mM) EDTA; pH was bu f f e r e d a t 7.0 ± 0.15 w i t h HEPES
using NaOH t o make the i n i t i a l pH adjustment.
Two d i f f e r e n t media were used f o r c u l t u r i n g s t r a i n s i s o l a t e d from
high Zn s i t e s ; both c o n t a i n low l e v e l s o f phosphate:
( i ) Chu 10D + N medium i s described by S i n c l a i r and Whitton 1977:
as Chu 10-D), but the v e r s i o n used here was b u f f e r e d a t a lower pH
value. The n i t r a t e - f r e e v ersion (Chu 10-N) was made by supplying Ca
as CaCl 2.
( i i ) ADM medium i s a m o d i f i c a t i o n of the medium of A l l e n and Arnon
(1955), w i t h phosphate reduced t o (0.445 mg 1 * P); the l e v e l of Fe
and c h e l a t i n g agent was reduced by a f a c t o r of 2 ( t o 2 mg 1 * Fe;
10 mg 1 1 (= 0.034 mM) EDTA).
Both media were b u f f e r e d a t pH 7.0 w i t h 1200 mg 1 _ 1 (= 5.04 mM) HEPES.
39.
Table 2.1 Composition of media (mg 1 of s a l t s )
s a l t s
KN03
Ca(NO ) 2 > 4 H 2 0 K 2HP0 4
KH 2P0 4
MgSO .7H20 Na SiC>3.5H20 NaCl Na2HC03
CaCl .2H?0 FeCl 3.6H 20 Na2EDTA.2H20 Na c i t r a t e . 2 H ? 0 Fe 2(S0 4) 3.6H 20 MnCl 2.4H 20 MnS04-4H20 NaMo04.2H20 ZnS0 4.7H 20 CuS04.5H20 CoCl 2.6H 20 CoS04.7H20 H3BD3
NH 4V0 3
NaoW0„.2H O 2 4 2 NiS0 4.7H 20 Cr(S0 4) 3.K 2S0 424H 20 pH
ADM A l l e n and Chu 10-D Arnon (1955)
2.5
200
230
66.2 9.7
25 .4
0.5 0.19 0.05 0.02 0.01
0.5
0.01
7.0
348.9
246.5
73 .5
2.03 0.15 0.22 0.08
2.86 0.02 0.018 0.045 0.96
40
8.0 25 .0 11.0
16.0
2 .42 3.17
0.05
0.007 0.056 0.019
0.01 0.72
ACM Kratz and Myers (1955)
500
10
2 50
19.86 9.7 0.635
1 .81
0.027
0.08
0.04 2.86
1000 25
1000
250
165 4.0 1 .81
0.017 0.222 0.08
2.86
7.2-7.5 7.0 7.5-7.8
Table 2.2 Composition of media (mg 1 of elements)
element ADM(-N) A l l e n and Arnon (1955)
Chu 10-D ACM Kratz and Myers (1955)
N - - 69.29 -P 0. 445 61.9 1 . 78 1 .78 177.8 S 26 0 32 .0 3 25 32 .52 4.23 CI 171 . 4 177.3 28.018 -Na 90. 5 92 .0 8. 44 10.62 -K 112. 2 156 2 . 24 - 835.6 Ca 18. 1 20 9. 77 5.45 -Mg 19. 7 24.3 2. 47 24.65 24.65 Si - - 2 . 50 - -Fe 2. 0 4.0 0. 5 2.0 0.86 Mn 0. 12 0.5 0. 012 0.5 0.5 Mo 0. 08 0.1 0. 0025 0.01 0.01 Zn - 0.05 0. 012 - 0.05 Cu 0. 005 0.02 0. 005 0.02 0.02 Co 0. 0005 0.01 0. 002 0.008 0.008 B 0. 09 0.05 0. 125 0.50 0.50 V 0. 004 0.01 - -W 0. 005 0.01 - -Ni 0. 002 0.01 - - -Cr 0. 001 0.01 - - -EDTA 10. 0 7 2. 47 0.5 -
Table 2„3 Amount of zinc as i m p u r i t i e s i n the stock (mg 1 )
Zn (mg 1 1 ) i n stock
Zn (mg 1~ ) i n medium from stock
K 2HP0 4 0„ 18 0.000072
MgSO .7H 0 0,08 0.0004
NaCl 0.072 0,000096
CaCl 2.2H 20 0.08 0.0008
KN03 0.64 0.0168
Fe.EDTA 0.40 0.0002
EDTA 0„008 -
HEPES 0.003 0.0016
Trace-element 0,08 0.0008
NaOH 0. 24 0.0024
T o t a l 0.024
2.22 Bu f f e r
Attempts were made t o increase the b u f f e r i n g capacity of media. -1 -1 HEPES a t 1200 mq 1 (= 5.04 mM) and 600 mg 1 (=2.52 mM) were shown
t o r e s t r i c t pH v a r i a t i o n s w i t h i n 0.15 and 0.25 pH u n i t s , r e s p e c t i v e l y ,
over a 10 day pe r i o d w i t h o u t a f f e c t i n g the growth of Anacystis nidulans
(Table 5.26). HEPES was chosen as a b u f f e r i n g agent on the basis o f
the r e s u l t s of Good e t al. (1966). They reported t h a t HEPES has a
n e g l i g i b l e binding capacity f o r the metals Mg, Ca, Mn and Cu. They
c a l c u l a t e d the approximate values f o r the m e t a l - b u f f e r binding constants,
from the displacement of the pH t i t r a t i o n curve i n the presence of an
equivalent of the c h l o r i d e s a l t of the metal i n question. Smith and
Foy (1974) chose HEPES as a b u f f e r f o r freshwater a l g a l media because
of i t s favourable PKa of 7.55 and the n e g l i g i b l e metal b i n d i n g capacity
reported by Good et al.; no f u r t h e r experimental studies were made.
In a comparison of the e f f e c t s of HEPES, EDTA and TES
(N-Tris(hydroxymethyl)-methyl-2-aminomethanesulphonic acid) on the
t o x i c i t y of Cd t o Daphnia, T e v l i n (1978) found t h a t i n c o n t r a s t to
EDTA and TES, 0,001 M and 0.002 M HEPES d i d not reduce t o x i c i t y . He
r e l a t e d t h i s t o the absence of Cd complexing by HEPES.
I t was r e a l i z e d a t a l a t e stage of the present studies t h a t , i n
s p i t e of the previous r e p o r t s , HEPES i s i n f a c t l i k e l y t o act as a
c h e l a t i n g agent. The studies of Good et al. d i d not i n v e s t i g a t e a l l
the p o s s i b l e ways t h i s molecule can act as a c h e l a t i n g agent (M. K i l n e r ,
pers„ comnio ) . The s i t u a t i o n i s complicated because both EDTA and
HEPES were present simultaneously i n the medium, w i t h EDTA known t o
act as a strong c h e l a t i n g agent. The l e v e l s of EDTA and HEPES used
f o r various experiments are given i n Table 2.4.
HEPES was added t o the medium before a u t o c l a v i n g and a d j u s t i n g
c o
to 4-1 q E
•r-l U 0> O,
X
W
u > rH O
4-1
TS CD co 3
in a & w
TD C (0
<
E H
D
w
4-1
o
0) > (1)
r-H XI
• H o 1—1 C N o o o o O • M « • • • • t * • • U ro L O r~ CD
T C N C N C N C N CN CN C N CN CN O o i n in m i n in i n m i n m
L D L O CN C M C N C N CN C N C N CN CN
w ft w
*~ i o o o o o O O o o o O (—i o o o o o o O o o O o
C N CN 1X1 Hi ••D 'X? vo CN CM cn r-« »—'
£
r-- r~ r~ r> r> i n m *—\ .—i . — i * — i *—i i—i CO co o o o o o o o o o o o o o o o o o o o o
o o o o o o c o o o r^ E H Q
« 1
i—1 L O m i n m m m i n i n m
E o o o o o o o o CN
p 2 2 2 £— 3 s £ S S 2 £ 2 2 + 1 +
•rH u U u a u u u O 73 ft < < < < < o o o <1) r—i r-t t e 3 3 3
X X X O U U
4-1 c >. a) 4-) 4-> to • H M •rH 0
•rH 4J • H </) to X 0) 3\ 0
+J 0 4-1 C fa aj m R TS 6 0 *S c
rfl rH c 4H a> 0 0 01 Q< • H 0
4-1 w C 0) u c rrj
3 •rH U T> rrj 0> 0 U r-H U 4-> 0 Ch 10 4J
CP
3 u
o u
• H • H
(0 u
• H 4-1 o
XI • H
>i 4-1 •rH U
•rH X 0 •p
en c
• H 4-1 U CJJ
m m rrj
rH o •P o rd
i n
0) ' i rrj
\
0 £• 4-1 4-1
3 as
til 4J • 9 03 0 +J 3 CM Ot to <U
c W fB to W 3 CO U ra <u 0 CD 0 tJ c 0
• H 4-' e QJ 5 O q • H 0) 4-1 0 3 o u 4J •c: (0 > i •rH (1) •rH 0 • H rH 0) •u
r-H 4-1 t ) rC! t3 0 •rH 0 3 • H •rH 4J •rH -c: q E u E 0 E 0 4J 0) 5 3 •rH rH d S-H 0) 0 S to U X 0 0 0 q O 0 r-H ^ ! 3 l rrj • • CM rt) 4-i to CL to O O
the pH. In some cases the pH of the medium was adjusted a f t e r auto-
c l a v i n g t o i t s normal value by the aseptic a d d i t i o n of 0.2 N NaOH t o
each f l a s k or tube. pH v£ilues were measured using a model 7050
( E l e c t r o n i c Instruments Ltd) pH meter,
2.23 Chelating agent
0.5 mg 1 * EDTA (ethy l e n e d i a m i n e t e t r a - a c e t i c a c i d , disodium s a l t )
was added t o the medium.
An experiment was c a r r i e d out t o measure the i n f l u e n c e of EDTA
on the s o l u b i l i t y of Zn. In samples which had been passed through
g l a s s f i b r e f i l t e r s , the l e v e l of Zn increased w i t h i n c r e a s i n g EDTA
(Table 2.5).
2.24 M a t e r i a l s used f o r t o x i c i t y t e s t s
A d d i t i o n of metals
-1
zin c : added t o c u l t u r e medium as ZnSO .7H O from a 1000 mg 1 Zn
stock s o l u t i o n i n deionized d i s t i l l e d water.
copper: added t o c u l t u r e medium as CuSO^^H^O from a 1000 mg 1 *
stock s o l u t i o n i n deionized water.
cadmium: added t o the c u l t u r e s as CdSO^.SH^O from a 1000 mg 1 *
stock s o l u t i o n i n deionized water.
A d d i t i o n of other selected c a t i o n s and anions
A l i s t of other metals whose t o x i c i t y was compared w i t h t h a t of
Zn, together w i t h the f a c t o r s whose i n f l u e n c e on Z n - t o x i c i t y was st u d i e d ,
i s given i n Table 2.6, together w i t h the substances added t o the medium t o
br i n g about r e q u i r e d changes. The basal media l a c k i n g p a r t i c u l a r ions
r e q u i r e d as c o n t r o l s were obtained by s u b s t i t u t i n g complementary s a l t s ,
E C M
• H
T3 QJ -—i
e C M
• V
C M o>
C M X! cd
Q J 4-> <-\ X) 0 10 V) E - i — I
U S
0) C O 01 W to C M
W •
•p + c
01 6
o .—1 • vi r - 0 )
Q
33 X a < U
0 )
e 0
• H 4 -1
•D (1) (1) X) 6
S u
C M
c u • H 0
4-1
>1 + J T3 • H C •—1 ( 0 • H 4->
XI 0 ) p
o o -P 0 1
4-1 c 4-4 N 0 )
i—1
C 0 c
Q J
<c x: E-< 4-1 Q
W c
* 0 • a
a) O J
u > c a) r - 4
u i - i 0 4 4 4-> c 3
H
ctJ E-i
o o O C P 0 0 C O 0 0 • o o o C P C P C P
o . — I
rN c o
• H o o o O o o C O
4 - ' » o o o o o C P C P
rd o T—1 T—;
4 J
i - l • H o o C O 0 1 o m 1 C P o C P C P C P C P C P
• — i LT) T-H
ai 1 M i—1 O i n o o CTv cr> C M C M co
4-1 en « o o C P C P C P co 0 1 £ C M
X! — ,
ss C M in in c •
• o o C O o C O
w i—H o o o C O C P C O
( 0 T-H
0 1
S
c i n o o C M >X> C M o C O
N • o o C P C P C P C P C O
o T—1 <*>
o oo C P i n r - * r o C P C P C P C P C P C P C O
o o o C P >x> o r -• o Q C P C P C P t-- i n
o r-H r—i
c C M
0 • H
4-1 o O o C P C P o r—t 1X3 « o o C O V O i n i£) i n !M o ,—1 T—1
4-> f-t •H . — . 4-1 T-H o o r-H —1 C M C M C O
1 • C P C O in i n M r H
0 ) -P C P 4-1 in <r C M C O vo C M C M
rd i n ro cn C"j C M
u a O in C M ro O C P
3 w • n m C O m r~i C M
W T—<
(TJ OJ e un C M C O C M r - o C O
C O m C M
c CD N
tip o i n m m i n C P C M C M C M C M C M C M C M
o o O O o o o •—'
r - 4 1 (XJ r H
C M en i n o
a •H CP CP S
• r 4
o c tSJ
Table 2.6 Factors i n f l u e n c i n g Z n - t o x i c i t y
s a l t used as a source f o r c omp1emen ta ry s a l t used i f
f a c t o r s element omitted
t o x i c i t y t e s t basal medium
Na NaCl NaCl Mg MgCl 2. 2H 20 and MgS04 7H 20 MgS04-7H2O Na 2S0 4
CI NaCl NaCl -K KCl KNC> and K HPO,, 3 2 4 NaNO and
Na 2HP0 4
Ca CaCl 2. 2H 20 CaCl 2-2H 20 -Mn MnCi 2. 4H 20 MnCl 2.4H 20 -Fe FeCl^. 6H 20 Fe.EDTA Na EDTA Ni N i C l 2 . 6H 20 - -Co CoSO„. 4 7H 20 CoSO„.7H 0 4 2 Cu CuSO„. 4 5H 20 CuS04-5H20 Na 2S0 4
Zn ZnS0„. 4 7H O ZnSO .7H 0 Na 2S0 4
ca CdSO„. 4 8H 20 -Hg HgCl 2 -Pb Pb(N0 3 >2 -NO -N KN03 KN°3 KCl PO 4-P K 2HP0 4 K 2HP0 4 KCl s o 4 - s Na 2S0 4 MgS04-7H20 MgCl 2.2H 20 EDTA EDTA Fe.EDTA FeCl 3.6H 20
47.
A d d i t i o n of a n t i b i o t i c s
p e n i c i l l i n - G : supplied as benzyl p e n i c i l l i n sodium s a l t (Sigma Products,
1669 u n i t s mg * ) .
p o l y m i x i n B: supplied as the sulphate (Sigma Products, 7700 mg S.
streptomycin: supplied as sulphate w i t h 3.0 H O per mole (Sigma Products
745 u n i t s mg * ) .
Stock s o l u t i o n s were prepared i n deionized d i s t i l l e d water,
s t e r i l i z e d by f i l t r a t i o n (2.91 ( i i i ) ) and appropriate d i l u t i o n s were
added t o c o l d , s t e r i l e medium. S t e r i l e s o l u t i o n s of these a n t i b i o t i c s
were stored a t 4°C i n the dark.
2.3 Subculturing
Subculturing was c a r r i e d out w i t h standard aseptic techniques, the
s u b c u l t u r i n g process t a k i n g place i n a h o r i z o n t a l laminar flow cabinet
(confirming t o B.S. 5295 class 1 ) . This takes i n a i r a t the top through
a f i l t e r which f i r s t l y removes the large p a r t i c l e s and then passes the
a i r through a high e f f i c i e n c y p a r t i c u l a t e a i r f i l t e r , out h o r i z o n t a l l y
across the work surface i n a laminar flow p a t t e r n . On the day of sub
c u l t u r i n g , the inoculum m a t e r i a l was checked m i c r o s c o p i c a l l y f o r con
tamination and i n a d d i t i o n four p e t r i dishes, each w i t h a d i f f e r e n t
b a c t e r i a l t e s t medium (2.423), were p l a t e d w i t h a suspension of the
Anacystis nidulans before being used as the inoculum f o r the experiment.
2.31 L i q u i d
No d i f f i c u l t y was found i n o b t a i n i n g a uniform inoculum of
Anacystis nidulans due t o the f a c t t h a t t y p i c a l l y the alga does not clump and
suspends homogeneously. Experiments were commenced w i t h a d e n s i t y of
48.
(see B 52) 2 x 10^ u n i t s ml A l l t o x i c i t y t e s t s were c a r r i e d out i n 10 ml of
experimental s t e r i l i z e d medium i n b o i l i n g tubes. In some cases when
c h l o r o p h y l l a was used as a growth c r i t e r i o n , or i n the case of metal
uptake, 50 ml medium was used i n a 100 ml c o n i c a l f l a s k .
2.32 S o l i d
I n order t o o b t a i n i n d i v i d u a l c o lonies, Anacystis was p l a t e d on
t o an agar surface. For t h i s purpose, both low concentrations of agar
( 1 % W/V: A l l e n 1968) and EDTA (instead of c i t r a t e as a c h e l a t i n g agent:
Van Baalen 1965) were used.
The agar was mixed w i t h the mineral medium before autoclaving.
The inoculum was spread on the surface of the p l a t e s w i t h an alcohol
s t e r i l i s e d glass spreading rod.
2.33 Incubation and l i g h t source
Experiments were c a r r i e d out e i t h e r i n a growth room or a tank
of water w i t h a shaking mechanism. Attempts t o increase metal resistance
and stock c u l t u r e s were made i n standing c u l t u r e i n the t h e r m o s t a t i c a l l y
c o n t r o l l e d growth room maintained a t 32°C. The c u l t u r e s were shaken
by hand once a day. Some other standing experiments were c a r r i e d out
i n a 25°C room. Inocula f o r experiments and t o x i c i t y t e s t s were grown
i n a t h e r m o s t a t i c a l l y c o n t r o l l e d tank of d i s t i l l e d water maintained
at 32°C. A shaking mechanism moved the vessels through a h o r i z o n t a l
distance of 33 mm about 72 times per minute. The f l a s k s were i l l u m i n a t e d
continuously from beneath w i t h warm white f l u o r e s c e n t tubes. The l e v e l
of r a d i a t i o n a t the surface of vessels was found t o vary between d i f f e r e n '
areas i n the tank, p a r t i c u l a r l y at the edges; i t was however more or less
constant i n the middle of the tank, so t o avoid these v a r i a t i o n s , no
experimental vessel was incubated near the edges. I n a d d i t i o n each
v e s s e l was moved around d a i l y . Tubes were h e l d a t a s l a n t e d a n g l e
i n a w i r e cage i n o r d e r t o p r o v i d e t h e c i r c u l a t i o n o f media d u r i n g
s h a k i n g . P h o t o s y n t h e t i c a c t i v e r a d i a t i o n as measured by a Macam
Quantum/Radi ometer/Photometer Model Q 101 (Macam P h o t o m e t r i e s L t d ) and
l i g h t i n t e n s i t y as measured w i t h an EEL l i g h t m a s t e r p h o t o m e t e r (Evans
E l e c t r o s e l e n i u m L t d ) are summarized below:
g r o w t h chamber
32 C g r o w t h room o
25 C g r o w t h room
s h a k i n g t a n k
s h a k i n g t a n k w i t h w i r e cage
-2 -1 source o f p o s i t i o n o f p.E m s
f l u o r e s c e n t i l l u m i n a t i o n l i g h t
w h i t e
w h i t e
warm w h i t e
warm w h i t e
above
above
below
below
25- 40
25- 40
120-155
75- 95
l u x
2000-2500
2000-2500
3000-4500
2500-3000
L i g h t measurements by two methods n o t n e c e s s a r i l y made a t same t i m e 2.4 A l g a l c u l t u r e s
2.41 O r i g i n s V
Anacystis nidulans was o b t a i n e d f r o m t h e Cambridge C u l t u r e C o l l e c t i o n
o f Algae and P r o t o z o a ( 1 4 0 5 / 1 ) ; i t has a Durham r e f . no. 33A. I t i s t h e
s t r a i n f i r s t c h a r a c t e r i z e d by K r a t z and Myers (1955)and i s c o n s i d e r e d
i d e n t i c a l t o Synechococcus sp. PCC 6301, ATCC 27144 (Rippka e t a1. 1979).
Anacystis nidulans was chosen because i t i s t h e b l u e g r e e n a l g a which has
been most used b o t h f o r e x p e r i m e n t a l s t u d i e s i n g e n e r a l and s t u d i e s o f
m u t a t i o n i n p a r t i c u l a r .
D e t a i l s o f o t h e r s t r a i n s from c u l t u r e c o l l e c t i o n s and ones i s o l a t e d
f r o m s i t e s w i t h e l e v a t e d Zn a r e g i v e n i n Tab l e 2.7.
2.42 I s o l a t i o n and p u r i f i c a t i o n
Algae were i s o l a t e d by p l a t i n g ; t h e i n i t i a l d r i e d sample c o l l e c t e d
by Dr B. A. W h i t t o n was s p r i n k l e d d i r e c t l y o n t o agar. ACM medium was
used f o r Synechococcus,. Gloeothece-and Phormidium, w h i l e ChulO-D was
Table 2.7 Sources o f c u l t u r e s
o r ganism Durham c u l t u r e no
source whether whether a x e n i c c l o n a l
low Zn
Anabaena cylindrica 2 Cambridge 1403/22 + +
Aphanothece castagnei 551 G. A. Codd, Dundee
Calothrix membranacea 179 Cambridge 1401/1 +
C. parietina 550 Sand S i k e , Enaland (Zn, x 0.069 ± 0.006: Holmes and W h i t t o n , i n p r e s s )
+ +
h i g h Zn
Calothrix sp. 184 f r o m Zn t a n k ( S i n c l a i r and W h i t t o n 1977) } s u b c u l t u r e d a t 8 mg l - 1 Zn
+ +
Calothrix sp. 473 "La C r o i s e t t e R uisseau", Rhone V a l l e y , France = Durham code 3027-50 (st r e a m d r y , b u t sediments w i t h e l e v a t e d z i n c )
+ +
Gloeothece sp. 562 E l v i n s pond, M i s s o u r i (Zn, c. 8 mg l - 1 : W h i t t o n et al.t i n pr e s s )
Phormidium autumnale 475 Riou M o r t , France = Durham code 3010-98 (Zn, c. 16 mg I - 1 )
+
Phormidium sp. 476 R. Etherow t r i b u t a r y England (Zn, c. 15 mg I - * : H a r d i n g e t al., i n p r e s s )
+
Synechococcus sp. 561 E l v i n s pond, M i s s o u r i (Zn, c. 8 mg 1 _ 1 : W h i t t o n et al., i n pr e s s )
51.
used f o r a l l Calothrix s t r a i n s , w i t h the e x c e p t i o n t h a t f o r Calothrix
D 473, 0.5 mg 1 ^ N i was added (Ni a p p a r e n t l y f a v o u r e d h e a l t h y g r o w t h
o f Calothrix D 473 on i n i t i a l i s o l a t i o n , though l a t e r e x p e r i m e n t s have
f a i l e d t o show t h i s ) . C y c l o h e x i m i d e was added t o t h e media t o g i v e
a c o n c e n t r a t i o n o f about 10 ,ug ml * . For agar media, t h e c y c l o h e x i m i d e
was d i s s o l v e d i n d i s t i l l e d w a t e r , and 0.1 ml o f the s o l u t i o n p i p e t t e d
on t h e t o p o f t h e a l g a l g r o w t h on t h e agar s u r f a c e . C y c l o h e x i m i d e i s
a c t i v e a g a i n s t a wide range o f f u n g i , y e a s t s , and most e u k a r y o t i c a l g a e ,
b u t i s i n a c t i v e a g a i n s t most b a c t e r i a (Kapoor and Sharma 1979). B l u e -
green a l g a e a re t o l e r a n t , a t l e a s t i n low c o n c e n t r a t i o n s .
2.421 P h y s i c a l
I s o l a t i o n was c a r r i e d o u t by s u c c e s s i v e t r a n s f e r on agar p l a t e s .
T h i s method had t h e advantage t h a t g l i d i n g tended t o s e p a r a t e new areas o f
g r o w t h f r o m the o r i g i n a l , c o n t a m i n a t e d i n o c u l u m . Areas c o n t a i n i n g t h e
r e q u i r e d algae c o u l d t h e n be c u t o u t and t r a n s f e r r e d f o r f u r t h e r s u b c u l t u r e .
I t was n o r m a l l y p o s s i b l e t o e s t a b l i s h u n i a l g a l c u l t u r e s a f t e r r e p e a t e d sub
c u l t u r e s . C l o n a l and b a c t e r i a - f r e e c u l t u r e s o f t h r e e s t r a i n s o f Calothrix
were s u c c e s s f u l l y e s t a b l i s h e d a f t e r many t r a n s f e r s as f o l l o w s . Algae
from a young, v i g o r o u s l y g r o w i n g c u l t u r e were i n o c u l a t e d o n t o t h e m i d d l e
o f an agar p l a t e , and i n c u b a t e d under normal g r o w t h c o n d i t i o n s . A f t e r a
week o r so, t h e r e was u s u a l l y a zone o f hormogonia around t h e i n o c u l u m ,
s u f f i c i e n t l y w e l l s e p a r a t e d f o r i n d i v i d u a l s t o be p i c k e d o f f . A s u i t a b l e
a r e a f o r hormogonia was l o c a t e d by u s i n g a b i n o c u l a r d i s s e c t i n g m i c r o s c o p e ,
and a s i n g l e hormogonium was p i c k e d o f f u s i n g a v e r y f i n e n e e d l e , and
t r a n s f e r r e d t o a f r e s h agar p l a t e . A f t e r s u c c e s s i v e t r a n s f e r s on aga r ,
a hormogonium was t r a n s f e r r e d t o f r e s h l i q u i d medium. When v i s i b l e g r o w t h
was w e l l e s t a b l i s h e d , t h e a l g a was t e s t e d f o r b a c t e r i a l c o n t a m i n a t i o n .
2.422 A n t i b i o t i c s
A t t e m p t s t o o b t a i n a x e n i c c u l t u r e s o f Phormidium and Calothrix
by means o f exposure t o v a r i o u s a n t i b i o t i c s m i x t u r e s based on some
work by Droop (1967) were u n s u c c e s s f u l .
2.423 T e s t s o f p u r i t y o f t h e c u l t u r e s
T e s t s have been made w i t h the f o l l o w i n g media:
( i ) b e e f peptone agar
( i i ) m a l t e x t r a c t agar
( i i i ) y e a s t e x t r a c t agar
( i v ) n u t r i e n t b r o t h
(v) SST
The c o m p o s i t i o n o f those media was d e s c r i b e d by Hoshaw and Rosowski
(1973). The most e f f i c i e n t medium f o u n d was t h e u s u a l a l g a l g r o w t h
medium, supplemented w i t h 0.02% casamino a c i d ( B a c t o - D i f c o ) and 2%
g l u c o s e and s o l i d i f i e d by a d d i t i o n o f 1% (W/V) agar.
2.5 P r e p a r a t i o n o f a l g a f o r assay
2.51 E s t i m a t i o n o f g r o w t h
2.511 U n i t c o u n t s and e s t i m a t i o n o f c e l l l e n g t h
Most u n i t c o u n t s were c a r r i e d o u t u s i n g a haemocytometer 0.1 mm
deep (Improved Neubaur r u l i n g ) w i t h cover g l a s s e s . A f t e r about 5 min
s e t t l i n g t i m e , n e a r l y a l l t h e u n i t s were i n f o c u s . I n most samples
Anacystis nidulans d i d n o t e x i s t as s i n g l e c e l l s , b u t as a m i x t u r e o f
r o d s , f i l a m e n t s and sometimes s u b s p h e r i c a l shapes. The t e r m ' u n i t ' i s
used here t o cover t h e range o f m o r p h o l o g i e s seen w i t h t h e l i g h t m i c r o
scope, i n c l u d i n g r o d s o f v a r i o u s l e n g t h s , f i l a m e n t s a p p a r e n t l y made up
o f c l o s e l y a t t a c h e d r o d s , f i l a m e n t s w i t h o u t o b v i o u s c e l l u l a r d i v i s i o n s
and s u b s p h e r i c a l s t r u c t u r e s .
53.
Most measurements o f t h e u n i t s were made u s i n g an X40 o b j e c t i v e l e n s and e y e - p i e c e o f X10 m a g n i f i c a t i o n , w i t h an X2 m a g n i f i c a t i o n l e n s i n t h e l i g h t p a t h . W i t h t h i s system t h e s m a l l e s t e y e - p i e c e g r a t i c u l e u n i t o f 0.01 mm was e q u i v a l e n t t o 1.5 |im.
2.512 E x t r a c t i o n and e s t i m a t i o n o f p i g m e n t s
( i ) C h l o r o p h y l l a
The a l g a l suspensions were made up t o t h e o r i g i n a l volume w i t h
d i s t i l l e d w a t e r , and were h a r v e s t e d by vacuum f i l t r a t i o n t h r o u g h Whatman
GF/F g l a s s - f i b r e paper (2.91 ( i ) ) . The a l g a and t h e s o l v e n t (95% methanol)
were t h e n p l a c e d i n 30 ml McCartney b o t t l e s and i n c u b a t e d f o r 10 min i n a
darkened w a t e r b a t h a t 70°C, and t h e n r e f i l t e r e d . The f i n a l f i l t r a t e was
made up t o a s t a n d a r d volume. The c h l o r o p h y l l peaks were r e a d i m m e d i a t e l y
a f t e r e x t r a c t i o n u s i n g an u l t r a v i o l e t - v i s i b l e s p e c t r o p h o t o m e t e r ( P e r k i n -
Elmer 4 0 2 ) . A b s o r o t i o n s p e c t r a were r e a d a t 665 nm and c o r r e c t e d f o r
t u r b i d i t y by s u b t r a c t i n g t h e absorbance a t 750 nm. E x t r a c t s were t h e n
a c i d i f i e d by a d d i n g one dr o p o f I n A n a l a r HC1 i n the o p t i c a l c e l l c a r e f u l l y
mixed i n w i t h a p a s t e u r p i p e t t e ; t he absorbance a t 665 nm was th e n r e a d a g a i n .
N e u t r a l i z a t i o n o f t h e e x t r a c t s w i t h magnesium c a r b o n a t e was n o t used
because n e u t r a l i z a t i o n by magnesium c a r b o n a t e on a b s o r p t i o n s p e c t r a shows
no e f f e c t (Marker 1972).
C h l o r o p h y l l a was c a l c u l a t e d from t h e f o r m u l a g i v e n by Marker ( 1 9 7 2 ) ,
b u t here a d i f f e r e n t " a c i d f a c t o r " has been d e r i v e d . T h i s f o r m u l a can
be w r i t t e n as f o l l o w s : V Chi a (ug/sample) = 2.61 (A - A ) x — x 13.1
d a J-
A^ = absorbance a t 665 nm b e f o r e a c i d i f i c a t i o n A = absorbance a t 665 nm a f t e r a c i d i f i c a t i o n a V = volume o f e x t r a c t (ml) 1 = l i g h t p a t h o f o p t i c a l c e l l (cm)
13.1 = c o n s t a n t , assuming a s p e c i f i c a b s o r p t i o n c o e f f i c i e n t o f C h i a i n 95% methanol o f 76.07 1 g 1 cm 1
2.61 = c o n s t a n t d e r i v e d f r o m an a c i d f a c t o r o f (x 1.60 + 0.2)
The a c i d f a c t o r was c a l c u l a t e d from 12 samples of t h e Anacystis
nidulans. I n t h e case o f Calothrix D184 and Anabaena cylindrica
an e x p e r i m e n t a l maximum a c i d f a c t o r o f 1.8 was d e t e r m i n e d . I t was
c a l c u l a t e d a c c o r d i n g t o the method o f Marker (1972) :
absorbance a t 665 nm b e f o r e a c i d i f i c a t i o n absorbance a t 665 nm a f t e r a c i d i f i c a t i o n
u s i n g t h e mean o f a c i d f a c t o r , a c o n s t a n t o f 2.61 and 2.28 were
d e r i v e d f o r use i n t h e Chi a e q u a t i o n . T h i s c o n s t a n t was d e r i v e d
as f o l l o w s :
a c i d f a c t o r 1.62 „ r , c o n s t a n t = • •• , : = — — — = 2.61
acxd f a c t o r - 1 1.62 - 1
( i i ) P hycocyanin
The lysozyme t e c h n i q u e t o be d e s c r i b e d below was employed
s u c c e s s f u l l y . A range o f p h y s i c a l t e c h n i q u e s were t r i e d :
s o n i c a t i o n , m o r t a r and p e s t l e w i t h a c i d washed sand,
r a p i d r e p e a t e d f r e e z i n g and t h a w i n g i n t h e presence of 0.05 M phosphate,
use o f l i q u i d n i t r o g e n . A l l were n o t e f f i c i e n t i n r e l e a s i n g
p h y c o c y a n i n c o m p l e t e l y . Comparisons are g i v e n i n T a b l e 2.8.
Lysozyme e x t r a c t i o n was o r i g i n a l l y based on work bv C r e s p i e t a 1 .
( 1 9 6 2 ) . The a l g a was c o l l e c t e d from g r o w i n g c u l t u r e s bv c e n t r i f u q a t i o n
f o r 30 min at3000 x g. The s u p e r n a t a n t was d i s c a r d e d ; t h e r e s i d u e was
washed t w i c e w i t h d i s t i l l e d w a t e r . P r i o r t o p h y c o c y a n i n e x t r a c t i o n ,
c h l a was e x t r a c t e d f i r s t w i t h 80% acetone and d i s c a r d e d ( i n t h i s
e x p e r i m e n t o n l y , because methanol was f o u n d t o e f f e c t p h y c o c y a n i n
e x t r a c t i o n ) . I f c h l a was r e q u i r e d f o r co m p a r i s o n , t h e sample was
d i v i d e d i n t o two 25 ml a l i q u o t s ; one was used f o r c h l a e x t r a c t i o n
( u s i n g 95% m e t h a n o l ) , and t h e second was used f o r p h y c o c y a n i n . The
a l g a l p e l l e t , which was b l u e i n c o l o u r , was i n c u b a t e d w i t h 1 mg 1 '
55.
Table 2.8 Comparison between methods used f o r p h y c o c y a n i n e x t r a c t i o n f rom Anacystis nidulans. Lysozyme e x p e r i m e n t s c a r r i e d o u t i n d a r k a t 25°C; pH 6.8.
method % c o n t r o l
1) lysozyme i n c u b a t e d f o r 96 h 100
2) lysozyme i n c u b a t e d f o r 96 h + 30 m i n u t e s s o n i c a t i o n
100
3) lysozyme i n c u b a t e d f o r 48 h 96.7
4) lysozyme i n c u b a t e d f o r 36 h 81 .2
5) lysozyme i n c u b a t e d f o r 24 h 70.9
6) lysozyme i n c u b a t e d f o r 12 h 35.4
7) s o n i c a t i o n f o r 60 min 71 .6
8) s o n i c a t i o n f o r 45 min 62 .0
9) s o n i c a t i o n f o r 30 min 45.8
10) acid-washed sand e x t r a c t i o n u s i n g m o r t a r and p e s t l e
14.6
11) f r e e z i n g and t h a w i n g w i t h l i q u i d n i t r o g e n
35 .6
12) in vivo (no s t e p f o r any e x t r a c t i o n )
21.5
56.
lysozyme (muramidase, mucopeptide N - a c e t y l m u r a m o y l h y d r o l a s e ; E.C. No. 3.2.1.17) i n 0.05 M phosphate b u f f e r , a t pH 6.8, i n c u b a t e d a t 25°C i n t h e d a r k . At i n t e r v a l s , t h e tubes were
moved f r o m t h e d a r k , c e n t r i f u g e d f o r about 10 min ( t o f a c i l i t a t e
f i l t r a t i o n ) , and then f i l t e r e d t h r o u g h g l a s s - f i b r e paper and t h e s o l u t i o n
made up t o a s t a n d a r d volume w i t h 0.05 M phosphate b u f f e r . I t was f o u n d
t h a t p h y c o c y a n i n s t a r t e d t o degrade w i t h i n 5 days a f t e r i t had been
r e l e a s e d ; i t produced an odour r e s u l t i n g f r o m lysozyme d e t e r i o r a t i o n ;
a d d i t i o n o f NaCl o r i n c u b a t i o n a t 4°C h e l p e d t o c o n t r o l t h i s p r o b l e m .
Comparisons a r e g i v e n i n Tab l e 2.9.
The a b s o r p t i o n peaks o f p h y c o c y a n i n were r e a d d i r e c t l y a t 618 nm.
The c o n c e n t r a t i o n o f p h y c o c y a n i n was c a l c u l a t e d f r o m t h e f o l l o w i n g
e q u a t i o n i n whic h c o r r e c t i o n i s made f o r c h l o r o p h y l l a b s o r p t i o n
(Myers and K r a t z , 1955):
OD ph y c o c y a n i n = 1.016^, n - 0.203 ODr„^ 618 b I I
p e r c e n t p h y c o c y a n i n was t h e n d e t e r m i n e d by d i v i d i n g t h e c o r r e c t a b s o r p t i o n
o f 618 nm by 0.073, t h e e x t e n s i o n c o e f f i c i e n t o f C r a i g and C a r r ( 1 9 6 8 ) .
These v a l u e s were t h e n n o r m a l i s e d t o a mg d r y w e i g h t a l g a b a s i s .
2.513 Dry w e i g h t
A l g a l m a t e r i a l was s e p a r a t e d f r o m t h e g r o w t h medium by c e n t r i f u g a t i o n
f o r 30 min at3C0C x g, i n a c i d washed c e n t r i f u g e d t u b e s , washed t h r i c e
w i t h EDTA, f o r a c c u m u l a t i o n e x p e r i m e n t s . The l i q u i d was decanted o f f and
k e p t f o r f u r t h e r a n a l y s i s . The washed a l g a l p e l l e t was t r a n s f e r r e d t o
acid-washed snap-top g l a s s v i a l s ( p r e v i o u s l y d r i e d a t 105°C) and d r i e d
f o r 48 h a t 105°C. On removal f r o m t h e oven, t h e v i a l s were p l a c e d
i m m e d i a t e l y i n a d e s i c c a t o r t o p r e v e n t a b s o r p t i o n o f w a t e r as t h e y c o o l e d
t o ambient t e m p e r a t u r e . For a c c u m u l a t i o n s t u d i e s (Chapter 6) the a l g a
was weighed t o g e t h e r w i t h the N u c l e p o r e f i l t e r ( F i l t e r s w i t h o u t a l g a
were shown t o have areasonably C o n s t a n t 3ry w e i g h t , A f t e r d r y i n g
4-1 0
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c o •H -P rtJ
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58.
a t 105°C fo r 48 h, x = 3.2 mg + 0.068, n = 2 0 . ) .
2.514 T u r b i d i t y
In applying t h i s method i t i s n e c e s s a r y t h a t the c u l t u r e s are
w e l l shaken and evenly d i s t r i b u t e d . For t h i s reason a Vortex s t i r r e r
( G r i f f i n ) was used. F a c t o r s such as p r e c i p i t a t i o n of the medium, a i r
bubbles, contamination of the a l g a l suspension with b a c t e r i a can
i n t e r f e r e with the r e s u l t s . P r e c a u t i o n s were taken to avoid these
f a c t o r s . Clean tubes of uniform diameter were washed w i t h d i s t i l l e d
water a f t e r each s i n g l e r e a d i n g ; blanks were used f o r both autoclaved
and non-autoclaved media fo r i n d i v i d u a l sample. The d e f l e c t i o n caused by
the t e s t tube and media, which was estimated before the c u l t u r e s t a r t e d ,
was s u b t r a c t e d from the a c t u a l r e ading. High t u r b i d i t y was found a t
h i g h e r c o n c e n t r a t i o n s of phosphate, calcium, manganese or z i n c i n
autoclaved media. Comparisons are given i n Tables 10a and 10b. The a l g a was
checked a t the end of each experiment to be sure no b a c t e r i a l con
tamination had o c c u r r e d . Measurements were made with a Unigalvo
Nephelometer (Evans E l e c t r o s e l e n i u m L t d ) . The sample was i l l u m i n a t e d
with a l i g h t source, of the same colour as t h a t of the sample by u s i n g
a OGRI f i l t e r and p h o t o e l e c t r i c d e t e c t o r with a readout d e v i c e to
i n d i c a t e the i n t e n s i t y of s c a t t e r e d l i g h t .
A standard r e p r o d u c i b l e c a l i b r a t i o n curve was prepare!! ( F i g 2.1)
showing the r e l a t i o n s h i p between p o p u l a t i o n d e n s i t y and
t u r b i d i t y s c a l e s on the nephelometer. The p l o t was l i n e a r up to 8 -1 1 — 1 1.6 x 10 u n i t s ml f o r the w i l d - t y p e and U p to 7 x 10 u n i t s ml
fo r the Z n - t o l e r a n t s t r a i n s . I n a l l cases of doubt a d i r e c t count was
a l s o made, us i n g a haemocytometer.
2.52 Techniques used f o r comparison of t o x i c i t i e s
E s t i m a t e s of t o x i c i t y to a p a r t i c u l a r s t r a i n were made i n batch
c u l t u r e s on growth, l a g and i n some ca s e s f i n a l y i e l d .
T a b l e 2.10b. I n f l u e n c e o f Ca as CaC.l on t u r b i d i t y o f ACM medium
(see 2.514).
, - 1 , Zn (mg 1 ) 0 2 .5 5 .0
Ca
10
(mg
20
r S 40 80 160 320
0 2.0 2 .0 2 . 2 2.5 2 . 5 2.6 2.8 3.4 3.5
1.0 2.0 2 .0 2 . 5 2.5 2 . 5 2.5 2.8 3.4 3.5
1-.5 2.0 2 .2 2 .6 2.8 2 .5 2.8 2.8 3.6 3.6
2.0 4.0 4 .0 4 .0 4.0 4 . 5 4.6 4.5 4.5 4.8
2.5 4.5 4 .7 4 .8 4.6 4 .7 4 .8 4.8 6.0 6.0
3 .0 5.0 5 .5 5 .6 5.8 5 .7 5.7 5.8 6.2 6.2
3.5 5.0 6 .0 5 .8 6.0 6 .2 6.2 6.2 6.4 6.6
4.0 6.0 7 .0 7 .2 7.0 7 .0 7.0 7.5 7.5 7.5
4.5 6.2 7 .5 7 .5 7.6 7 .6 7.8 7.6 8.1 8.2
5.0 6.5 8 .0 8 .2 8.1 8 r 8.6 8.5 9.0 9.5
T a b l e 2 . 1 0 a . i n f l u e n c e o f Mn as MnCl^ on t u r b i d i t y o f ACM medium
(see 2.514).
Zn (mg 1 ) 0 0.05 0.10 0 5
Mn
1 .0
(mg 1
1 .5
l)
2 .5 5 10 15 20
0 1.2 2.0 2.0 2 .0 2.0 3.0 3 .0 4.0 5.0 5.0 6.0
1.0 1.2 2.0 2.0 2 .0 2.5 3.0 3 .0 4.0 5.0 5.0 6.0
1. 5 1.5 2.0 2.5 3 .0 3.0 3.0 4 .0 4.0 5.0 5.0 6.0
2.0 1 . 6 4.0 4.0 4 .0 4.0 4.0 5 .0 5.0 6.0 6.0 7.0
2.5 1 .8 4.5 4.0 5 .0 4.0 4.0 5 .0 5.0 6.0 7.0 7 .0
3 . 0 2.5 5.0 5.2 5 .6 5 . 6 5.6 6 .0 6.0 7 .0 7 . 4 7.5
3 . 5 3.0 5.0 5.0 6 .2 6 , 5 6.5 6 .5 6 . 6 7.0 7 . 4 7 . 4
4 . 0 3 . 5 6.0 6.0 6 .2 6 . 6 6 . 6 6 . 5 6,7 7 . 4 7 . 6 7 . 8
4 . 5 4.0 6.2 6.5 6 ,5 6 . 6 6 , 6 6 . 5 7,2 7 , 8 8 . 0 8 . 0
5 . 0 4 . 6 6 . 5 6 . 5 6 . 5 6 . 6 6 . 6 6 . 5 8 . 0 8 . 0 8 . 0 8 . 0
0>
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CM 00 i n oo fa OLxuw sjiun
A s e r i e s o f g r o w t h c u r v e s was made f o r d i f f e r e n t m e t a l l e v e l s
and d a t a f r o m t h e s e were used t o p l o t a f u r t h e r g r a p h f r o m w h i c h t h e
v a r i o u s i n d i c e s o f t o x i c i t y c o u l d be e s t i m a t e d .
( i ) T o x i c i t y based on g r o w t h r a t e i s e x p r e s s e d as t h a t c o n c e n t r a t i o n
o f m e t a l l e a d i n g t o a 50% r e d u c t i o n i n r a t e o f e x p o n e n t i a l g r o w t h as
compared w i t h a c o n t r o l grown i n b a s a l medium. I n most cases p a r t o f
t h e g r o w t h c u r v e was i n f a c t e x p o n e n t i a l , b u t when t h i s was n o t so, an
e s t i m a t e o f t h e f a s t e s t g r o w t h o b t a i n e d was used f o r t h e p r e s e n t
c a l c u l a t i o n .
( i i ) T o x i c i t y based on l a g i s expressed as t h e c o n c e n t r a t i o n o f
m e t a l l e a d i n g t o an i n c r e a s e d l a g (as compared w i t h t h a t o f t h e
c o n t r o l ) e q u a l t o t h e d o u b l i n g t i m e d u r i n g e x p o n e n t i a l g r o w t h o f t h e
c o n t r o l .
( i i i ) T o x i c i t y based on f i n a l y i e l d i s e x p r e s s e d as t h e c o n c e n t r a t i o n
o f m e t a l p e r m i t t i n g 12.5% o f t h e y i e l d o b t a i n e d w i t h t h e c o n t r o l .
( I n p r a c t i c e , y i e l d was e s t i m a t e d as t h e number o f u n i t s x mean l e n g t h
o f u n i t s ; t h i s n e g l e c t s p o s s i b l e changes i n c e l l d i a m e t e r . )
Other p o t e n t i a l l y ambiguous terms needed t o summarize t h e r e s u l t s
a r e d e f i n e d as f o l l o w s . A s t r o n g l y i n h i b i t o r y l e v e l o f a t o x i c
a gent r e f e r s t o t h a t l e v e l w h i c h j u s t p e r m i t s d e t e c t a b l e g r o w t h ; a
s l i g h t l y h i g h e r l e v e l k i l l s a l l c e l l s u n l e s s m u t a t i o n o c c u r s . An
e s t i m a t e o f t h e s t r o n g l y i n h i b i t o r y l e v e l can o n l y be a p p r o x i m a t e , b u t
p r o v i d e s a much q u i c k e r means o f making a comparison t h a n t h e t i m e
consuming methods d e s c r i b e d above. R e s i s t a n t and t o l e r a n t a r e used
i n t h e manner adopted by many w o r k e r s on h i g h e r p l a n t s . A r e s i s t a n t
o r g a n i s m i s one w h i c h can w i t h s t a n d r e l a t i v e l y h i g h l e v e l s o f a
p o t e n t i a l l y t o x i c a g e n t , w h a t e v e r t h e mechanism by w h i c h i t does t h i s ;
a t o l e r a n t s t r a i n i s one w h i c h s u r v i v e s h i g h e r l e v e l s due t o g e n e t i c
d i f f e r e n c e s from o t h e r s t r a i n s .
( i v ) T.I.C. ( T o l e r a n c e Index C o n c e n t r a t i o n ) . The assay was a
r e f i n e m e n t o f t h a t d e s c r i b e d by W h i t t o n (197Ca). Growth i n t h e f l a s k s
o r t u b e s was compared v i s u a l l y on days 2, 4, 6, 8 b o t h a g a i n s t
p r e s e r v e d r e p l i c a t e s o f t h e o r i g i n a l i n o c u l a and a l s o w i t h each t u b e
one a g a i n s t t h e o t h e r . O b s e r v a t i o n s were r e c o r d e d on each o c c a s i o n
as f o l l o w s :
I Maximum c o n c e n t r a t i o n o f m e t a l c a u s i n g no l a g i n a l g a l g r o w t h
I I Maximum c o n c e n t r a t i o n o f m e t a l c a u s i n g i n h i b i t i o n
I I I Maximum c o n c e n t r a t i o n o f whi c h a l g a i s a l i v e
IV Maximum c o n c e n t r a t i o n o f m e t a l r e q u i r e d t o k i l l a l g a
The T o l e r a n c e Index C o n c e n t r a t i o n was c a l c u l a t e d as = ( I . I I . I I I . IV)
2.6 P r o d u c t i o n o f r e s i s t a n t Anacystis s t r a i n s
2.61 NTG
The p r o c e d u r e was based on work o f Kumar (1968) , w i t h a n t i b i o t i c s
I n p r e p a r a t i o n f o r t h i s e x p e r i m e n t , e x p o n e n t i a l grown u n i t s were
h a r v e s t e d by M i l l i p o r e f i l t r a t i o n , washed w i t h M/20 T r i s - m a l e i c b u f f e r
o f pH 8.0, and suspended i n 20 ml o f T r i s - m a l e i c b u f f e r ( m i x t u r e o f
T r i s + m a l e i c a c i d , pH 5 . 0 ) . A l i q u o t s o f 0.2 ml fr o m a p p r o p r i a t e l y
d i l u t e d s u s p e n s i o n s were spread on agar p l a t e s , s o l i d i f i e d by
( 1 % W/V) a g a r , t o serve as c o n t r o l s . F r e s h l y p r e p a r e d N-methyl-N 1-
n i t r o - N - n i t r o s o g u a n i d i n e i n T r i s - m a l e i c b u f f e r (pH 5.0) was added
i n t o t h e r e m a i n i n g s u s p e n s i o n t o o b t a i n a f i n a l NTG c o n c e n t r a t i o n o f
about 400 ug ml *. The r e a c t i o n m i x t u r e was shaken f o r some t i m e
a f t e r m i x i n g . One ml a l i q u o t s were w i t h d r a w n a f t e r t i m e i n t e r v a l s
between 0 - 6 0 m i n u t e s , t h e n t h e u n i t s were washed t w i c e w i t h s t e r i l e
ACM (pH 7.0) on M i l l i p o r e f i l t e r s . The washed u n i t s were t h e n
suspended i n ACM medium t o o b t a i n t h e same u n i t c o n c e n t r a t i o n as i n
u n t r e a t e d c o n t r o l s , and 0.2 ml a l i q u o t s spread on agar p l a t e s . A l l
p l a t e s were s e a l e d w i t h p a r a f i l m and i n c u b a t e d a t 32°C f o r 10 days.
They were t h e n o v e r l a i d w i t h a t h i n l a y e r o f agar (0.7 W/V) c o n t a i n --1
i n g d i f f e r e n t Zn c o n c e n t r a t i o n s (2 up t o 12 mg 1 ) .
2.62 S e r i a l s u b c u l t u r e
see 3.2
2.7 Study o f e n v i r o n m e n t a l f a c t o r s
2.71 F a c t o r s i n f l u e n c i n g t o x i c i t y
D u r i n g t h e s t u d i e s o u t l i n e d below, t h e a d d i t i o n o f t h e s a l t s o f
th e i o n s , whose e f f e c t upon t o x i c i t y were b e i n g t e s t e d , caused
s i m u l t a n e o u s a d d i t i o n o f v a r y i n g l e v e l s o f Na, C I o r SO^. A s e r i e s
o f t o x i c i t y t e s t s were t h e r e f o r e p e r f o r m e d upon Anacystis nidulans
w i t h v a r y i n g l e v e l s o f NaCl o r Na^SO^ i n t h e medium.
The i n i t i a l pH i n a l l e x p e r i m e n t s t e s t i n g these f a c t o r s was k e p t
w i t h i n l i m i t s 7.0 ± 0.1 pH u n i t s u s i n g NaOH t o make t h e a d j u s t m e n t ,
e x c e p t on s t u d y o f e f f e c t o f Na on t o x i c i t y , when KOH was used. For
p r a c t i c a l purposes the e x p e r i m e n t a l p r o c e d u r e f o r i n v e s t i g a t i o n o f
each f a c t o r a f f e c t i n g m e t a l t o x i c i t y was v a r i e d s l i g h t l y .
( i ) Zn t o x i c i t y : i n o c u l a f o r each f a c t o r t e s t e d were o b t a i n e d from
c u l t u r e s i n c u b a t e d i n a medium c o n t a i n i n g a v e r y low l e v e l o f t h a t
f a c t o r ( i . e . i f t h e f a c t o r t o be t e s t e d was Mg, t h e a l g a l s t o c k was
i n c u b a t e d i n a medium w i t h 0.25 rag 1 1 Mg f o r 24 - 48 h p r i o r t o
i n o c u l a t i o n ) . For s t u d i e s on t h e e f f e c t o f phosp h a t e , c u l t u r e s t o be
used as i n o c u l a f o r t h e s t a n d a r d t o x i c i t y t e s t s were i n c u b a t e d i n
phosphate-free medium f o r four days p r i o r to the experiment. Two
other experiments were c a r r i e d out to demonstrate the e f f e c t or
or g a n i c phosphate on Z n - t o x i c i t y . S t e r i l i z e d ^ - g l y c e r o p h o s p h a t e
and <K -D glucose-l-phosphate (Sigma products) were added to s t e r i l e
medium a f t e r a u t o c l a v i n g to provide the same l e v e l of P as i n i n o r g a n i c
phosphate t e s t e d .
( i i ) Cu t o x i c i t y : The procedure used for Cu c l o s e l y followed t h a t of
Zn but due t o the g r e a t e r t o x i c i t y found with Cu a lower range of l e v e l s
was used i n the assay. Addition of Cu a t the c o n c e n t r a t i o n s used
caused no s h i f t i n the pH of ACM medium.
( i i i ) Cd t o x i c i t y : As with Cu, the procedure c l o s e l y followed t h a t
used with Zn, but again a lower range of Cd c o n c e n t r a t i o n s was used.
No pH s h i f t was encountered.
2 . 7 2 Inoculum s i z e
The s i z e of the inoculum on Z n - t o x i c i t y was t e s t e d only f o r the
w i l d - t y p e . The i n v e s t i g a t i o n s were c a r r i e d out by u s i n g a s e r i e s of
d i l u t i o n s of a l g a l suspension, with s t e r i l i s e d ACM medium as d i l u e n t .
Both c h l a and u n i t counts were used as c r i t e r i a f o r growth. The - 1 4 f i n a l u n i t s ml i n each v e s s e l a t the beginning of c u l t u r e were 1 0 ,
1 0 ^ , 1 0 ^ , and u n i t s ml
2 . 7 3 F a c t o r s i n f l u e n c i n g Zn s o l u b i l i t y
pH The experiment was c a r r i e d out simul t a n e o u s l y t o i n v e s t i g a t e
the e f f e c t s of v a r i o u s pH l e v e l s , on both the t o x i c i t y and the
s o l u b i l i t y of Zn. Four r e p l i c a t e s i n 1 0 0 ml f l a s k s were s e t up a t
d i f f e r e n t pH l e v e l s , i n c r e a s i n g by 0 . 5 u n i t s i n the range 6 . 0 to 8 . 0 ,
a t s i x l e v e l s of Zn, range 1 - 1 0 mg 1 *. The medium was b u f f e r e d and
a d j u s t e d to the r e q u i r e d pH, u s i n g 0 . 2 N NaOH or 0 . 2 N H C 1 . The f l a s k s
were incubated under the same c o n d i t i o n s as used for the a c t u a l a ssay,
A f t e r a p e r i o d o f t i m e each f l a s k was f i l t e r e d and t h e l e v e l o f Zn
was measured. The c r i t i c a l range o f pH f o r s i g n i f i c a n t p r e c i p i t a t i o n
o f f i l t e r e d Zn i n ACM medium was pH 6.5 - 8.0. The r e s u l t s a r e g i v e n
i n T a b l e 2.11.
EDTA: see (2.23)
C a l c i u m The p r o c e d u r e was r e p e a t e d w i t h f o u r Ca l e v e l s . Changes
i n t h e l e v e l o f Ca i n t h e range 5 - 1 0 0 mg 1 * has a v e r y s l i g h t e f f e c t
on t h e l e v e l o f f i l t r a b l e Zn i n ACM medium (Table 2.12).
Phosphate The e x p e r i m e n t was p e r f o r m e d s i m u l t a n e o u s l y w i t h t h e
i n v e s t i g a t i o n o f t h e e f f e c t o f d i f f e r e n t c o n c e n t r a t i o n s o f phosphate on
th e Zn t o x i c i t y . A w h i t e t o y e l l o w i s h p r e c i p i t a t e appeared i n most
h i g h phosphate c o n c e n t r a t i o n s w i t h Zn a t above 5 mg 1 S t h i s
p r e c i p i t a t e may perhaps be due t o t h e w h i t e Zn-phosphate p r e c i p i t a t e
Zn (PO.)„ w h i c h i s i n s o l u b l e i n w a t e r :
T h i s p r e c i p i t a t e was n o t v i s i b l e i f t h e phosphate was added a f t e r
a u t o c l a v i n g . Comparisons o f a u t o c l a v e d and n o n - a u t o c l a v e d media are
g i v e n i n T a b l e 2.14. Samples were a n a l y s e d f o r Zn, i n d i c a t i n g t h a t
70 - 75% o f Zn was i n s o l u t i o n a f t e r f i l t r a t i o n i n n o n - a u t o c l a v e d
medium i n a comparison o f t h a t 46 - 50% i n a u t o c l a v e d medium.
2.8 E x p e r i m e n t a l p r o c e d u r e f o r a c c u m u l a t i o n s t u d i e s
2.81 EDTA washing -1
I n a p r e l i m i n a r y e x p e r i m e n t i t was f o u n d t h a t 40 mg 1 EDTA
was t h e most e f f i c i e n t l e v e l f o r removing a l l the absorbed Zn from
t h e s u r f a c e o f t h e c e l l s . Comparisons a r e g i v e n i n Table 2.15.
An u p t a k e e x p e r i m e n t was c a r r i e d out, i n which Anacystis nidulans
3 4 2
3ZnS0..7H.O + 2K HPO 4 » Z n 3 ( P Q 4 ) 2 + 2K 2S0 4 + H 2S0 4 + 7 ^ 0
Table 2.11 I n f l u e n c e o f pH on z i n c s o l u b i l i t y on ACM medium
(10 mg 1 _ 1 EDTA + HEPES : Table 2.2); medium a u t o c l a v e d
and t h e n l e f t t o s t a n d f o r 24 h b e f o r e e x p e r i m e n t .
(See a l s o Tables 2.5, 2.12, 2.13, 2.14).
o r i g i n a l Z J I (mg 1 *)
1 .0
2 .0
4.0
6.0
8.0
10.0
6 .0
99
100
97
95
93
90
Zn measured a f t e r f i l t r a t i o n
pH v a l u e s
6.5 7.0 7.5 8.0
99
100
90
88
85
78
100
99
68
56
50
46
94
88
55
42
34
26
90
72
38
24
18
10
67.
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T a b l e 2.14 I n f l u e n c e o f a u t o c l a v i n g and n o n - a u t o c l a v i n g on
Zn s o l u b i l i t y i n ACD medium (pH 7.0 + HEPES + 10 mg 1 _ 1
EDTA: Tabl e 2 . 2 ) ; medium a u t o c l a v e d and l e f t t o s t a n d f o r
24 h b e f o r e e x p e r i m e n t . (See a l s o Tables 2.5, 2.11, 2.12,
2.13, 2.14).
o r i g i n a l % Zn measure
a f t e r f i l t r a t i o n % Zn
b e f o r e measure f i l t r a t i o n
Zn (mg 1 1 ) a u t o c l a v e d
non-a u t o c l a v e d a u t o c l a v e d
non-a u t o c l a v e d
r.o 100 + 3.8 100 + 2 .6 100.1 + 4 . 2 98 + 8.2
2.0 99 + 1 . 5 98 + 2 .6 100. 5 ± 2. 5 100 ± 1.6
3.0 80 ± 2.4 95 + 3 . 7 100 ± 7 . 5 97 + 4.4
4.0 68 + 1 .7 94 ± 2 . 5 100.3 ± 7 . 6 100 + 2.5
5.0 57 ± 4.7 77 + 5 .6 100 X 2. 0 100 ± 9.6
6.0 58 + 4.7 84 + 1 .3 99 + 1 . 1 100 + 1.4
7.0 51 ± 1. 3 77 + 8 .2 99 ± 1 . 3 99 ± 1 .5
8.0 50 ± 4.7 69 ± 8 .2 99 ± 2 . 8 98 ± 1.3
9.0 50 + 4.7 66 + 1 .4 99 ± 1. 8 100 + 1.3
10.0 46 + 4.7 65 ± 8 .2 98 ± 4. 3 99 + 9.6
70.
T a b l e 2.15 I n f l u e n c e o f EDTA (pH 5.4) on removal o f Zn from Anacystis
nidulans; a l g a s t i r r e d f o r 5 min i n c o n t a c t w i t h 10 ml o f
EDTA a t 4°C, th e n c o l l e c t e d by c e n t r i f u g a t i o n .
EDTA Zn l e f t Zn washed d r y w e i g h t ji.g Zn g (mg l - 1 ) i n t h e media w i t h EDTA (mg 1 _ 1 ) d r y w e i g h t
<mg l " 1 ) (mg l " 1 )
0 0.41 30 19400 0.40 52 11360 0.45 96 6072.9 0.39 174 3030
0.5 0.41 0.12 36 12638 0.39 0.14 52 8884.6 0.39 0.09 94 5691.5 0.38 0.10 178 2520.2
5 0.38 0.33 32 11625 0.38 0.28 50 6720 0.36 0.16 100 4120 • 0.37 0.14 172 2427
10 0.38 0.37 32 8375 0.40 0.32 50 6320 0.40 0.26 98 3440 0.39 0.21 172 2213.5
20 0.38 0.40 32 6812.5 0.39 0.35 52 4923 0.40 0.26 100 3250 0.39 0.22 170 2026.3
40 0.40 0.58 30 400 0.37 0.60 56 446.43 0.38 0.54 96 645.8 0.38 0.48 174 701.03
80 0.40 0.58 30 366.6 0.38 0.56 50 480 0.38 0.60 98 489.8 0.37 0.56 176 494.74
71 .
c e l l s w h i c h had t a k e n up Zn were suspended i n 40 mg 1 EDTA (pH 5.4).
The a l g a l c e l l s were s t i r r e d f o r 5 min i n c o n t a c t w i t h measured
volume o f EDTA a t 4°C, by u s i n g a v o r t e x s t i r r e r . The c e l l s were
c o l l e c t e d by c e n t r i f u g a t i o n w i t h c o o l i n g u s i n g a MSE MISTRAL 4 1
c e n t r i f u g e . The p r o c e d u r e was r e p e a t e d t w i c e o r sometimes t h r i c e ,
t h e s u p e r n a n t s b e i n g d e c a n t e d each t i m e i n t o a c i d washed s n a p - t o p
v i a l s and s t o r e d i n a r e f r i g e r a t o r , w i t h a d r o p o f A r i s t a r HNO^ u n t i l
a n a l y s i s ( u s u a l l y w i t h i n t h e p e r i o d o f t h e e x p e r i m e n t ) .
2.82 A c i d d i g e s t i o n
A l l samples o f t h e a l g a were d r i e d f o r 48 h a t 105°C i n a c i d
washed snap-top g l a s s v i a l s and c o o l e d i n a d e s i c c a t o r . D i g e s t i o n i n 1 ml
b o i l i n g AristarHNO^ was t h e n c a r r i e d o u t i n t h e same v i a l s f o r a b o u t
20 m i n , i n w h i c h c l e a r s o l u t i o n was n e a r l y formed. D i g e s t s o l u t i o n s
were made up t o 5 ml i n volume i n a c i d washed v o l u m e t r i c f l a s k w i t h
d e i o n i z e d d i s t i l l e d w a t e r , s t o r e d i n t h e same v i a l s .
The r e a s o n f o r a l l t h e s t e p s o f d i g e s t i o n b e i n g i n t h e same v i a l
was t o a v o i d any chance o f c r o s s c o n t a m i n a t i o n o f Zn from one c o n t a i n e r
t o a n o t h e r . Three b l a n k s were i n c l u d e d w i t h each new b a t c h o f d i g e s t s :
( i ) b o i l i n g A r i s t a r HNO^ o n l y i n snap-top v i a l s ;
( i i ) d e i o n i s e d d i s t i l l e d water.;
( i i i ) f i l t e r d i g e s t e d w i t h b o i l i n g A r i s t a r HNO^.
2 .9 S p e c i a l i z e d t e c h n i q u e s
2.91 F i l t r a t i o n
Three t y p e s o f f i l t r a t i o n were used:
( i ) F i l t r a t i o n t h r o u g h Whatman GF/C o r GF/F g l a s s - f i b r e paper mounted i n t o
s i n t e r e d g l a s s d i s c s o f u l t r a - f i n e p o r o s i t y , with a s u i t a b l e h o l d e r ,
f i l t e r e d , under p r e s s u r e o r p a r t i a l vacuum. I t i s the s i m p l e s t method
and e f f i c i e n t f o r p i g m e n t e x t r a c t i o n .
( i i ) F i l t r a t i o n u s i n g a range o f membrane po r e s i z e s (0.22 urn,
0.45 urn M i l l i p o r e f i l t e r s , 0.2 am Nuclepore f i l t e r ) and Whatman GF/C
g l a s s - f i b r e paper was t e s t e d f o r m e t a l a n a l y s i s . A c i d washed d i s
p o s a b l e p l a s t i c s y r i n g e s were used t o pass medium or sample t h r o u g h
th e f i l t e r , w i t h 20 ml o f d e i o n i z e d d i s t i l l e d w a t e r b e i n g t h r o u g h
and d i s c a r d e d , b e f o r e t h e c o l l e c t i o n o f sample i n a c i d washed snap-
t o p v i a l s .
( i i i ) A s e p t i c f i l t r a t i o n t e c h n i q u e was based on ( i i ) . A f t e r mount
i n g a f i l t e r w i t h s m a l l pore s i z e ( u s u a l l y 0.22 um M i l l i p o r e ) i n
Swinnex p l a s t i c h o l d e r , t h e whole i t e m was a u t o c l a v e d . S t e r i l i z e d
d i s p o s a b l e p l a s t i c s y r i n g e s were used. The f i l t e r e d sample was
c o l l e c t e d i n a s t e r i l e empty f l a s k .
2.92 Atomic a b s o r p t i o n s p e c t r o p h o t o m e t e r
The a n a l y s i s o f m e t a l i o n s i n s o l u t i o n was d e t e r m i n e d by t h e
flame Atomic A b s o r p t i o n S p e c t r o p h o t o m e t e r ( P e r k i n - E l m e r 403) . The
aqueous samples are c o n v e r t e d i n t o t h e i r a t o m i c vapour by a s p i r a t i o n
i n t o a flame t h r o u g h w h i c h i s passed a beam o f l i g h t e n e r g y o f t h e
same ele m e n t b e i n g d e t e r m i n e d .
The atoms i n t h e flame absorb energy f r o m t h e beam and t h i s
a b s o r p t i o n i s r e l a t e d q u a n t i t a t i v e l y t o t h e c o n c e n t r a t i o n o f t h e
m e t a l i o n s p r e s e n t i n t h e sample.
Sta n d a r d s o l u t i o n s were i n c l u d e d a t t h e b e g i n n i n g and end o f a
run and a l s o p e r i o d i c a l l y d u r i n g l o n g e r r u n s . A b l a n k was r u n
n o r m a l l y between each sample o r s t a n d a r d t o v e r i f y b a s e l i n e s t a b i l i t y .
2.93 A c e t y l e n e r e d u c t i o n assays
A x e n i c c u l t u r e s o f Calothrix D 184 f r o m t h e h i g h z i n c s i t e and
Anabaena cylindrica f r o m an e n v i r o n m e n t l a c k i n g Z n - enrichment were
u s e d - i n a c e t y l e n e r e d u c t i o n s t u d i e s . The algapwere m a i n t a i n e d i n
l i q u i d medium (ADM, n i t r o g e n - f r e e ) ; 8.0 mg 1 1 Zn was i n c l u d e d i n t h e
medium f o r Calothrix D184. A l l e x p e r i m e n t s were i n c u b a t e d i n a s h a k i n g o — 2 — 1
t a n k m a i n t a i n e d a t 25 C and w i t h an i l l u m i n a t i o n about 120-155 pJEm s /
3000 - 4500 l u x (see 2.33). Algae i n the e x p o n e n t i a l g r o w t h phase were
used i n a l l e x p e r i m e n t s and Zn was added as ZnSO^.lH^O.
N i t r o g e n a s e a c t i v i t y was assayed u s i n g t h e a c e t y l e n e r e d u c t i o n
t e c h n i q u e d i s c u s s e d by Hardy e t al. ( 1 9 7 3 ) . Both a l g a e were c e n t r i f u g e d
under a x e n i c c o n d i t i o n s and t h e s u p e r n a t a n t decanted? t h e a l g a l p e l l e t
was t h e n washed t w i c e w i t h ADM and t h e suspension homogenized by
p a s s i n g t h e a l g a e g e n t l y two o r t h r e e t i m e s t h r o u g h a s t e r i l i z e d
s y r i n g e . A s t a n d a r d volume o f each a l g a was re-suspended i n f r e s h ADM
medium w i t h the r e q u i r e d c o n c e n t r a t i o n s o f z i n c . The algae were
i n c u b a t e d f o r 24 h, and sampled a t 2 h o u r l y i n t e r v a l s . Zero t i m e and
d a r k c o n t r o l s were i n c l u d e d i n a l l assays. A l i q u o t s (2 ml) o f a l g a e
were p l a c e d i n 7 ml serum b o t t l e s and t h e n s e a l e d w i t h p e r f o r a t e d
serum cap f i t t e d w i t h r u b b e r l i n e r s . 1 ml a c e t y l e n e (BOC) was i n j e c t e d
t h r o u g h t h e serum l i n e r w i t h a s y r i n g e . A n o t h e r s y r i n g e was used t o
e q u a l i z e t h e p r e s s u r e by v e n t i n g t h r o u g h t h e l i n e r . The serum b o t t l e s
were shaken w e l l t o a i d t h e d i s s o l v i n g o f t h e gases p r i o r t o p l a c i n g o 2 — 1 i n a s h a k i n g t a n k m a i n t a i n e d a t 25 C w i t h a 120-155 u.Em s /
3000 - 4500 l u x . F i v e r e p l i c a t e s were used i n each case. The b o t t l e s
were i n c u b a t e d f o r 90 m i n u t e s a f t e r t h e a d d i t i o n o f a c e t y l e n e . A t t h e
end o f t h e e x p e r i m e n t a l p e r i o d , gas samples were removed w i t h m u l t i p l e -
sample v a c u t a i n e r needles (Becton and D i c k i n s o n L t d ) and s t o r e d i n
n o n - s i l i c o n e c o a t e d , 5 ml draw v a c u t a i n e r s (Becton and D i c k i n s o n L t d ,
A3206, f o r m u l a 134).
A gas sample (1 ml) f r o m t h e s e a l e d v a c u t a i n e r was i n j e c t e d i n t o
a V a r i a n Aerograph s e r i e s 1200 gas chroraatograph equipped w i t h a
hydrogen flame i o n i z a t i o n d e t e c t o r , and a "Poropak" R (100/120 mesh)
1/8 i n c h by 6 f e e t (ca 3.0 mm x 1.7 m), s t a i n l e s s s t e e l column. The
o p e r a t i n g c o n d i t i o n s were as f o l l o w s : d e t e c t o r temperature 150°C;
column temperature 40°C? hydrogen flame r a t e 30 ml min S a i r 300 ml
min *; and n i t r o g e n 30 ml min *. Eth y l e n e peaks were i d e n t i f i e d on
r e c o r d e r t r a c e s by the r e t e n t i o n time and q u a n t i f i e d with standard
c u r v e s . The chromatograph was c a l i b r a t e d u s i n g d i l u t i o n s of high
p u r i t y e t h y l e n e (99.9% A i r Products Ltd) prepared using a Hamilton
gas s y r i n g e . A l i q u o t s of the standards i n c l u d i n g b l a n k s , were
i n j e c t e d i n t o the serum b o t t l e s c o n t a i n i n g an e q u i v a l e n t l i q u i d phase,
and incubated along with the experiment. At the end of the experiment
the standards were evacuated u s i n g v a c u t a i n e r s and then run through
the gas chromatograph. I n t h i s way any d e v i a t i o n i n the draw of a
batch of v a c u t a i n e r s i s e l i m i n a t e d .
The r e s u l t s of the a c e t y l e n e r e d u c t i o n a s s a y s were expressed as
the nMC H produced ug c h l a * min
75.
CHAPTER 3
PRODUCTION OF RESISTANT STRAINS OF ANACYSTIS NIDI!LANS
3,1 I n t r o d u c t i o n
A r e l a t i v e l y l a r g e number o f a n t i b i o t i c and d r u g - t o l e r a n t m u t a n t s
have been i s o l a t e d i n b l u e - g r e e n a l g a e , e s p e c i a l l y Anacystis nidulans
( s e c t i o n 1.3). On t h e o t h e r hand no such m e t a l t o l e r a n t s t r a i n s have
been r e p o r t e d .
3„2 NTG
The use o f NTG as a mutagenic agent ( s e c t i o n 2.6) f a i l e d t o l e a d
t o any d e t e c t a b l e i n c r e a s e i n t h e r a t e o f m u t a t i o n f o r Zn t o l e r a n c e . g
NTG a l s o f a i l e d t o l e a d t o d e t e c t a b l e m u t a t i o n (1 i n 5 x 10 u n i t s ) i n
c u l t u r e s l a c k i n g Zn e n r i c h m e n t .
3.3 S e r i a l s u b c u l t u r i n g
3„31 M e t a l s
I t p r o v e d easy t o i n c r e a s e t h e r e s i s t a n c e o f Anacystis t o a l l f i v e
heavy m e t a l s s t u d i e d (Co, N i , Cu, Zn, Cd) by r e p e a t e d s u b c u l t u r e s b e i n g
made fr o m a s t r o n g l y i n h i b i t o r y l e v e l t o a l e v e l j u s t l e t h a l t o ( w i l d -
type) Anacystis. Four f l a s k s were used f o r each s u b c u l t u r e i . e .
7
in o c u l u m = 2 x 10 u n i t s . I n any f l a s k o f t h e l a t t e r showing g r o w t h
a t l e a s t one f l a s k each t i m e was used as an i n o c u l u m f o r f u r t h e r
s e r i a l s s u b c u l t u r e . I n i n s t a n c e s where more t h a n one o f t h e f l a s k s
showed g r o w t h , t h e c u l t u r e was chosen w h i c h grew most r a p i d l y . The
pr o c e s s was r e p e a t e d many t i m e s , l e a d i n g t o g r a d u a l l y i n c r e a s i n g
' s t r o n g l y i n h i b i t o r y ' l e v e l s . For i n s t a n c e , t h e l e v e l o f Zn a t which
s t r o n g i n h i b i t i o n o c c u r r e d was r a i s e d f r o m 1.45 t o 16.5 mg 1 * a f t e r
75 s u b c u l t u r e s ( T a b l e 3.1). The l a g p e r i o d i n most cases was v e r y
76.
T a b l e 3.1 R e s i s t a n c e o b t a i n e d (so f a r ) on r e p e a t e d s u b c u l t u r e o f
Anacystis nidulans t o p r o g r e s s i v e l y h i g h e r m e t a l
c o n c e n t r a t i o n s . (Four f l a s k s used f o r each s u b c u l t u r e
i . e . i n o c u l u m = 2 x 10 u n i t s )
m e t a l no. s u b c u l t u r e s s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l (mg 1
Co
Zn
if
N i
Cu
Cd
25
25
75
25
25
25
w i l d - t y p e
0.32
1 .45
II
0.16
0.15
0.55
most t o l e r a n t s t r a i n
2 .45
5.5
16.5
1 .30
0.55
2.5
l o n g , sometimes more t h a n 48 h between each s u b c u l t u r e , b u t i t was
Shorty by r e p e a t e d s u b c u l t u r i n g a t l e a s t s i x t i m e s a t t h a t l e v e l
w h i c h caused t h e l a g .
D e t a i l s o f t h e maximum r e s i s t a n c e o b t a i n e d are g i v e n i n T a b l e 3.1
and t h e r a t e a t w h i c h r e s i s t a n c e to Zn was o b t a i n e d i s shown i n
F i g . 3.1. Colony f o r m a t i o n on agar w i t h d i f f e r e n t l e v e l s o f Zn
( T a b l e 4.2) showed t h a t t h e a c q u i s i t i o n o f i n c r e a s e d r e s i s t a n c e was
due t o t h e p r o d u c t i o n o f m u t a n t s .
The f i r s t m utants i s o l a t e d f r o m a w i l d - t y p e p o p u l a t i o n i n response
t o s t r o n g l y i n h i b i t o r y Zn had an i n c r e a s e i n t o l e r a n c e , based on t h e
c r i t e r i o n o f e x p o n e n t i a l g r o w t h , o f about 0.25 mg 1 * Zn. The mutants
c o r r e s p o n d t o t h o s e r e g a r d e d as spontaneous elsewhere i n t h e b l u e - g r e e n
a l g a l l i t e r a t u r e , b u t c r i t i c a l e x p e r i m e n t s d i d n o t r u l e o u t t h e
p o s s i b i l i t y t h a t Zn i t s e l f has a r o l e as a mutagenic agent. I t
p r o v e d i m p o s s i b l e t o d e m o n s t r a t e t h e presence o f m u t a n t s when making
s u b c u l t u r e s from medium l a c k i n g Zn e n r i c h m e n t . S u b c u l t u r e s o f 20
f l a s k s each w i t h 5 x 10^ u n i t s f r o m a l g a grown i n Z n - f r e e medium t o
a l e v e l j u s t l e t h a l t o ( w i l d - t y p e ) Anacystis, f a i l e d t o l e a d t o any g
i n c r e a s e i n t h e r a t e o f 'spontaneous' m u t a t i o n (1 i n 1 x 10 u n i t s )
f o r Z n - t o l e r a n c e .
A l l t h e m e t a l - t o l e r a n t s t r a i n s s t i l l grow w e l l i n b a s a l medium.
There was no i n d i c a t i o n t h a t t h e y now r e q u i r e h i g h e r l e v e l s o f these
m e t a l s f o r optimum g r o w t h . Only v e r y s l i g h t changes were d e t e c t a b l e
i n t h e l a g , e x p o n e n t i a l g r o w t h r a t e and y i e l d as compared w i t h t h e
w i l d - t y p e . Two o f t h e m u t a n t s w i t h h i g h t o l e r a n c e f o r Zn
78.
s V)
03 0) 0) 13
u Si
<0 0)
a) 3 o 0) 0
u
(/) (0
CO <U
tn
CP ro o (0 ro 8
M
ID in (Nl
d - i 6 u J ) u z
( Z n - t 5 . 0 , Z n - t l 2 . 0 mg 1 ) and one f o r each o f t h e o t h e r m e t a l s were
chosen f o r c o m p a r a t i v e s t u d i e s .
The c o m p a r a t i v e response o f t h e v a r i o u s s t r a i n s t o a p a r t i c u l a r
m e t a l was i n g e n e r a l q u i t e s i m i l a r whether judged by g r o w t h r a t e ,
l a g , o r y i e l d (Table 3.2); w i t h the c r i t e r i a used ( 2 . 5 2 ) , t h e e f f e c t
on y i e l d was g r e a t e s t . I t can be seen from Table 3.2 t h a t t h e p a r t i a l l y
s u b j e c t i v e e s t i m a t e s o f ' s t r o n g l y i n h i b i t o r y 1 g r o w t h a r e q u i t e s i m i l a r
t o t h e o b j e c t i v e e s t i m a t e s o f t o x i c i t y based on a 50% r e d u c t i o n i n
g r o w t h r a t e f o r f o u r o f the s i x m u t a n t s . I n d i v i d u a l g r o w t h curves f o r
t o l e r a n t s t r a i n s are g i v e n below; those f o r w i l d - t y p e Anacystis are
i n c l u d e d i n S e c t i o n 4.
C o - t l . 8 F i g . 3.2 and T a b l e A3.1
N i - t l . O 3.3 A3.2
Cu-t0.5 3.4 A3.3
Zn-t5.0 3.5 A3.4
Z n - t l 2 . 0 3.6 A3.5
Cd-t2.0 3.7 A3.6
S t a b i l i t y o f Z n - r e s i s t a n c e
S t r a i n s o f Anacystis r e s i s t a n t t o 5.0 and 12.0 mg 1 * Zn were
s u b c u l t u r e d i n t h e absence o f the m e t a l f o r 72 and 96 g e n e r a t i o n s
r e s p e c t i v e l y , and t h e n i n o c u l a t e d i n t o t h e i r r e s p e c t i v e Zn medium.
Growth curves a r e shown i n F i g s 3.8 and 3.9, w h i l e t h e r e s u l t s a re
summarized i n T a b l e 3.3. These g r o w t h c u r v e s may be compared w i t h
those i n F i g s 3.5 and 3.6, i n which a l g a e were s u b c u l t u r e d from a
s t r o n g l y i n h i b i t o r y l e v e l o f Zn. The Zn-t5.0 and Z n - t l 2 . 0 s t r a i n s
grew e x p o n e n t i a l l y a f t e r a l a g o f about 24 and 48 h, r e s p e c t i v e l y .
T h i s suggests t h a t r e s i s t a n c e t o Zn i s a s t a b l e t r a i t .
80.
T a b l e 3.2 T o l e r a n c e o f mutants used f o r e x p e r i m e n t s t o t h e m e t a l s
used i n t h e i r i s o l a t i o n . (For d e t a i l s o f a s s a y s , see
M a t e r i a l s and Methods)
s t r a i n
C o - t l . 8
B i - t - 1 .0
Cu-t0.5
Zn-t5.0
Zn-t-12.0
Cd-t2.0
t o x i c c o n c e n t r a t i o n o f m e t a l (mg 1 *) a c c o r d i n g t o d i f f e r e n t c r i t e r i a
l a g g r o w t h
1.9 1.1
0.95 0.82
0.35 0.45
4.1 4.8
8.8 11.2
1.5 1.35
y i e l d
0.30
0.20
0.12
4.0
6.0
0.50
24 48 72 96 120 144 160
time (h)
2 I n f l u e n c e o f Co on g r o w t h o f C o - t l . 8 ; i n o c u l u m was
t a k e n f r o m s t r o n g l y i n h i b i t o r y l e v e l s o f Co.
control . 0-2 mg r* 0-4 0- 8 1- 0
24 48 72 96 120 144 168
time (h)
I n f l u e n c e o f N i on g r o w t h o f N i - t l . O j i n o c u l u m was
t a k e n f r o m s t r o n g l y i n h i b i t o r y l e v e l s o f N i .
i o 5 - i
2U LA 72 96 120 1 U 168
time (h)
F i g . 3.4 I n f l u e n c e o f Cu on g r o w t h o f Cu-t0.5; i n o c u l u m was
t a k e n f r o m s t r o n g l y i n h i b i t o r y l e v e l s o f Cu.
-o control . 2 mg 1-1 Zn
U
1U 168
time (h)
F i < ? - 3.5 I n f l u e n c e o f Zn on g r o w t h o f Z n - t 5 . 0 ; i n o c u l u m was
t a k e n f r o m s t r o n g l y i n h i b i t o r y l e v e l o f Zn.
83.
^control ^ - A mg 1-1 Zp
^ 1 1 8 10
12
D 10
10
10
1 i i i i i i i i i 0 Ik L8 72 96 120 144 168 192 226
time (h)
F i g . 3,6 I n f l u e n c e of Zn on growth of Zn-tl2.0f inoculum was
taken from s t r o n g l y i n h i b i t o r y l e v e l of Zn.
control - * 0-5 mg I 8 TO
•0
1-5
3 10
10
10
0 24 48 72 96 120 144 168
time (h)
x 5 - 3.7 I n f l u e n c e of Cd on growth of Cd-t2.0; inoculum was
taken from s t r o n g l y i n h i b i t o r y l e v e l of Cd.
84.
o control
8 10
3 10
10
10
control 2 mg I
4
5
0 24 48 72 96 120 144 168
time (h)
F i g . 3.8 I n f l u e n c e of Zn on growth of Zn-t5.0, grown f o r
72 c e l l g e n e r ations i n b a s a l medium.
control e 10 4 mg I 8 10 11 12
D 10
10
10
0 24 48 72 96 120 144 168
time (h)
F i g . 3,9 I n f l u e n c e of Zn on growth of Z n - t l 2 . 0 , grown f o r
96 c e l l g e n e r a t i o n s i n b a s a l medium.
>1 X) C s 0
JC w
r-l
•P 0 Cr> C 0 in c 0 •H 4-1 • m 1 4-1 rH c Q) B o c w 0 4-> u •H
C c 3 to W
rd C rt) TS 0> U) M c 3 0 W •H id 4-1 d) •H 6 T3 C x: 0 4-1 U 0 X3 u 4-> 0
• tn o
CN 0 r H
•H •p M 1 Oj c
N M-4 0 TS
C QJ rO U C O d) » 3 in
i—1 +J m i C c H tsj
xi rtJ Eh
03
3 u O C
Uh o & o
r - l
o
a .
I
6
C N
r - l 3 U O C
• r |
144 O QJ U u 3 0
C • r l
113
00
C c (0 x: 4J M d) c 0 > 1
r - l 4-1 x: CP •H
(1) c o
o in
4J I C
C
4-1 H 0) CP C O
4-) X! •H
C O
in CN
r H
W A sl
A
| i i r- in i d
m r H
*x> CN
| i i *X> oo »-h CN
o O
CM •cf o *r CN CN r - l
CO CO CN
o r- ro H
r H CN in in CO CN ro n
CO CM in to I 1 ^>
CM m CN CO r H
in in N c c 0 0 1 •
S •H •H 1 3 4-> •p rH •r-l rd rd -a rl rl CP
0) 0) 6 s c C Q) o rH Cn CP • rrj CN 0) co •£i t-H (0 CO a> XI e XI rl o
o 0 rl H 144 mH En
4-1 I c CS3
86.
3.32 A n t i b i o t i c s
A s i m i l a r p r o c e d u r e was used t o i s o l a t e s t r a i n s t o l e r a n t t o
3 mq 1 ' s t r e p t o m y c i n , and 1.25 mg 1 * p e n i c i l l i n ; t h i s t ook about 4
and 11 s u b c u l t u r e s r e s p e c t i v e l y . A t th e s e c o n c e n t r a t i o n s t h e
r e s i s t a n t s t r a i n s d i d however show a 50% r e d u c t i o n i n g r o w t h r a t e .
The presence o f Zn a t a s u b - i n h i b i t o r y l e v e l d i d n o t l e a d t o any Q
i n c r e a s e i n t h e r a t e o f 'spontaneous' m u t a t i o n (1 i n 1 x 10 u n i t s )
f o r r e s i s t a n c e t o e i t h e r a n t i b i o t i c .
3 .4 Morphology
M o r p h o l o g i c a l changes were n o t e d a t t h e h i g h e r c o n c e n t r a t i o n s
o f m e t a l s and i n some cases t h e r e was c o n s i d e r a b l e d i v e r s i t y w i t h i n
a s i n g l e f l a s k . The changes were n o t f o l l o w e d i n d e t a i l b u t r e p r e s e n
t a t i v e forms are shown i n F i g . 3.10. No marked d i f f e r e n c e i n c e l l
l e n g t h were observed i n w i l d - t y p e w i t h any m e t a l up t o t h e s i x t h day
a f t e r i n o c u l a t i o n . I n o l d e r c u l t u r e s , however, t h e average c e l l l e n g t h
o f m e t a l - t r e a t e d a l g a was s l i g h t l y g r e a t e r t h a n t h a t o f t h e c o n t r o l , and
a t t h e s t r o n g l y i n h i b i t o r y l e v e l o f Cu, s u b s p h e r i c a l u n i t s were o f t e n
formed. The Co, N i , Zn and C d - t o l e r a n t s t r a i n s i n g e n e r a l showed a
s i m i l a r b e h a v i o u r when grown i n t h e absence o f m e t a l , a l t h o u g h t h e
average l e n g t h o f t h e u n i t s o f b o t h Z n - t 5.0 and Zn-t 12.0 was g r e a t e r .
A t t h e l e v e l s o f m e t a l s s u b - i n h i b i t o r y f o r each o f these s t r a i n s ,
f i l a m e n t s were p r o d u c e d , o c c a s i o n a l l y r e a c h i n g 100 x o r i g i n a l l e n g t h
i n Z n - t 5.0 and Zn-t 12.0. C y t o l o g i c a l s t u d i e s were n o t made t o
e s t a b l i s h t h e e x t e n t t o whic h these were t r u l y m u l t i c e l l u l a r o r o n l y
c o e n o c y t i c l i k e t he mutants d e s c r i b e d by Kunisawa and Cohen-Bazire (1970) .
The d i s t r i b u t i o n o f these u n i t s i n t h e f l a s k s a t v a r i o u s i n t e r v a l s d u r i n g
g r o w t h ( i n b a t c h c u l t u r e ) a r e g i v e n i n F i g s 3.11, 3.12, 3.13 and
Tab l e s A3.7, A3.8, A3.9.
88 Zn-t 120 Zn- t 5 0 A Wild-type
B
Cu-t 0 50 o 10 urn
100 u ^ 80 cr
U. 60
40 20
/ /
/
Wild- type 24 h
7~ / /
/ / / / / / / / / / / / -•' /
004 0-5 0-75 VO Zn (mg
1-25 1-1)
100 o 5 80 cr
i t 60
48 h.
40
20
7 / / / / / / / / / / / / / /
/ / / / /
0-04 0-5 075 1-0 Zn (mg
1-25 -1)
u c ^ 80 c r CD
L L 60 40
20
/
0-04 0-5 075 1-0
• 3«6pm 0 6S9pm 0 9 s 12pm
Zn (mg
12 5 24pm 24 S 36pm 36 S 48 pm
96 h
1-25 -1)
100 o § 80 O"
60 40
20
004
P7]
j i 0-5 075
I 1-0
192 h
1-25 Zn (mg M)
F i g - 3.11 I n f l u e n c e o f Zn and t i m e o f h a r v e s t i n g i n b a t c h
c u l t u r e on l e n g t h o f Anacystis.
7 n « 5 - 0 *.-.>.••
CD
| T 60
I n 40
20
/
0 0 4 1-0 2-0 3-0 4-0 5-0
Zn (mg H)
48h 100
0) 80 c r PI
l T 60
/, 40
20 ^5
c i 3 0-04 1-0 2-0 3-0 4-0 5-0
Zn (mg M)
96 h 100
0) 80
IT 60
40
20
0-04 1-0 2-0 3-0 4-0 5-0
Zn (mg M) 0 3«6>jm 0 12«24>jm Q 6S9) jm S 24S36>jm 0 9«12pm Q 36«48^jm
192 h 100
5i 80
2! 2 3
7 40 0
20
. 2 0-04 1-0 20 3-0 4-0 50
Zn (mg H )
F i g . 3.12 I n f l u e n c e o f Zn and t i m e o f h a r v e s t i n g i n b a t c h
c u l t u r e on l e n g t h o f Z n - t 5 . 0 ; i n o c u l u m from b a s a l medium.
24 h ion
0> I I I ! >
40
J3 20
2 0<I4 1-0 2-0 3-0 4-0 5-0
Zn (mg H)
48h 100
80
I T 50
40
20
4-0 5-0 3-0 2-0 •0 00 4
Zn (mg H)
^ 1 0 0 u c ^ 80 cr
i t 60
- ° ° 40
20
96 h
9 / / J
mm 0 0 4 1-0 2-0 30
D 3«6>jm Q 12S24>jm
Q 6 S 9 j j m i 24S36pm
Q 9S12>jm 1 36S48pm
0-04 1-0
4-0 5-0
Zn (mg H )
192 h
3-0 4-0 5-0
Zn (mg (-1)
F i g . 3.13 I n f l u e n c e o f Zn and t i m e o f h a r v e s t i n g i n b a t c h
c u l t u r e on l e n g t h o f Zn-t 5 . 0 ; i n o c u l u m f r o m
s t r o n g l y i n h i b i t o r y l e v e l o f Zn.
The morphological response to Cu was d i f f e r e n t . I n h i b i t o r y
l e v e l s of Cu, the w i l d - t y p e , Cu-t 0.5 and both Z n - t o l e r a n t s t r a i n s
a l l formed many s u b s p h e r i c a l u n i t s , q u i t e d i f f e r e n t i n shape from the
normal rods. I n most cases the populations were markedly heterogeneous,
with rods about 3.2 u.m to 6.3 urn, s u b s p h e r i c a l u n i t s , and f i l a m e n t s
about 30 urn to 100 urn, but a t l e a s t i n the case o f Cu-t 0.5 the sub
s p h e r i c a l u n i t s often formed the bulk of the p o p u l a t i o n . Although
unequivocal evidence was not obtained, i t i s almost c e r t a i n t h a t the
u n i t s were capable of growth i n t h i s form. A s u b s p h e r i c a l form
s i m i l a r to those found a t high l e v e l s of Cu was noted a l s o with
Co-t 1.8 grown a t high Co l e v e l s , but here these u n i t s formed l e s s
than 1% of the p o p u l a t i o n .
CHAPTER 4
TOLERANCE AND TOXICITY STUDIES ON ANACYSTIS NIDULANS STRAINS
4.1 M e t a l s
Comparative s t u d i e s were made o f the r e l a t i v e t o x i c i t y o f Co, N i
Cu, Zn, Cd, Hg and Pb t o Anacystis nidulans s t r a i n s , namely w i l d - t y p e
C o - t l - 8 , N i - t l . O , Cu-t0.5, Z n - t 5 . 0 , Z n - t l 2 . 0 and Cd-t2.0. T o x i c i t y
t e s t s were p e r f o r m e d under c o n t r o l l e d l a b o r a t o r y c o n d i t i o n s u s i n g
g r o w t h r a t e and sometimes a l s o l a g ( s e c t i o n 2.52) as i n d i c a t i o n o f
responses.
The i n f l u e n c e o f each m e t a l on t h e g r o w t h o f a l l v a r i o u s s t r a i n s
( w i l d - t y p e , C o - t l . 8 , N i - t l . O , Cu-t0.5, Z n - t 5 . 0 , Z n - t l 2 . 0 and Cd-t2.0)
i s shown i n a s e r i e s o f f i g u r e s .
Two sources o f i n o c u l u m were used f o r each m e t a l i n v e s t i g a t e d :
a) f r o m b a s a l medium; b) from s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t • i
w h i c h s t r a i n was adapted.
4.11 C o b a l t The i n f l u e n c e o f v a r i a t i o n i n l e v e l o f e n v i r o n m e n t a l Co
on t h e g r o w t h o f w i l d - t y p e F i g . 4.1 a, b Table A4.1
N i - t l . O 4.2 a, b " A4.2
Cu-t0.5 4.3 a, b " A4.3
Zn-t5.0 4.4 a, b " A4.4
Z n - t l 2 . 0 4.5 a, b " A4.5
Cd-t2.0 4.6 a, b " A4.6
4.12 N i c k e l w i l d - t y p e 4.7 a, b " A4.7
C o - t l . 8 4.8 a, b " A4.8
Cu-t0.5 4.9 a, b " A4.9
Zn-t5.0 4.10 a, b " A4.10
Z n - t l 2 . 0 4.11 a, b " A 4 . l l
Cd-t2.0 4.12 a, b " A4.12
control 0-02 mg I"1 Co 0-04 e 10 0-08 0-10
0-16 ZJ 10
0-20
0-32
10
10 5 \
0 24 48 72 96 120 144 168
time (h)
control 0-02 mg n Co 0-04 0-08 8 10 0-10
0-16
0-20 o 10
0-40
10
1 0 5 -
i i i i ' i ' 0 24 48 72 96 120 144 168
time (h)
F i g . 4 . 1 I n f l u e n c e o f Co on g r o w t h o f w i l d - t y p e Anacystis;
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f Co
control O05 mg [~> Co 0-10 0-20 8 10
0-40
D 10 0-60
10
0-80
10
I I I I I I I
0 24 48 72 96 120 144 168
time (h)
control 0^05 mg H Co
8 10 040 0-20
0-40 ZJ 10
0-60 10
10
0 24 48 72 96 120 144 168
time (h)
F i g . 4.2 I n f l u e n c e o f Co on g r o w t h o f N i - t l . O ;
a) i n o c u l u m from b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f
m e t a l a t whic h adapted
control 0-05 mg H Co
8 10 0-10 0-20
0-40
0-60
10 - A —
1 0 5 -
0 24 48 72 96 120 144 168
t ime (h)
-o control
- a 0-05 mg I'1 Co 8 10
0-10 10
D 10 0-20
0-40
10
0-06 10
i • 1 : , , f = _ ,
0 24 48 72 96 120 144 168
t ime (h)
F i g . 4.3 I n f l u e n c e o f Co on g r o w t h o f Cu-t0.5;
a) i n o c u l u m from b a s a l medium
b) i n o c u l u m from s t r o n g l y i n h i b i t o r ' / l e v e l o f m e t a l a t which adapted
97.
control —o 0-05 mg I
0-10
— a 0-20
0-30
o-vo
2U £8 72 96 120 1 U 168
time (h)
control -a 0-05 mg M
V.U 168
time (h)
4,4 I n f l u e n c e o f Co on g r o w t h o f Z n - t 5 . 0 ;
a) i n o c u l u m from b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f
m e t a l a t w h i c h adapted
b
control
0-05 mg |-1 Co 8 10 0-10
0-20
=J 10
0-30
10 - e 0<0
A
10
I I 1 I I I I 0 2U L8 72 96 120 UA 168
time (h)
o control
8 0-05 mg M Co 10
0-10
0-20 3 10
0-30
0-40 10
1 0 5 -
i • ' i l l • I I 0 2e> W 72 96 120 168
time (h)
F i g . 4.5 I n f l u e n c e o f Co on g r o w t h o f Z n - t l 2 . , 0;
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t w h i c h a d a p t e d
9 9 .
o control o 0-2 mg I
10 8
0-6
D 10
0-8
10
10
24 48 72 96 120 144 168
time h)
-o contra
0-2 mg I e 10 8*
08 r> 10
1-0
10
10
0 24 48 72 96 120 144 168
time (h)
F i g . 4.6 I n f l u e n c e o f Co on g r o w t h o f C d - t 2 . 0
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f
m e t a l a t w h i c h a d a p t e d
1 (JO.
control
O 0 2 mg I"" Ni 8 10 0-04
0-08
0-10
r j 10 0-16
0-20 10
10
0 24 48 72 96 120 144 168
time (h)
contro
0-02 mg 1-1 N 0-04 8 10
0-08
0-10 I/)
0-16 D 10
0-20
10
10
0 24 48 72 96 120 144 168
time (h)
F i g . 4.7 I n f l u e n c e o f N i o n g r o w t h o f w i l d - t y p e
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f N i
l O ) .
control
0-05 mg I 8 0-10 10
0-15
Z3 10
0-20
10
10
7U £8 72 96 120 1 U 168
time h)
control 0-05 mg I
0-10 e 10
0-15
• 10
0-20
10
10
i 2U 72 96 120 168
time h
F i g . 4.8 I n f l u e n c e o f N i o n g r o w t h o f C o - t l . 8
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f
me L a i a t w h i c h a d a p t e d V
102.
control
0-05 mg 1-1 Ni 8 10
0-10
0-15
O 10
0-20
10
10
I
0 24 48 72 96 120 144 168
time (h)
10°
£ to
D 10'
10° -
10b
time (h)
o control
0-05 mg M Ni
0-20
F i g . 4.9 I n f l u e n c e o f N i on g r o w t h o f C u - t 0 . 5
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f
m e t a l a t w h i c h a d a p t e d
103.
control
O05 mq I 8 10
0-10
=) 10 0-20
0-22
10 0-25
10
168 144 96 120 72 24 48
time h)
control
0-05 mq I 8 10
0-10 to D 10
0-20
10 - A 0-22
A
10
0 96 120 144 24 48 72 168
time h
F i g . 4.10 I n f l u e n c e o f N i on g r o w t h o f Z n - t 5 . 0
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t w h i c h a d a p t e d
104.
-o control
0-05 mg M Ni 8 10
0-10
D 10 0-15
0-20 10
—A
10
0 24 72 96 20 48 144 168
time h
T 10 8
e
c => 10 7
10b
10b
-o control
O05 mg l"1 Ni
0-10
0-15
144 168
time (h)
F i g . 4.11 I n f l u e n c e o f N i on g r o w t h o f Z n - t l 2 . 0
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t w h i c h a d a p t e d
105.
contra 0 05 mg H Ni
8 0-10 10
0-15
=> 10
0-20
10
, , , i— 1 i 1 0 24 48 72 96 120 144 168
time (h)
control
10 0-20
=> 10 0-25
10 0-30
ioM * 1 r- ( • i ' r — — = i 0 24 48 72 96 120 144 168
time (h)
g. 4.12 I n f l u e n c e o f N i on g r o w t h o f C d - t 0 . 2
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t w h i c h a d a p t e d
106.
4.13 C o p p e r w i l d - t y p e F i g . 4.13 a, b T a b l e A4.13
C o - t l . 8 4.14 a, b " A4. 14
N i - t l . 0 4.15 a, b " A4. 15
Z n - t 5 . 0 4.16 a, b " A4.16
Z n - t l 2 . 0 4.17 a, b " A4.17
C d - t 5 . 0 4.18 a, b " A4. 18
4.14 Z i n c w i l d - t y p e 4.19 a, b " A4.19
C o - t l . 8 4.20 a, b " A4.20
N i - t l . 0 4.21 a, b " A4.21
C u - t 0 . 5 4.22 a, b " A4.22
C d - t 2 . 0 4.23 a, b " A4.23
4.15 Cadmium w i l d - t y p e 4.24 a, b " A4.24
C o - t l . 8 4.25 a, b " A4.25
N i - t l . 0 4.26 a, b " A4.26
C u - t 0 . 5 4.27 a, b " A4.27
Z n - t 5 . 0 4.28 a, b " A4.28
Z n - t l 2 . 0 4.29 a, b " A4.29
I n f l u e n c e o f Zn on g r o w t h o f w i l d - t y p e a nd Z n - t l 2 . 0 a r e g i v e n l a t e r
i n F i g s 6.1 and 6.2 r e s p e c t i v e l y ; d r y w e i g h t was u s e d as a g r o w t h
c r i t e r i o n .
4.16 M e r c u r y a n d l e a d Two f u r t h e r m e t a l s , Hg a n d Pb, a l t h o u g h n o t
u s e d f o r t h e m a j o r i t y o f s t u d i e s w e r e t e s t e d f o r t h e i r t o x i c i t y t o t h e
w i l d - t y p e ( F i g s 4.30, 4 . 3 1 : T a b l e A 4 . 3 0 , A4.31) r e s p e c t i v e l y . A
m a r k e d i n f l u e n c e o f Hg was n o t e d a t v e r y l o w c o n c e n t r a t i o n s ; f o r
- 1
i n s t a n c e t h e a l g a was k i l l e d a t 0.06 mg 1 Hg; i n c o n t r a s t Pb was n o t
t o x i c a t l e v e l s u p t o 30 mg 1 ^ Pb.
The d a t a f o r c r o s s - r e s i s t a n c e o f s t r a i n s t o l e r a n t t o one p a r t i c u l a r
m e t a l t o t h e o t h e r f o u r m e t a l s a r e s u m m a r i z e d l a t e r ( T a b l e 8 . 1 ) .
control a 0-01 mg I
0-02 A 0-04
0-08
0-10
0-12
0-16
48 72 96 120 144 168
time (h)
control 0-01 mg l" 1
0-02 0-04 0-08
0-10
0-12
0-16
i i 1 — r ' 48 72 96 120 144 168
time (h)
f l u e n c e o f Cu o n g r o w t h o f w i l d - t y p e Anacystis
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f Cu
1 Ofi.
o control
0 0 5 mg I"1 Cu 8 > 10 0-10
7 => 10 0-15
10
10
168 96 120 144 72 24 48
time (h
control
0O5 mg 1-1 Cu
0-10
0-15
144 168
time (h)
F i g . 4.14 I n f l u e n c e o f Cu o n g r o w t h o f C o - t l . 8
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t w h i c h a d a p t e d
control
10 0-05 mg I
0-10
r> 10
0 - 5
10
0-20
* 0
24 48 72 96 120 144 168
time h)
control
8 10 0-05 mg I
3 10
0-10
10
A 0-15
10
' 1 I 1 I I I I
0 24 48 72 96 120 144 168
time (h)
F i g . 4.15 I n f l u e n c e o f Cu on g r o w t h o f N i - t l . O
a) i n o c u l u m f r o m b a s a l m e dium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f
m e t a l a t w h i c h a d a p t e d
110.
-o control -a 0-05 m 05 mg C 1 Cu
0-10 e 10
to 0-20
-> 10
0-25
10
10
0 24 48 72 96 120 144 168
time (h)
control
0-05 mg r 1 Cu e 10
0-10
D 10 0-20
0-25 10
ioM * — - — — - i 1 i - i — • • 1 1 1 0 24 48 72 96 120 144 168
time (h)
I'-'iq. -1 . H. I n f l u e n c e o f Cu on g r o w t h o f Z n - t 5 . 0
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t w h i c h a d a p t e d
1 1 1 .
o control e 10 0-05 mg I"1 Co
0-10
to 0-20
O 10
0-25
10
10
2U US 72 96 120 1 U 168
time h)
control
8 10 0-05 mg f 1 Cu t
0-10
D 10
0-20 6 10
10
0 2L i.8 72 96 120 )U 168
time (h)
F i g . 4.17 I n f l u e n c e o f Cu on g r o w t h o f Z n - t l 2 . 0
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f
m e t a l a t w h i c h a d a p t e d
1 1 2 .
trot 8 com 0-05 mg H Cu
8 10 0-10
10 0-15
0-20 10
10
0 24 48 72 96 120 144 168
time (h)
_ 10 8 -
E
§ io 7 H
10 B -
1 0 5H
24 48 72 96
i 120
control
0-10 mg 1-1 Cu
0-15
0-20
0-25
0-30
144 168
time (h)
F i g . 4.18 I n f l u e n c e o f Cu on g r o w t h o f C d - t 2 . 0
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f
m e t a l a t w h i c h a d a p t e d
! 1 3
—o cont ro
0-5 mg l"1 Zn
1-0
1-25 8 10
1-5
Z> 10
10
10
0 2L 4& 72 96 120 H A 168
time (h)
control
0-5mg I"1 Z n
1-0 1-25
8 10
1-5
3 10 1-75
10
10
0 2 U « 72 96 120 \LL 168
time (h)
F i g . 4.19 I n f l u e n c e o f Zn o n g r o w t h o f w i l d - t y p e Anacystis
a) i n o c u l u m f r o m b a s a l medium
b ) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f Zn
114.
— 10° i E i / )
-*—*
C 7
r j 107 H
106
105
r 2U 48
-o control
—i
72 — r -96
1-5
I l 1
120 1U 158
time (h)
^ 108 H
1
C 7
=3 107 J
10"
10s
24 48 — r -72 96
-o control
-t> 0-5 mg I
1-0
1-25
120 144 168
time (h)
F i g . 4.20 I n f l u e n c e o f Zn on g r o w t h o f C o - t l , 8
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t which adapted
! 1 ~>.
c o n t r o l 0-5 mg r 1 Zn
10 8
1-0 I/)
r j 10
1-5
10
10
168 24 96 120 48 72 144
time h)
control
144 168
time (h)
F i g . 4,21 I n f l u e n c e o f Zn on g r o w t h o f N i - t l . O
a) i n o c u l u m from b a s a l medium
b) i n o c u l u m from s t r o n g l y i n h i b i t o r y l e v e l o f
m e t a l a t whic h adapted
10"
—r— 2L 72 96
— i 1 1
120 1 U 168
time (h)
^- 10
- o control
0-5 mg H Zn
time (h)
F i g . 4.22 I n f l u e n c e o f Zn on g r o w t h o f Cu-t0.5
a) i n o c u l u m from b a s a l medium
b) i n o c u l u m from s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t w h i c h adapted
10 5 H
- r -2U
—r~ 48 72
"T" 96 120 1U 168
time (h)
— 10
control 1-0 mg 1-1 Zn
time (h)
F i g , 4.2 3 I n f l u e n c e o f Zn on g r o w t h o f Cd-t2.0
a) i n o c u l u m from b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f
m e t a l a t w h i c h adapted
118.
control 0-15 mg |-1 Cd s
8 10 0-30
0-35
0-40 5 10 0-50
0-60
10
10
24 48 72 96 120 144 168
time h)
control . 0-15 mg 1-1 Cd
0-30
040
0-50
0-60
24 i
48 72 96 120 144 168
time (h)
F i g . 4.24 I n f l u e n c e o f Cd on g r o w t h o f w i l d - t y p e Anacystis
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f Cd
119.
o control 0-05 mg I e 10 0-10
0-20 0-30
D 10
u-40 10
10
24 48 72 96 120 168 144
time h)
-o control
0-05 ma M
144 168
time (h)
F i g . 4.25 I n f l u e n c e o f Cd on g r o w t h o f C o - t l . 8
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m from s t r o n g l y i n h i b i t o r y l e v e l o f
m e t a l a t whic h a d a p t e d
120.
control 0-05 mg [-' Cd 10
0-10
0-15 3 10
0-20
10
10
0 2U 96 120 LB 72 168
time h)
control
, - 108
I E
E 107
106
105
0-05 ma H Cd
UA 168 time (h)
F i g . 4.26 I n f l u e n c e o f Cd on g r o w t h o f N i - t l . O
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f
m e t a l a t w h i c h a d a p t e d
121.
control 8 10 0-05 mg I
0-1
D 10
10
10
1 1 1 1 I I =i
0 2U 48 72 96 120 %U 168
time (h)
control
005 mg
time h
F i g - 4.27 I n f l u e n c e o f Cd on g r o w t h o f Cu-t0.5
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f
m e t a l a t whic h adapted
122.
control
* r 10 0-20 mg H Cd
1 U 168
time (h)
control
* 0-20 mg M Cd 0-30
(WO 0-50 0-60
0-80
1-0
1 U 168
time (h)
F i g . 4.28 I n f l u e n c e o f Cd on g r o w t h o f Zn-t5.0
a) i n o c u l u m f r o m b a s a l medium
b) i n o c u l u m f r o m s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t w h i c h adapted
2U 48 72
96 120
4.29 Influence of Cd on growth of Zn-tl2.0
a) inoculum from basal medium
b) inoculum from strongly inhibitory l e v e l of metal at which adapted
1 U 168
time fh)
124.
control
O01 mg I 8 10
-o f>02
D 10 0-03
O04 0-05
10
10
0 24 48 72 96 120 144 168
time (h)
4.30 I n f l u e n c e o f Hg on g r o w t h o f w i l d - t y p e Anacystis.
o control
10 s
e 10 mg r 1
15
20 3 10
30
6 10 35
10
24 48 72 96 120 144 168
time (h)
F i g . 4.31 I n f l u e n c e o f Pb on g r o w t h o f w i l d - t y p e Anacystis.
125
A comparison was made o f t h e t o x i c i t y o f Zn t o Anacystis i n
l i q u i d and i n agar. As a p r e l i m i n a r y e x p e r i m e n t showed t h a t t h e
c o l o n y f o r m a t i o n on p l a t e s was dependent on t h e g r o w t h s t a g e o f t h e
a l g a ( T a b l e 4 . 1 ) , u n i t s were t a k e n from g r o w t h s t a g e B. The a l g a was
s l i g h t l y l e s s t o l e r a n t t o Zn i n l i q u i d t h a n w i t h agar. For i n s t a n c e ,
i n l i q u i d t h e a l g a was i n h i b i t e d s t r o n g l y a t 1.5 nig 1 1 Zn and d i e d
a t 1.75 mg 1 ' Zn; i n s o l i d medium, t h e c o m p a r a t i v e v a l u e s were -1 -1 2.25 mg 1 Zn and 2.5 mg 1 Zn, r e s p e c t i v e l y . The f u l l r e s u l t s a r e
g i v e n i n Table 4.2.
A comparison was a l s o made o f t h e t o x i c i t y o f Zn t o Anacystis i n
l i q u i d s h a k i n g ( F i g . 3.6) and s t a n d i n g c u l t u r e s ( F i g . 4.32). The
a l g a had m a i n l y t h e same t o l e r a n c e t o Zn i n l i q u i d i n b o t h c o n d i t i o n s ,
e x c e p t t h a t , s h a k i n g c u l t u r e was f a s t e r t h a n s t a n d i n g c u l t u r e . The
g e n e r a t i o n t i m e o f s h a k i n g c u l t u r e was 9.7 h i n comparison t o t h a t o f
16.5 h i n s t a n d i n g c u l t u r e .
Summary t o 4.1
The r e s u l t s p r e s e n t t h u s f a r i n d i c a t e marked d i f f e r e n c e s i n t h e
t o x i c i t y o f m e t a l s t o t h e t e s t Anacystis s t r a i n s ( T a b l e 4.3). Exposure
o f a c u l t u r e t o a p a r t i c u l a r m e t a l o f t e n l e d t o a s l i g h t i n c r e a s e i n
t o l e r a n c e t o t h a t m e t a l d u r i n g t h e n e x t s u b c u l t u r e , even w i t h o u t m u t a t i o n
I n t h e case o f w i l d - t y p e s l i g h t decreases i n l a g were n o t e d w i t h a l l
seven m e t a l s and i n c r e a s e s i n g r o w t h r a t e w i t h Co and Zn. A l l m e t a l s
e x h i b i t e d s i m i l a r i n h i b i t o r y e f f e c t s w i t h i n c r e a s i n g c o n c e n t r a t i o n s .
These i n c l u d e d t h e d e p r e s s i o n o f g r o w t h r a t e , i n c r e a s i n g l a g , some
m o r p h o l o g i c a l changes i n u n i t s , and e v e n t u a l l y d e a t h . I n g e n e r a l t h e
o r d e r o f m e t a l t o x i c i t y f o r each s t r a i n f r o m t h e p r e v i o u s r e s u l t s a r e :
T a b l e 4.1 I n f l u e n c e o f g r o w t h stage on c o l o n y - f o r m i n g a b i l i t y
o f w i l d - t y p e Anacystis nidulans on 1% agar p l a t e s ; n = 5
g r o w t h s t a g e
A young a l g a ( l a g phase)
B i n c r e a s i n g g r o w t h r a t e
C e x p o n e n t i a l phase
D o l d c u l t u r e
p o p u l a t i o n u n i t s c o l o n i e s d e n s i t y o f p e r p e r o r i g i n a l p l a t e p l a t e c u l t u r e s ^
( u n i t s m l - )
s.d.
2.0 x io~
5.2 x 10
9.6 x 10
1.8 x 10
20
50
100
19.5
51 .5
88.7
180 125
+ 1.3
+ 5.6
± 4.6
r e c o v e r y
97.5
± 3 . 4 103
88.7
69.4
3 O O c
• H
i—I <C • H P • H C • H
W <D •P US
r H
a
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r H 0
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W P r H P to (1) rJ
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ft in P • H c D
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en a
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-a r H • H 3 1 0
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in m cn O vO • CO oo 0 0 VD VD CM n vo LTl VD VO in m > ^ o
IS) + 1 + I -H -H + 1 44 + 1 - H
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I X X X X X X X X X o in L O L D m CO i n in i n
C I o-i CM CM CM CM
r H 0 r~ r- r- r- CO vO
4 H M o 0 -P o vO *—t vo C N CM m C o r» in CM
o « 0 *—i o
in oo p- m in • • • • • • > « • o vD oo vo co CO VD ro • *—< CM CM
1 0
- H - H + 1 • H - H - H - H -ft
0 0 O co in m O n • • o m P- CO CTi 0 0 r- O CO
r~ in vo o VO
o m o O r - f
in CM
m o CM
m CM m
CM
128.
«- CD
CM
1 // / / /
CN CN
CN •
CP
00 T3 ( 0
O
4-1
CN
CP
CD
CM
CN CO
Cn
CM
00
,.]uu sjiun
CO d>
r-H X! <d
I t s 0
01 c
•rH m N
P wi P c 3 aj o > 1
• P > 1 r-l i r-H 0
r H P i d c •rH
. 0 X! 0 d) W -rH
e oi 4 J X i d to c d ) •r-l
0 U u
01 Q) 0) r H r-l U i d <d c 01
4-) i d i d d ) P XI 6 01
•rH
u 01 0! > 1
x : M > i -p i—i 0 0 C D> p
• H C •r-l 0 0 XI
P QJ dJ u •rH tn 01 4-) X!
d ) C i d 01 C u i d • H
c X i d 0 u
+ J c r H 0) •r-l i d
•rH 01 oi i d d ) XI rH
c E - H D
r-H . — 3 o O
0 •p c
• H
in 0 4H
0 M 0 x : -p • P o 3 <d o
UH M cn
i d w
C 0 i d I H
4-1 T3 dJ
M 01 d ) 3
• P i d 01 3
T3 ~-— d )
6 oi <D Cn C c i d • H x ; id u i n
p 0)
u
c
U
C
U
O U
• H 2
O U
3 U
• H
o u
0 u
co
p I o u
o
p i I
•rH
z
o p
I 4J
I c to
o CNI p
I
u
d )
W i l d - t y p e Hg> N i > Cu> Co> Cd > Zn > Pb
C o - t l . 8 Cu> N i > Cd> Zn
N i - t l . 0 Cu> Cd > Co> Zn
Cu-tO.5 N i > Cd> Co > Zn
Zn-t5.0 N i = Cu > Co > Cd
Z n - t l 2 . 0 N i > Cu> Co> Cd
Cd-t2.0 N i > Cu> Co> Zn
4.2 A n t i b i o t i c s
A b r i e f a t t e m p t was made t o compare t h e t o x i c e f f e c t s o f t h r e e
a n t i b i o t i c s , p e n i c i l l i n , p o l y m y x i n and s t r e p t o m y c i n on t h e g r o w t h o f
w i l d - t y p e , Zn-t5.0 and Z n - t l 2 .0 Anacystis s t r a i n s . I t was c l e a r
f r oro t h e r e s u l t s ( T a b l e 4.4) t h a t r e l a t i v e l y low c o n c e n t r a t i o n s o f
a n t i b i o t i c s were t o x i c t o a l l t h e Anacystis s t r a i n s t e s t e d . The w i l d -
t y p e was however i n each case more s e n s i t i v e .
4.3 NTG
The g r o w t h response o f w i l d - t y p e Anacystis t o NTG ( F i g 4.33)
d i f f e r e d a c c o r d i n g t o whether p o p u l a t i o n d e n s i t y o r C h i a was used as
a c r i t e r i o n o f g r o w t h . The r e d u c t i o n i n g r o w t h was more pronounced
i n t h e l a t t e r ; f o r i n s t a n c e a t 2.0 mg 1 1 NTG, t h e c e l l s s t i l l d i v i d e d ,
and gave a s l i g h t l y l o n g e r u n i t (4 x 10 u n i t ml) t h a n u n t r e a t e d a l g a ,
w h i l e c h l a was reduced t o about 0.5 mg 1 The c o l o u r o f the a l g a l
s u s p e n s i o n a t h i g h e r NTG l e v e l s was p a l e y e l l o w .
4.4 Pigment c o m p o s i t i o n
D u r i n g t h e r o u t i n e e x p e r i m e n t a l s t u d i e s , no s h i f t s i n t h e
a b s o r p t i o n maxima o f t h e methanol e x t r a c t i o n s (430 nm, 665 nm) were
observed as a r e s u l t o f t r e a t m e n t w i t h t h e heavy m e t a l s . There was
131.
T a b l e 4.4 Comparison o f response o f w i l d - t y p e and Z n - t o l e r a n t s t r a i n s
t o p e n i c i l l i n , p o l y m i x i n and s t r e p t o m y c i n . T o x i c i t y e s t i m a t e d
as c o n c e n t r a t i o n l e a d i n g t o a 50% r e d u c t i o n i n g r o w t h r a t e .
s t r a i n t o x i c a g e n t
w i l d - t y p e
Zn-t5.0
Z n - t l 2 . 0
p e n i c i l l i n p o l y m i x i n s t r e p t o m y c i n -1 -1 -1
(u.g ml ) (|ig ml ) (ug ml )
0.01 0.0! 0.01
0.06 0.065 0.06
0.08 0.058 0.06
Ta b l e 4.5 Comparison o f Chi a c o n t e n t p e r u n i t l e n g t h o f a l g a
i n Z n - t r e a t e d c u l t u r e s ; n = 8.
Chi a -1 s t r a i n s C h i a (mg 1 )
X s .d.
w i l d - t y p e 3.41 + 0.65
Zn-t5.0 2.86 + 0.52
Z n - t l 2 . 0 2.65 + 0.48
( H 6ui) 5 i o o o CSI ^- o 7 CT) /
(0
0> 4J CO
i n ID
(0 0) a.
T3 0)
•a
CO
-a
(SI ft
en
00 p~ ID ID o o o o [»|uu sjiun
however a p p a r e n t l y a decrease i n c h l a c o n t e n t p e r u n i t l e n g t h o f a l g a
i n Z n - t r e a t e d c u l t u r e s ( T a b l e 4„5)„ I n a d d i t i o n no changes i n phyco:
c h l a r a t i o were o b s e r v e d as a r e s u l t o f Z n - t r e a t m e n t . I n C d - t r e a t e d
a l g a , however, v i s u a l changes i n t h e c o l o u r o f t h e a l g a were o b v i o u s
i n e a r l i e r s t a g e s o f g r o w t h . The c u l t u r e s became an i n t e n s e b l u e
c o l o u r ; t h e s e l a t e r r e v e r t e d t o g r e e n . T h i s sequence t o o k p l a c e
i m m e d i a t e l y a t lo w e r Cd l e v e l s (0.005 mg 1 1 ) b u t a t h i g h e r l e v e l s
(0„25 mg 1 *) t h e r e was an i n i t i a l p e r i o d when t h e y remained g r e e n
( i . e . g r e e n — > b l u e g r e e n ) . Pigment e x t r a c t s ( F i g , 4.34; T a b l e A4„32)
c o n f i r m e d t h a t t h e s e changes were t h e r e s u l t o f marked d i f f e r e n c e s i n
p h y c o : c h l a r a t i o .
4.5 Zn r e q u i r e m e n t f o r g r o w t h
An e x p e r i m e n t was d e s i g n e d t o d e t e r m i n e t h e minimum and maximum
c o n c e n t r a t i o n s o f Zn f o r t h e optimum g r o w t h o f w i l d - t y p e and two
Z n - t o l e r a n t s t r a i n s o f Anacystis ( F i g . 4„35). The l o w e s t p o s s i b l e
l e v e l o f Zn o b t a i n e d i n ACM medium was 0„04 mg 1 1 , d e r i v e d as
c o n t a m i n a n t s ( s e c t i o n 2 02)„ T h i s e x p e r i m e n t f a i l e d t o d e m o n s t r a t e
any s t i m u l a t i o n o f g r o w t h e i t h e r a t t h a t minimum c o n c e n t r a t i o n t e s t e d , -1 -1 0„04 mg 1 Zn, o r a t maximum c o n c e n t r a t i o n s up t o 1.0 mg 1 Zn.
I n c r e a s i n g t o x i c i t y however d e v e l o p e d f o r w i l d - t y p e a t i n c r e a s i n g
c o n c e n t r a t i o n s o f Zn above 0.5 mg 1 I
0 C h l Q
Q phycocyanin
0 0 Cd mg 1-1 80
— 70 CT>
- § 6 0 I/) | 50
.5? Q- 4 0
30
20 •
ro
2 1 — d l 10 12 14
time (d)
80 N
— 70 CD
^ 6 0 in <D 50 E a. to
30
20
10
0 05 Cd mg H
DO.
7 / / • / /
/
/ / /
10 12 H
time (d)
0 -15 Cd mg H
y / / /
10 12 14
time (d)
0-25 Cd mg H
80
T. 7 0 -
2 fro
S 50 -
£ CL 4 0 -
30 -
20
10
2
P
/
/ / / / /
10 12 14
time (d)
F i g . 4.34 Pigment changes d u r i n g g r o w t h o f w i l d - t y p e An&cystis
at d i f f e r e n t Cd c o n c e n t r a t i o n s i n b a t c h c u l t u r e .
ooooo oo U3
14-1
en 03
v,
0) U 4-1
73
M-l (0
CD
(0 O CP
CM 4->
14-1
OO a) in
in CM
0>
1/1
(..]uj) sjiun
CHAPTER 5
ENVIRONMENTAL FACTORS AFFECTING TOXICITY OF METALS
TO ANACYSTIS STRAINS
5.1 I n t r o d u c t i o n
I t seemed p r o b a b l e f r o m a knowledge o f t h e l i t e r a t u r e (see 1.3)
t h a t t h e t o x i c i t y o f m e t a l s i n t h e f i e l d and l a b o r a t o r y may be reduced
by t h e c o n c e n t r a t i o n o f o t h e r substances p r e s e n t . I n o r d e r t o
e s t a b l i s h c l e a r l y what i n f l u e n c e , i f any, o t h e r f a c t o r s had upon t h e
t o x i c i t y o f Zn, Cu and Cd i n t h e l a b o r a t o r y , a s e r i e s o f e x p e r i m e n t s
were p e r f o r m e d upon Anacystis. I n o r d e r t o i n v e s t i g a t e whether t h e
e f f e c t s d i f f e r e d between s t r a i n s w i t h v a r y i n g g e n e t i c t o l e r a n c e s t o
m e t a l s , a l l t h e e x p e r i m e n t s were p e r f o r m e d s i m u l t a n e o u s l y on c u l t u r e d
m a t e r i a l o f w i l d - t y p e and two Z n - t o l e r a n t s t r a i n s (Zn-t5.0; Z n - t l 2 . 0 ) ,
and on C u - t o l e r a n t s t r a i n ( C u - t . 0 . 5 ) . Most o f t h e r e s u l t s a r e
summarized i n T a b l e s 5.1 and 5.2; d a t a i n d i c a t i n g no d e t e c t a b l e
i n f l u e n c e on g r o w t h o r t o x i c i t y a r e g i v e n i n Appendix A5. Assays
were c a r r i e d o u t b o t h i n b a s a l medium and i n t h e s u b - i n h i b i t o r y l e v e l
o f t h e m e t a l under t e s t . The r e s u l t s a r e based on t u r b i d i m e t r i c -1 7
measurements, c o n v e r t e d t o u n i t s ml x 10 ( s e c t i o n 2.514); o t h e r
e x p e r i m e n t a l methods a re g i v e n i n s e c t i o n s 2.2, 2.3
5.2 I n f l u e n c e o f complementary c a t i o n s and a n i o n s
I n o r d e r t o t e s t t h e e f f e c t s o f o t h e r e l e m e n t a d d i t i o n t o b a s a l
medium on m e t a l t o x i c i t y , i t i s n e c e s s a r y t o add s a l t s , which l e a d
t o t h e a d d i t i o n o f f u r t h e r i o n . As f a r as p o s s i b l e Na +, K +, C I and
SO^ were used as complementary i o n s . P r e l i m i n a r y t e s t s were t h e r e
f o r e c a r r i e d o u t on t h e i n f l u e n c e o f NaCl, KC1 and Na^SO^ on m e t a l
t o x i c i t y .
137.
~ 0 -< 2 £<§
C + I
O I I
3 + C + I
0/ c
t -
o a
o a
13?
T a b l e 5.2 Key e n v i r o n m e n t a l f a c t o r s c h a n g i n g t o x i c i t y o f m e t a l s
t e s t e d , based on e n t r i e s i n T a b l e 5.1 d i f f e r i n g by a t
l e a s t two s c o r e s .
Reduced t o x i c i t y
Cu on w i l d - t y p e Fe pH (-)
Cu on Cu-t0.5 Fe pH (-)
Zn on w i l d - t y p e Ca Mn Fe P
Zn on Zn-t5.0 Mg Ca Mn Fe P pH (-)
Zn on Z n - t l 2 . 0 Mg Ca Mn Fe P pH (-)
Cd on w i l d - t y p e Ca Zn Fe P
I n c r e a s e d t o x i c i t y
Zn on Zn-t5.0 N i
Zn on Z n - t l 2 . 0 N i
Cd on w i l d - t y p e Pb
139.
Na (2.5 - 300 mg 1 ) l e d t o no d e t e c t a b l e change i n t h e t o x i c i t y
o f Cu o r Cd t o w i l d - t y p e ( T a b l e 5.1), o f Zn t o Z n - t o l e r a n t or o f Cu
t o C u - t o l e r a n t s t r a i n . Na a t h i g h e r c o n c e n t r a t i o n s d i d however
decrease t h e t o x i c i t y o f Zn t o w i l d - t y p e s t r a i n (Table 5.3). T h i s
i n f l u e n c e can n o t be due t o i n c r e a s i n g c l l e v e l s i n t h e medium, s i n c e
a d d i t i o n o f s i m i l a r l e v e l s o f C l as KC1 p r o v e d no response (Table 5 . 4 ) .
V a r i a t i o n i n the l e v e l s o f K (5 - 500 mg 1 S as KC1 had no
d e t e c t a b l e e f f e c t upon any o f t h e s t r a i n s t e s t e d ; examples are g i v e n
(Tables A5.2, A5.3). SO -S (2.5 - 320 mg l " 1 ) had no d e t e c t a b l e
e f f e c t on g r o w t h o f any s t r a i n t e s t e d i n m e t a l - f r e e medium. SO^-S
had a l s o no d e t e c t a b l e e f f e c t on t h e t o x i c i t y o f Zn t o e i t h e r w i l d -
t y p e o r t h e two Z n - t o l e r a n t s t r a i n s , o f Cu t o w i l d - t y p e o r Cu-t0.5
(Table 5 . 1 ) . On t h e o t h e r hand t h e r e was a s l i g h t i n c r e a s e i n t h e
t o x i c i t y o f Cd t o w i l d - t y p e ( T a b l e 5.5) when SO^-S c o n c e n t r a t i o n s
were i n c r e a s e d f r o m 2.5 - 320 mg 1 *.
5.3 I n f l u e n c e o f m a j o r c a t i o n s
Magnesium
T h i s i n h i b i t e d g r o w t h o f t h e w i l d - t y p e and Cu-t0.5 Anacystis
s l i g h t l y i n b a s a l medium a t c o n c e n t r a t i o n s above 160 mg 1 ^ Mg,
a l t h o u g h t h e two Z n - t o l e r a n t s t r a i n s were n o t a f f e c t e d (Table 5.1).
The i n f l u e n c e o f Mg (0.25 - 200 mg 1 *) on t h e t o x i c i t y o f Zn t o
w i l d - t y p e and t h e two Z n - t o l e r a n t s t r a i n s i s shown i n T a b l e s 5.6,
5.7, 5.8. There was a d e t e c t a b l e i n c r e a s e i n t o l e r a n c e o f t h e w i l d -
t y p e up t o 2.5 mg 1 * Mg (Table 5 . 9 ) , b u t l i t t l e change t o o k p l a c e a t
h i g h e r c o n c e n t r a t i o n s . I n t h e two Z n - t o l e r a n t s t r a i n s , t h e t o x i c i t y
was s l i g h t l y l o w e r a t h i g h e r c o n c e n t r a t i o n s . Mg had no d e t e c t a b l e
e f f e c t on Cu t o x i c i t y t o e i t h e r w i l d - t y p e ( T a b l e 5.10) o r Cu-t0.5
140.
Table 5.3 I n f l u e n c e o f Na on 7n t o x i c i t y t o w i l d - t y p e Anacystis;
-1 7
t h e r e s u l t s based on u n i t s ml x 10 ; acje o f t h e a l g a
5 days; d = d i e d .
Zn (mg 1 ) Na (mg 1 )
0-04
0.25
0 .50
0.75
1 .0
1 .25
1 .5
2.0
2.5
7.2
7.0
6.4
3.4
2.5
d
d
7.2
7.0
6.5
4.1
2.4
d
d
10 20 30 40 50 100 200 300
7.3
7.2
7.0
5.8
5.4
3.2
d
d
7 .5
7 .4
7.5
6 .6
5 .7
3.6
d
d
7.5
7.4
7.4
6.8
1.2 8.
8.2
8.1
7.7
6.0 7.5 7.8
8.6
8.6
8.0 8.4
7.8 8.2
1.2
5.2 7.2 7.5 7.8
0.77 0.45 4.2 . 6.9
d d 2.5 3.2
8.8
.8 8.9
8.6
8.4
7.9
7.1
3.6
8.7
8.6
8.1
7.4
4.6
Tabl e 5.4 I n f l u e n c e o f CI as KCl on Zn t o x i c i t y t o w i l d - t y p e -1 7
Anacystis; t h e r e s u l t s based on u n i t s ml x 10 ;
age o f t h e a l g a 5 days; d = d i e d . Zn (mg 1 -1
0.04
0.10
0.25
0.50
0.75
1.0
1 .25
1 .50
2.0
CI (mg l " 1 )
10 20 40 100 200
9.2 9.6 9.4 9.6 9.4
9.6 9.5 9.5 9.8 9.4
9.4 9.6 9.5 9.7 9.4
8.5 8.5 8.4 8.7 8.4
7.6 7.E 7. 7.5 7.7
7.7 7.4 7.4 7.4 7.6
7.5 7.2 7.2 7.2 7.5
0.05 0.02 0.03 0.04 0.04
d d d d d
141.
Table 5.5 I n f l u e n c e o f SO -S on Cd t o x i c i t y t o w i l d - t y p e Anacystis;
-1 7
t h e r e s u l t s based on u n i t s ml x 10 ; age o f t h e a l g a
5 days; d = d i e d .
Cd (mg 1 1 ) SO -s 4 -1
(mg 1 )
2.5 5.0 10 20 40 80 160 320
0 .04 8.6 8.6 8.2 8.6 8.4 8.8 8.4 8.2
0.15 7.0 6.8 7.0 7.0 6.5 5.6 4.4 4.2
0.30 4.8 4.8 4.6 4.4 4.5 3.8 3.0 3.2
0.45 4.2 4.4 4.2 3.8 3.4 3.2 2.8 2.6
0 .60 2.5 2.8 3.4 2.0 1 .2 1 .4 1.3 1.2
0.90 d d d d d d d d
1.20 d d d d d d d. d
Ta b l e 5.6 I n f l u e n c e o f Mg on Zn t o x i c i t y t o w i l d - t y p e Anacystis;
( h i g h Mg l e v e l s ) ; t h e r e s u l t s based on u n i t s ml 1 x 10 ;
age o f t h e a l g a 5 days; d = d i e d .
Zn (mg 1 ) Mg (mg r 1 )
2.5 5 10 20 40 80 100 200
0.04 9.1 9.3 9.7 9.5 9.5 9.3 8.4 0.05
0.25 8.9 9.4 9 . 3 9.5 9.8 9.2 7 .8 0.05
0.50 8.8 9.2 9.3 9.4 9.6 9.2 7.8 0.05
0.75 8.7 9.2 8.8 9.2 9.4 9.1 7 .6 5.0
1 .0 8.7 8.8 8.7 8.2 9.2 8.8 7.4 4.6
1 .25 7.6 8.2 8.12 8.1 8.6 8.6 7.0 4.6
1 .50 d 0.26 d 0.13 d d 0.07 d
2.0 d d d d d d d d
Table 5.7 I n f l u e n c e o f Mg on Zn t o x i c i t y t o Zn-t5.0 Anacystis;
t h e r e s u l t s based on u n i t s ml x 1 0 7 , age o f t h e a l g a
5 days; d = d i e d .
Zn (mg 1 1 ) Mg (mg 1
1.0 2.5 5 10 20 40 80 160 200
0 .04 8.2 8.0 8.3 8.0 8.2 8.3 8.4 • 8.2 8.2
2 7.6 7.6 7.8 7.8 7.8 7.8 8.0 7.8 7.6
4 5.4 5.2 5.4 5.4 5.5 5.4 5.6 5.5 5.4
5 1.4 1 .8 2.0 1 .8 2.2 2.8 3.4 4.2 4.4
6 d d d d d d 0.05 3.2 3.2
8 d d d d d d d 0.05 0.05
10 d d d d d d d d d
T a b l e 5.8 I n f l u e n c e o f Mg on Zn t o x i c i t y t o Z n - t l 2 . 0 Anacystis;
-1 7
t h e r e s u l t s based on u n i t s ml x 10 ; age o f t h e a l g a
5 days; d = d i e d .
Zn (mg 1 1 ) Mg (mg r 1 )
2.5 5 10 20 40 80 160 200
0.04 7.8 8.3 8.0 8.2 8.3 8.4 8.2 8.2
4.0 7.6 7.8 7.8 7.8 8.0 8.0 7.8 7.6
8.0 5.2 6.4 6.5 6.6 6.4 6.5 6.6 6.8
10.0 4.8 5.4 5.8 5.4 5.8 5.4 5.8 6.0
12.0 2.2 2.3 2.6 3.2 3.6 3.8 4.5 4.6
16.0 d d d d d 0.5 3.2 3.2
20.0 d d d d d d 0.5 0.5
24.0 d d d d d d d d
Table 5.9 I n f l u e n c e o f Mg on Zn t o x i c i t y t o w i l d - t y p e Anacystis;
(lev; Mg l e v e l s ) ; the r e s u l t s based on u n i t s ml * x 1"";
age o f the a l g a 5 days; d = d i e d .
Zn (mg r 1 ) Mg (mg r 1 )
0.25 0.5 0.75 1 .0 1 .25 1 .5 1 .75 2.0
0 04 8.5 8.8 9.4 9.5 9.2 9.4 9.5 9.4
0. 25 8.5 8.6 9.0 9.2 8.8 9.0 9.3 9.4
0. 5 7.5 8.8 8.8 8.5 8.6 8.6 8.6 8.8
0. 75 7 . 4 7.6 7.5 7.8 8.5 8.6 8.4 8.5
1. 0 6.4 7 . 2 7 .1 7.6 7.2 7.8 7.8 8.7
1. 25 0.4 0.6 0.8 1 .6 3.4 4.5 6.5 7.8
1 . 5 0.05 0.05 0.05 0.05 0.06 0.05 0.04 0.05
2. 0 d d d d d d d d
Table 5.10 I n f l u e n c e o f Mg on Cu t o x i c i t y t o w i l d - t y p e Anacyst
Cu (mg 1 )
0
0 .02
0.04
0.08
0 .10
0.16
0 .20
0 .24
0.30
t h e r e s u l t s based on u n i t s ml ^ x 10^; age o f t h e a l g a
5 days; d = d i e d .
-1 Mg (mg 1 )
0.25 2.5 5 10 20 40 80 160 200
7.5 1.0 8.0 10 10 6.5 7.0 4.0
6.3 7.2 6.8
5.0 6.0 6.0
2.3 2.4 2.6
7.0 6.3 5.8 4.6 1.6 1.4
5.4 5.0 3.6 2.1 0.65 d
2.4 2.3 1.5 0.8 0.03 d
0.62 0.72 0.71 0.65 0.71 0.32 0.06 d d
0.05 0.06 0.04 0.05 0.06 0.04 d d d
d d d d d d
d d d d d d
d d d d d d
0.02 d
d d
d a
144.
s t r a i n ( T a b l e 5.11). The i n f l u e n c e o f Mg on Cd t o x i c i t y t o w i l d - t y p e
was s i m i l a r t o t h a t o f Zn, i . e . a s l i g h t i n c r e a s e i n t o l e r a n c e up t o
2.5 rag 1 _ 1 Mg (Table 5.12).
C a l c i u m
An i n c r e a s e i n Ca f r o m 2.5 t o 200 mg 1 * b r o u g h t about a s l i g h t
i n c r e a s e i n t h e g r o w t h o f Anacystis s t r a i n s i n b a s a l medium. The
i n f l u e n c e o f Ca on Zn t o x i c i t y t o w i l d - t y p e and t h e two Z n - t o l e r a n t
s t r a i n s i s shown i n F i g s 5.1, 5.2, 5.3; T a b l e s A5.4, A5.5, A5.6. A
marked a m e l i o r a t i n g e f f e c t was e v i d e n t i n a l l s t r a i n s up t o t h e -1
h i g h e s t l e v e l (200 mg 1 Ca) i n v e s t i g a t e d , even a l l o w i n g f o r t h e f a c t
t h a t Ca has some e f f e c t i n b a s a l medium. The response o f w i l d - t y p e
and Z n - t o l e r a n t s t r a i n s was s i m i l a r . I n c r e a s i n g Ca caused a s l i g h t
i n c r e a s e i n t o l e r a n c e o f Cu t o w i l d - t y p e and Cu-t0.5. I t was more
pronounced t o w i l d - t y p e (Table 5.13) t h a n Cu-t0.5 (Table 5.14). The
i n f l u e n c e o f i n c r e a s i n g Ca on t h e t o l e r a n c e o f Cd t o w i l d - t y p e i s shown
i n F i g . 5.4; T a b l e A5.7. A marked i n c r e a s e i n t o l e r a n c e o f a l g a was
f o u n d as t h e Ca was i n c r e a s e d f r o m 2.5 t o 200 aig 1
Manganese
Mn (0.05 - 20 mg 1 S had no d e t e c t a b l e e f f e c t upon t h e g r o w t h o f
w i l d - t y p e i n b a s a l medium, w h i l e a s l i g h t decrease i n t h e g r o w t h o f
t h e two Z n - t o l e r a n t s t r a i n s o c c u r r e d . The i n f l u e n c e o f i n c r e a s i n g
Mn on Zn t o x i c i t y t o t h e w i l d - t y p e and Z n - t o l e r a n t s t r a i n s d i f f e r e d .
I n t h e case o f w i l d - t y p e i n c r e a s i n g Mn up t o 20 mg 1 * reduced t h e
t o x i c i t y o f Zn (Table 5.15), w h i l e i n t h e case o f the Z n - t o l e r a n t
s t r a i n s , i n c r e a s e i n t o l e r a n c e was f o u n d as Mn was i n c r e a s e d from
0.05 t o 5.0 mg 1 ~ 1 ( F i g s 5.5, 5.6; T a b l e s A5.8, A5.9). The e f f e c t
was more pronounced on Zn-t5.0 t h a n t h e Z n - t l 2 . 0 s t r a i n . Mn had
n e g l i g i b l e e f f e c t on Cu t o x i c i t y t o e i t h e r w i l d - t y p e o r Cu-t0.5. On
t h e o t h e r hand i n c r e a s e d Mn caused a v e r y s l i g h t decrease i n Cd
t o x i c i t y t o w i l d - t y p e (Table 5.16).
T a b l e 5 .11 I n f l u e n c e o f Mg on Cu t o x i c i t y t o Cu - t 0 . 5 Anacystis;
t h e r e s u l t s based c m u n i t s -1 ml " x 1 0 7 ; age o f the
5 days; d = a i e d .
Cu (mg 1 1 ) Mg (mg r 1 )
0 .25 2 . 5 5 10 20 40 80 160
0 6 . 2 5.8 5 .0 6.3 5 .8 6 .0 6 .2 3 .0
0 . 2 6 . 0 3 .6 4 . 1 5 .0 3 .0 4 . 6 5 .0 1.0
0 . 4 3 .6 2 . 5 3.5 4 . 8 4 . 6 4 . 8 4 . 8 0 . 6
0 .5 0 . 4 0 . 4 0 .3 0.3 0 . 8 8 1 .0 3 .6 0 .02
0 . 6 d d d r\ r\r\ \J . U O 0 .2 d
0 .8 d d d d d d 0.04 d
1.0 d d d d d d d d
1. 5 d d d d d d d d
Table 5 .12 I n f l u e n c e o f Mg on Cd t o x i c i t y t o w i l d - t y p e Anacystis
the r e s u l t s based on u n i t s ml 1 x 1 0 7 ; age o f the a l g
5 days; d = d i e d .
Cd (mg 1 Mg (mg 1 * )
0
0 .15
0 .30
0 .45
0 . 6 0
0 . 9
1.2
0 .25 2 .5
9 .8
7 .8
5 .0
4 .5
3 .8
d
d
9 .6
7 . 6
4 . 8
4 . 4
3 .6
d
d
9 .2
7 .8
4 . 9
4 .5
3 .2
d
d
10 20 40 80 100 200
9 .6
7 .4
4 .6
4 . 0
2 . 8
d
d
9 . 0
6 . 4
4 . 8
4 . 2
3 . 0
d
d
0 . 4
6 .6
4 . 8
4 . 0
3 .2
d
d
7.5
5 .0
4 . 1
3.2
2 .3
d
d
4 . 0 0 .05
3 .2 1.8
0 .03 d
0 .04
0 .05
d
d
d
d
d
d
Wild-type Ca (mg H)
o zoo 80 «100
60 A
2 5 S 5 0
1 j.r-
2 5
Zn (mg
F i a . 5. 1 I n f l u e n c e o f Ca on Zn t o x i c i t y t o w i l d - t y p e Anacystis.
10 Zn-t 50 Ca (mg H)
8 200
\
\ \ \ \
\ \ \ 80 & 100
\ \
2 5 40
10 20
7 4 6 8 10 12
Zn (mg H )
F i g . 5 _ 2 I n f l u e n c e o f Ca on Zn t o x i c i t y t o Zn-t5.0 Anacystis
s t r a i n .
o
6 H •t— ' c
\\\
\\ \
Zn-t 120 Ca (mg H )
\ 8 0
12 16
^ 100 & 200
20 2U 28
Zn (mg H )
F i g . 5.3 I n f l u e n c e o f Ca on Zn t o x i c i t y t o Z n - t l 2 . 0 Anacystis
s t r a i n .
100
Wild- typG Ca (mg M ) \ \ \ 8 0
K V) \ \
\ V 6 0 \ \ \ \ \
oo a 200 80 4 0
\
\ 2 0 10 a 20 \ \ 4 0 V
\
2 5 4 5
0 0-1 0 3 0 6 0 9 1 2 1 5
Cd (mg 1-1)
F l 9 . 5.4 I n f l u e n c e o f Ca on Cd t o x i c i t y t o w i l d - t y p e Anacystis.
Table 5.13 I n f l u e n c e o f Ca on Cu t o x i c i t y t o w i l d - t y p e Anacystis;
-1 1
the r e s u l t s based on u n i t s ml x 10 ; age o f t h e a l g a
5 days; d = d i e d .
Cu (mg r 1 ) Ca (mg l " 1 )
2.5 5 1 0 20 40 80 160 200
0. 0 7.2 7 .4 8. 1 9.0 9.3 10.2 10.4 10.6
0. 02 2.4 6.3 7.0 7.2 8.0 10 8.8 8.8
0. 04 1 .3 5 .0 5.8 6 .0 7.3 8.0 8.0 8.1
0. 08 0.6 2.3 2.3 4.2 6.8 8.0 7.5 7.4
0. 10 0 .42 0.71 0.72 1.3 6.2 6.8 7.1 7.0
0. 16 d 0.06 0 .06 0.86 2 .0 6.0 7.0 6.8
0 . 20 d d d 0.06 1 .0 6 .1 5.8 6.1
0. 24 d d d d 0.45 3.4 4.6 5.2
0 . 30 d d d d d 0 .03 0.02 0.4
T a b l e 5.14 I n f l u e n c e o f Ca on Cu t o x i c i t y t o Cu-t0.5 Anacuxtj s •„
-1 7
t h e r e s u l t s based on u n i t s ml x 10 ; age o f the a l g a
5 days; d = d i e d .
Cu (mg 1 l ) Ca (mg r 1 )
2.5 5 10 20 40 80 160 200
0.0 4.0 4.0 5.2 5.6 6.0 6.3 8.8 9.4
0.2 2.2 2.3 3.4 3.8 3.6 4.4 6.5 8.6
0.4 0.8 1 .0 2.6 3 .2 4.2 4.6 5.2 6.3
0.5 0.07 0.85 2.1 3 .4 3.6 3 .0 6 .0 6.2
0.6 d d 0.03 0. 38 1 .8 0.8 0.45 0. 50
0.8 d d d 0.02 0.66 d d d
1 .0 d d d d 0. 52 d d d
1 . 5 d d d d 0.45 cl d d
149.
Table 5.15 Tnf luence o f Mn on Zn t o x i c i t y t o w i l d - t y p e Anacystis;
t l e r e s u l t s base d on u n i t s ml 1 x 1 0 7 ; age o f the a l g a 5 days; d = d i e d
Zn (mg r 1 ) Mn (mg r 1 )
0.05 0.10 0.5 1.0 1.5 2.5 5 10 15 20
0 . 04 8.8 8.9 8.8 8.9 8.8 8.4 8.4 8.6 8.0 8. 6
0. 25 8.9 8.8 8.6 8.7 8.8 8.3 8.2 8.4 8.3 8. 8
0. 50 8.7 8.8 8.5 8.6 8.7 8.2 8.0 8.5 8.4 8. 8
0. 75 8.0 7.9 8.0 8.3 8.5 8.0 8.2 8.6 8.4 8. 6
1 . 0 8.2 8.3 7.9 8.3 8.0 8.1 7,8 8.5 8.3 8. 5
1 . 25 7.6 7.1 7.2 8.0 8.0 7.7 7.8 7.6 7.9 7 . 5
1 . 5 0.05 0.05 0.06 0.04 0.05 0.40 7.7 7.6 7.6 7. 8
2. 0 d cl d d d 0.60 7.6 7.7 7.5 7. 5
Table 5.16 I n f l u e n c e o f Mn on Cd t o x i c i t y t c w i l d - t y p e Anacystis ;
t h e r e s u l t s based on -1 u n i t s mi x 10 ; age o f t h e a l g a
5 days ; d = d i e d •
Cd (mg 1 1 1 Mn (mg 1 *)
0 .05 0.10 0.50 1.0 2. 5 5.0 10 20
0.0 9.8 9.0 8.8 8.8 8. 5 8.4 8.6 8.2
0.15 8.4 8.0 7.6 7.5 6. 5 6.2 5.6 5.1
0.30 5.6 5.4 5. 4 5.5 5. 2 4.8 4.5 3.5
0.45 4.8 4.6 4 . 5 4.4 4. 2 3.8 3.5 3.2
0.60 1 .2 1 .8 1 . 5 1.6 1 . 2 1.3 1.4 1 .5
0.90 d d d d d d d d
1 . 20 d d d d d d d d
t- 10
o
E
c 3
6 -l
\
X
Zn~t 5 0 i !
Mn (mg H )
20 0
10 0
\ - 6 -
0 05,01 & 0 5
2 5
1 0
^ 5 0
5 6 10 12
Zn (mg [-1)
F i g . 5.5 I n f l u e n c e o f Mn on Zn t o x i c i t y t o Zn-t5.0 Anacystis
s t r a i n .
O 10
Zn-t 120 Mn (mg
c
6 A
2 i - > 2-5 1-0 5-0 0-05.0-1 ft 0-5
12 16 20 U
Zn (mg H ) 28
F i < ? - 5.6 I n f l u e n c e o f Mn on Zn t o x i c i t y t o Z n - t l 2 . 0 Anacystis
s t r a i n .
151 .
I r on
T h i s caused a s l i g h t decrease i n the g r o w t h o f Anacystis s t r a i n s -1
i n b a s a l medium w i t h i n c r e a s i n g c o n c e n t r a t i o n s from 0.05 t o 20 mg 1 - 1
( T a b l e 5.1). The i n f l u e n c e o f Fe K).05 - 20 mg 1 ) on Zn t o x i c i t y
t o w i l d - t y p e and Z n - t o l e r a n t s t r a i n s i s summarized i n Ta b l e 5.2. S l i g h t
d ecreases i n t h e t o x i c i t y were e v i d e n t , the e f f e c t b e i n g more pronounced
w i t h t h e w i l d - t y p e (Table 5.17). Fe a l s o b r o u g h t about a s l i g h t
decrease i n Cu t o x i c i t y t o b o t h w i l d - t y p e (Table A5.12) and Cu-tO.5
s t r a i n s ( T a b l e A5.1?), and decrease i n Cd t o x i c i t y t o w i l d - t y p e
(Table 5.18).
5.4 I n f l u e n c e o f major a n i o n s
Phosphate-P
I t was e v i d e n t t h a t i n c r e a s i n g PO^-P from 1.75 mg 1 * t o
112 mg 1 ^ had a s l i g h t i n f l u e n c e on t h e g r o w t h r a t e o f a l l s t r a i n s
t e s t e d i n b a s a l medium. The i n f l u e n c e o f PO^-P on Zn t o x i c i t y on
w i l d - t y p e and the two Z n - t o l e r a n t s t r a i n s was v e r y s i m i l a r . Both
showed a marked Zn t o l e r a n c e a t h i g h e r P c o n c e n t r a t i o n s ( F i g s 5.7,
5.8, 5.9; T a b l e s A5.14, A5.15, A5.16). An i n v e s t i g a t i o n on the
i n f l u e n c e o f PO-P on the t o x i c i t y o f Zn t o w i l d - t v r j e was c a r r i e d o u t 4
u s i n g b o t h PO^-P-starved i n o c u l a ( T a b l e 5.19a) and PO^-P-rich i n o c u l a
(Table 5.19b); a v e r y s l i g h t d i f f e r e n c e i n t o x i c i t y o c c u r r e d . The
i n f l u e n c e o f PO-P was t e s t e d a l s o by i n t r o d u c i n a t h e PO-P l a t e r , 4 J " 4
i . e . 10 min a f t e r c o n t a c t o f t h e in o c u l u m w i t h Zn i n the medium. The
r e s u l t s (Table 5.20) show t h a t PO^-P i n t r o d u c e d a f t e r a l g a l
i n o c u l a t i o n s had much l e s s e f f e c t i n r e d u c i n g Zn t o x i c i t y , i n
comparison t o phosphate i n t r o d u c e d b e f o r e i n o c u l a t i o n (Table 5.19).
The i n f l u e n c e o f PO^-P on t h e t o x i c i t y o f Cu t o w i l d - t y p e and Cu-t0.5
was moderate (Tables 5.21, 5.22) w h i l e i t had a marked e f f e c t on
152.
T.-ibl< I n f l u e n c e of Fe on Z r t o x i c i t y t o w i l d - t y p e Anacustis;
-1 the r e s u l t s based on u n i t s nil
=; days ; = d i e d .
x !0 ; age o f t h e a l g a
Zn (mq 1 1 )
0 . 04
0.2 5
0 .50
0.75
1 .0
1 .25
1 . 5
? n
Fe (mg 1 )
0.05 0.10 0.50 i.O 1.5 2.5 10
5.5 7.6 7.8 7.8 7.8 7.8 6.9 6.5
5.8 7.6 3.0 7.8 7.8 7.9 6.8 6.3
5.6 7.5 7.6 7.5 7.6 7.6 6.8 6.5
5.4 7.2 7.5 7.4 7.5 7.7 6.9 5.0
5.2 6.4 7.3 7.3 7.4 7.5 6.8 5.8
d 0.3 0.40 4.2 6.5 6.8 4.01 5.6
d d d d 0.05 0.06 0.05 5.2
d d d d d d d 4.6
15 20
5.2
5.4
5.3
5.0
4.8
4.6
3.8
4.8
5.2
5.1
4.6
4.3
4.2
4.1
3.8
[•able 5.18 I n f l u e n c e o f Fe o n u c i t o x i c i t y t o w i l d - t y p e Anacystis;
-1 7 t h e r e s u l t s based on u n i t s ml * x 10 ; age o f t h e a l g a
days; d = d i e d .
Cd (mg 1 l )
0 .0
0.15
0.3 0
0.45
0.60
0.90
1 . 2
f e (mg 1 *)
0.05 0.10 0.50 1.0
8.5 8.8
6 .6 7 .8
3.3 4.2
2.1 3.8
0.05 1.4
d d
d d
8.8
7.6
8.9
7.6
.5 4.5
4.4 4 . 5
2.6
d
d
.5 5.0 10 20
9.2
7.8
4.8
4.2
2.8
d
d
9.4
7 . 5
4.5
4.4
3.2
d
7.4
6.0
4.2
3.0
1 .5
d
d
6.8
1 .4
0.05
0.04
0.05
d
d
10
b X j
1: 8 . "——o "" -
^ 8 '
^ - - " ^
«/> 8
uni
6 •
4 1
V,
w
i\ '
1-75 v<
26
i l l
Wild-type P 0 4 - P (mg 1-1)
'-o 112
to 56
4 5
Zn (mg H)
F i g « 5./ I n f l u e n c e o f PO -P on Zn t o x i c i t v t o w i l d - t y o e Anacustis. 4
10
o x
E
c D
1-75 \ \ x 3-5
\
Zn-t 5-0 P°i-P (mg 1-1)
- e 56 6 112
-o 28
\ 7 - 0
10 12
Zn (mg H )
Fi<J. 5.8 I n f l u e n c e o f PO -P on Zn t o x i c i t y t o Zn-t5.0 Anacystis
s t r a i n .
to 1 Zn-t 12 0 P 0 4 - P (mg 1-1)
8 56 & 112 \ 28 \
V \ \
v \ \
\ 1-75 & 3-5
7-0
\ 1 T
0 8 12 16 20 2L 28
Zn (mq H )
F i a I n f l u e n c e o f fQ^-P on Zn t o x i c i t y t o Z n - t l 2 . 0 Anacystis
r a i n
10-0 Wild-type
P V P (mg 1-1) 9-0
\ 8-0
\ O—
\ 3 7-0 • 2 \
6-0
V - o 5 6 5-0
\ \ \ ^ ® 2 . 3 \
30 v
\ ^ 1 \7-0 2-0 \
1 75 a 3 5 \ \ *0
\
I I 0 0-1 0-3 0-6 0-9 1-2
Cd (mg 1-1)
9- 5.10 I n f l u e n c e o f PC^-P on Cd t o x i c i t y t o w i l d - t y p e Anacystis.
T a b l e 5.19(a) I n f l u e n c e o f PO^-P on Zn t o x i c i t y t o w i l d - t y p e Anaciistis;
s t a r v e d i n o c u l u m ; t h e r e s u l t s based on u n i t s ml ' x 10 ;
age o f t h e a l g a 5 days; d = d i e d .
Zn (mg 1 ^ ) 1 P 0 4 " P (mg 1 ')
1 .75 3.5 7 14 28 56 112
0-04 . 8.4 8.6 8.8 9 .6 10.0 9.8 9 .6
0.5 8.2 8.3 8.4 9.2 9.4 9.3 9.4
1 .0 6.4 6.8 7.0 8.0 8.2 8.5 8.6
1 .25 4.4 4.8 5.5 7.6 7.6 8.1 8.0
1 .5 0.06 0 .32 2 .1 4.6 8.0 7.8 7.8
2.0 . d d d 1 .4 5.6 7.2 7.8
3.0 d d d d 4.6 6.8 7.0
4.0 d d d d 1 .4 4.8 6.8
^ b l e 5.19(b) I n f l u e n c e o f PO -P 4 on Zn t o x i c i t y t o w i l d - t y p
r i c h i n o c u l u m ; the r e s u l t s based on u n i t s ml
age o f the a l g a 5 days; = d i e d -
-1 Zn (mg 1 ) P°4" -P (mg l " - 1)
1 .75 3.5 7 14 28 56 112
0 .04 8.4 8.6 8.8 8.4 9.2 11 .0 10.0
0.5 8.0 8.5 8.6 9.2 8.5 9.8 9.6
1 .0 6 .4 6.8 7.0 7.8 8.4 8.8 9.2
1 .25 4.8 5.0 5.5 7.6 8.2 8.6 9.0
1 .5 0 .50 2.4 4.2 7 .0 8.0 8.3 8.8
2.0 d d d . 4.2 5.6 7 .4 8.4
3.0 d d d d 4.8 6.2 8.0
3.5 d d d d d 5.4 8.0
4.0 d d d d d d 7.5
T a b l e 5.20 I n f l u e n c e o f PO^-P on Zn t o x i c i t y t o w i l d - t y p e Anacystis;
(PO^-P was i n t r o d u c e d a f t e r a l g a l i n o c u l a t i o n and c o n t a c t -1 7
w i t h Zn f o r 10 m i n . ) ; the r e s u l t s based on u n i t s ml x 10 ;
age o f t h e a l g a 5 days; d = d i e d .
Zn (mg 1 1 ) PO P 4- (mq r 1 )
1 7 \ > r 7 ( i 1 4 56 1 1 /
0.04 8 0 0 9.6 9.6 9.8 9,0 9.8 9„8
0.5 4,5 5.2 6.0 6.5 6.8 7.2 7t,8
0.75 1.8 1.8 4.8 4.8 5.2 7.2 7.8
1 = 0 d rj ;3 2.4 7.3 8.0
1.25 d d d d 1=8 7.1 7.2
1.5 d d d d d 6,. 2 7.2
2.0 d d d d d 5.4 6.8
3.0 d d d d d 2.6 7,0
Table 5.21 I n f l u e n c e o f PO^-P on Cu t o x i c i t y t o w i l d - t y p e Anacystis;
t h e r e s u l t s based on u n i t s ml * x 10^; age o f t h e a l g a
5 days; d = d i e d .
-1 :u (mg 1 ) PO -P
4 (mg 1 l)
1 .75 3.5 7 14 28 56 112
0 7.2 7.4 8.1 9.0 9.3 10 10
0.02 6.4 6.5 7.2 7 .8 8.1 8.6 9.0
0.04 4.8 5.0 6.0 6.8 8.0 8.2 8.8
0.08 2.2 2.4 2.5 5.0 7.3 8.2 8.0
0.10 0.72 0.85 0.70 1 .6 7.0 7.8 7 . 5
0.16 0.06 0.05 0.06 0.7 6.8 7.1 7.6
0.20 d d d 0.06 6.2 7.0 5.6
0.24 d d d 0.05 2.0 2.6 3.1
0.30 d d d d d 0.02 0.02
T a b l e 5.22 I n f l u e n c e o f PO^-P on Cu t o x i c i t y t o Cu-t0.5 Anacystis;
-1 7
th e r e s u l t s based on u n i t s ml x 10 ; age o f t h e a l g a
5 days; d = d i e d .
Cu (mg 1 1 ) PO -P 4 (mg r 1 )
1 .75 3.5 7 14 28 56 112
0.0 5.0 5.2 6.2 6.3 6.0 8.0 3.6
0.2 3.0 3.4 4.5 6.8 6.2 6.4 2.5
0.4 1 .5 2.0 3.6 4.2 5.4 6.0 0.7
0.5 0.52 1.2 5.5 3.1 3.2 1.5 0.02
0.6 d d d d 0.64 0.38 d
0.8 d d d d 0.24 0.05 d
1 .0 d d d d d d d
1.5 d d d d d d d
158.
r e d u c i n g Cd t o x i c i t y t o w i l d - t y p e ( F i g 5.10; Tabl e A5.17).
Two q u a n t i t a t i v e e x p e r i m e n t s (Based on Chi a and u n i t s ml
were c a r r i e d o u t t o de m o n s t r a t e t h e e f f e c t o f o r g a n i c p hosphate on
Zn t o x i c i t y . F i r s t l y i t was shown t h a t ^ - g l y c e r o p h o s p h a t e
(Tables 5.23 a, b) and <K - D - g l u c o s e - l - p h o s p h a t e (Tables 5.24 a, b)
can a c t as a P source f o r Anacystis. At low e r l e v e l s o f P ( 14 mg 1 S
these sources o f phosphate l e d t o s l i g h t l y s l o w e r g r o w t h r a t e s t h a n
i n o r g a n i c phosphate, b u t a t ^ 28 mg 1 * P t h e r e was no d e t e c t a b l e
d i f f e r e n c e i n g r o w t h r a t e . I n c o n t r a s t however t h e two o r g a n i c phosphate
had much l e s s e f f e c t i n r e d u c i n g Zn t o x i c i t y (Tables 5.23 a, b;5.24 a, b ) .
N i t r a t e - N
A s l i g h t e f f e c t upon g r o w t h o f d i f f e r i n g NO^-N l e v e l s was e v i d e n t
i n m e t a l - f r e e c o n t r o l s The i n f l u e n c e o f i n c r e a s i n g NO^-N c o n c e n t r a t i o n s
( f r o m 10 t o 400 mg 1 S on t h e t o l e r a n c e o f Zn t o t h e w i l d - t y p e (Table A5.18)ani
two Z n - t o l e r a n t s t r a i n s and Cu t o w i l d - t y p e and Cu-t0.5 was v e r y
s i m i l a r . A s l i g h t decrease i n t h e t o x i c i t y was f o u n d i n a l l cases.
NO^-N caused a s l i g h t r e d u c t i o n i n t o x i c i t y o f Cd t o w i l d - t y p e
(Table 5.25).
5.5 I n f l u e n c e o f o r g a n i c compounds
E x p e r i m e n t s were c a r r i e d o u t t o i n v e s t i g a t e t h e i n f l u e n c e o f
EDTA on Zn, Cu and Cd t o x i c i t y t o a l l Anacystis s t r a i n s and o f HEPES
on Zn t o x i c i t y t o w i l d - t y p e .
EDTA
EDTA (0.5 - 32 mg 1 _ 1 = 0.0017 - 0.11 mM) l e d t o marked r e d u c t i o n
i n t h e t o x i c i t y i n a l l cases ( F i g s 5.11, 5.12, 5.13, 5.14; T a b l e s A5.19.,
A5. 20, A5.21 , A5.22). For i n s t a n c e i n t h e case o f Zn o r Cd t o x i c i t y
t o w i l d - t y p e Anacystis, t h e presence o f 32 mg 1 * EDTA, l e d t h e a l g a t o
grow a t 2.0 mg 1 1 Zn o r 0.45 mg 1 * Cd, s i m i l a r l y t o t h e c o n t r o l .
159.
Table 5.23a I n f l u e n c e o f PC^-P (as y O - g l y c e r o p h o s p h a t e ) on Zn t o x i c i t y
t o w i l d - t y p e Anacystis; u n i t s ml 1 was used as a g r o w t h
c r i t e r i o n .
Zn (mg 1 1 ) PO -P 4 (mg r 1 )
1 .75 3.5 7 14 28 56 112
0.04 6.2 7.6 7.6 8.2 9.4 9.8 9.5
0.5 d d a a 4.2 4 .8 7.8
1 .0 d d d d d d d
1 .25 d d d d d d d
1 .5 d d d d d d d
2.0 d d d d d d d
3.0 d d d d d d d
160.
fa
rH rC u .* 0) •H 4-) to 0 to
ype
d-t
rH •H
0 4-1
>1 4-1 -H u •H X 0
4->
rsi C 0
(U 4-J
£ ft w o A ft O H <U • O c >i 0 rH •H Cn !H 1 <U
it
w o rd
4-1
1 0 M O Cn
(0 m 0 « fC <u u t l c <D a) 3 D rH m Ul
c fO M 13
43
CN
in CD
•a EH
o Cm
CP
e
co o o o
CN T—1 +1 +1 T—1
CO CO r o
r-H
CM VO o
o o +1 +1
un co
CO
< *—i
in 03
o O 00 -H + 1 CN
r o o> en
-H o
di O > • •H O rH
(0 *r -H rH CN rH
•rH • 4->
0)
o D > • •H
o rH fO
r > •H rH
CO rH •—1 •H • 4->
in
co o o
LD • +t T J
co "H_
o o
r-• -H
<o o> o
o
T3
T3
LO O
T3
13
CN
T3
T3
T3
-0
T3
T3 13
T3
T3
-0
O CN
O CO
161.
T a b l e 5.24a I n f l u e n c e o f PO -P ( °< -D g l u c o s e - l - p h o s p h a t e )
on Zn t o x i c i t y t o w i l d - t y p e Anacystis; u n i t s ml ^
was used as a g r o w t h c r i t e r i o n .
-1 Zn (mg 1 ) PO -P 4 (mg r 1 )
1 .75 3.5 7 14 28 56 112
0 .04 6.0 7.4 7.6 8.2 10.0 9.8 9.6
0.5 d 4.0 5 = 3 6.4 7.4 7.8 8.0
1.0 d d d d d d a
1 .25 d d d d d d d
1 .5 d d d d d d d
2.0 d d d d d d d
3.0 d d d d d d d
162.
to • H •U V) » 1 0 <B
^ ID
ft >, •P
1 T ) r H • H 5 o 4J
>1 4-1 •rH 0
• H X 0
4-»
c tsj
c 0
u 4-1 (0 •
c
ft 0 • H
0 u . c <D
ft +J 1 -rH
r H rH 1 u 0) 10 r c o •P 0 p 0
r H u Cn Cn
Q m
to ITS
— V CD
CM co 1 3
O tn CM i t!
MH o
Q) r H U c U 0> 3
r H 4-1 C
H
X !
CM
m QJ
r H -Q i d
E-i
e CM i o CM
E
N
00 c o a) 0) 0) o r H > > > > > • • • H • H •rH •rH • H o o i—i r H r H r H i-H
CN (0 (0 US (0 r H +1 - H r H r H r-H r H r-H r H
>£> r H r H r H r-H r-H • H •rH • H •rH •rH
• • 4-1 •P 4-> P -P r H r H to to CO CO to
o CN r H r H
o o
+1 +i n -a T3 -a •a >> CM
i n
r H r H
r-O
o O
CO +1 HH •a TS T J T3 CN
o o 00
n r H o
O o
- H HH •n T3 T3 CM
CD o
o
-H CO
CO CO
i n o
c +i
CTi
T3
r H O
CM 00 r H o
O o in • +1 +1
CO
r H CO r H o
CN O O
• +1
T3
T3
O i n
o
in CM i n o
r H t-H CN
O
CO
163.
T a b l e 2.25 I n f l u e n c e o f NO -N on Cd t o x i c i t y t o w i l d - t y p e Anacystis;
t h e r e s u l t s based on u n i t s m l " 1 7 x 10 ; age o f th e a l g a
5 days; d = d i e d •
Cd (mg 1 NC^-N (mg 1 1 )
5 10 20 40 50 100 200 400
0 3 .8 4.0 4.2 7.8 8.6 8.8 8.8 8.8
0.15 2.4 2.4 2.8 6.5 7.4 7.8 7.8 7.5
0.30 0.05 2.2 2.4 4.2 6.1 5.4 5.4 5.9
0.45 d d d 3.4 4.2 3 .8 3 .6 4.2
0.6 d d d d 1.6 1 .4 1 .5 1.5
0.9 d d d d d d d d
10 164 Wild-type EDTA (mg [-1)
8 \ \ \
\ 64
\ 32
16 0-5 8
T 1-0 2-0 30 4-0 5-0 6-0 8-0
Zn (mg H )
F i g . 5.11 I n f l u e n c e o f EDTA on Zn t o x i c i t y t o w i l d - t y p e Anacgstis.
10 Zn-t 5 0 EDTA (mg H)
8
64 \ \
32
\ N> 16 \ 05 \ 8
\ \
»
6 8 10 12 V.
Zn (mg |-1)
F i g - 5.12 I n f l u e n c e o f EDTA on Zn t o x i c i t y t o Zn-t5.0 Anacystis
s t r a i n .
10 0 165 Zn-t 120 o EDTA (mg [-1)
\ \ 80 \ \
\ \
\ \ \ \ 1 \ \
\ 60 \ \ \ \ \ \ \ 1 \
\ o 6L \ \
4-0 \ \ 2 5 \ \ \ \ \ 0 5 \ \ \
\ 20
\ 8 \ 0 32
\ 1
1 I
0 8 12 15 20 1U 28
Zn (mg H ) F i g 5.13 I n f l u e n c e o f EDTA on Zn t o x i c i t y t o Z n - t l 2 . 0 Anacgsti
s t r a i n .
Wild-type 10-0
EDTA (mg H) 9-0
8-0 I/) o 32-0 \
3 7-0 \
8-0 & 16-0 60 \ \ V \ 5-0
\
4-0 V
3-0 \ \ \ \ \ 2-0 2-0 0-5
1-0 4-0
0-2 0-6 0-8 1-0 1-2
Cd (mg |-1)
F i g 5.14 I n f l u e n c e o f EDTA on Cd t o x i c i t y t o w i l d - t y p e Anacystis
166.
HEPES
HEPES (0 - 1200 ing l " 1 = 0 - 5.04 mM) l e d t o a s l i g h t decrease
i n t h e t o x i c i t y o f Zn t o w i l d - t y p e Anacystis (Table 5.26).
I n t h e absence o f HEPES t h e r e were marked r i s e s i n t h e pH o f t h e
medium and a t 300 mg 1 ' HEPES, r i s e s o f up t o 0.8 u n i t s . As a r i s e
i n pH l e a d s t o a decrease i n Zn t o x i c i t y t o w i l d - t y p e ( s e c t i o n 5 . 7 ) ,
i n c r e a s e d b u f f e r i n g would be expe c t e d t o l e a d t o i n c r e a s e d t o x i c i t y .
The s l i g h t changes i n t o x i c i t y o b s e r v e d w i t h i n c r e a s i n g HEPES can
t h e r e f o r e n o t be e x p l a i n e d by the poor b u f f e r i n g a t low e r pH v a l u e s
and i t i s c o n c l u d e d t h a t t h e y are a d i r e c t e f f e c t o f t h e HEPES
m o l e c u l e , presumably due t o c h e l a t i o n ( s e c t i o n 8.31).
5.6 I n f l u e n c e o f o t h e r heavy m e t a l s
I n c r e a s i n g l e v e l s o f o t h e r heavy m e t a l s such as N i , Cu, Hg and
Pb a l l l e d t o i n c r e a s e d t o x i c i t y o f Zn (Tab l e s 5.27, 5.28, 5.29, 5.30)
and o f Cd t o w i l d - t y p e (Tables 5.31, 5.32, 5.33, 5.34). The i n f l u e n c e
o f Zn on t h e t o x i c i t y o f Cd was t e s t e d u s i n g e i t h e r Chi a o r p o p u l a t i o n
d e n s i t y as c r i t e r i a o f g r o w t h . A marked i n c r e a s e i n t o l e r a n c e o f Cd
by t h e w i l d - t y p e w i t h i n c r e a s i n g Zn fr o m 0.04 t o 1,0 mg 1 ' ( F i g . 5.15;
TablesA5.23, A5.24) and by t h e Zn-t5.0 s t r a i n ( F i g . 5.16; Tables A5.25,
A5.26). I n c o n t r a s t Cd b r o u g h t about no d e t e c t a b l e change i n t h e
t o x i c i t y o f Zn t o e i t h e r s t r a i n .
5.7 I n f l u e n c e o f pH
I n b a s a l medium g r o w t h o f a l l t h r e e s t r a i n s was l e s s a t pH 6.0
and 6.5 t h a n 7.0, 7.5 and 8.0; t h i s e f f e c t was more pronounced f o r
t h e w i l d - t y p e . The i n f l u e n c e o f pH on t h e t o x i c i t y o f Zn t o w i l d - t y p e
( F i g . 5.17? T a b l e A5.27) shows t h a t a marked decrease i n t o x i c i t y
o c c u r r e d when t h e pH was r a i s e d f r o m 6.5 t o 8.0. I n c o n t r a s t a s i m i l a r
167.
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r - 1 ^ r- r -
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c o r H r H CN
r H CN u n r ~ CO c n
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r ~ m i n CN u n i n a o CO en e n 2 • • • » •
o o o r H r H r H
CO o CO n u n 0 0
i£> r - c n o r H CN CN r H r H r H
r ~ r-- r H CN r n Q o m r H r ~ r H
2 o O r H O r H CN
o CO CN C£l O CN c n CN
r H r H
T a b l e 5.27 I n f l u e n c e o f N i on Zn t o x i c i t y t o w i l d - t y p e Anacystis;
-1 7
the r e s u l t s based on u n i t s ml x 10'; age o f t h e a l g a
5 days; d = d i e d .
Zn (mg 1 1 ) N i (mg 1
0 0 .02 0 .04 0.08 0.10 0.2 0.24
0 .04 9.6 9 .2 9 .5 9.4 9 .1 7.3 4.2
0.25 9.4 9 .0 9 .2 9.2 8.8 6 .2 3.4
0.50 9.4 9 .1 O . 8 8.6 8.8 5.8 2.3
0.75 9.2 8 .8 8 .6 7.2 6.3 4.4 d
1 .0 9.0 7 .9 6 .4 4.8 3 .6 2 .8 d
1 .25 8.0 6 .8 5 .2 2.8 2.6 1 .8 d
1 .50 3 .5 2 .4 1 .6 d d d d
Ta b l e 5.28 I n f l u e n c e o f Cu on Zn t o x i c i t y t o w i l d - t y p e Anacijstis;
-1 7
t h e r e s u l t s based on u n i t s ml x 10 ; age o f t h e a l g a
5 days; d = d i e d .
Zn (mg r 1 ) Cu (mg r 1 )
0 0 .02 0 .04 0 .06 0 .08 0.10 0 .16 0 .20
0 .04 9.6 9 .2 9 .5 9.2 8.8 8.8 4.6 2.5
0 .25 9.2 9 .0 9 .4 9.6 8.6 7 .8 1 .8 d
0 .50 9.2 8 .6 8 .2 8.2 8.0 4.4 d d
0 .75 9.4 8 .2 7 .8 4.8 2.8 2.1 d d
1 .0 9.0 8 .8 5 .8 2.2 d d d d
1 .25 8.0 8 .0 3 .6 d d d d d
1 .50 3.2 2 .5 1 .8 0.8 d d d d
T a b l e 5.29 I n f l u e n c e o f Hg on Zn t o x i c i t y t o w i l d - t y p e Anacystis;
- 1 7
t h e r e s u l t s based on u n i t s ml x 10 ; age o f the a l g a
5 days; d = d i e d .
- 1 Zn (mg 1 ) Hg (mg r 1 )
0 0 02 0 .04 0.08 0 .10 0.20 0.22 0.24
0 .04 9.2 8 8 8 .4 7.5 6.2 1 .6 0.10 d
0 .25 9.5 8 .8 8 .0 7.4 6.5 1 .8 d d
0 .50 9.4 9 .0 7 .8 7 .0 5.2 1 .5 d d
0.75 9.0 8 .8 6 .8 6 .8 4.6 d d d
1 .0 8.8 7 .8 6 .5 5.5 4.8 d d d
1 .25 7.8 7 .2 6 .0 4.2 2 .6 d d d
1 .50 3.1 2 .7 1 .4 d d d d d
T a b l e 5.30 I n f l u e n c e o f Pb on Zn t o x i c i t y t o w i l d - t y p e Anacystis;
t h e r e s u l t s based on u n i t s m l " 1 x 1 0 ? ; age o f t h e a l g a
5 days; d = d i e d .
- h Zn (mg 1 ) Pb (mg 1 1 )
0 1 2 5 10 20 40
0.04 9.3 9.5 9.4 9.3 9.2 8.5 1.2
0.25 9.5 9.4 9.3 9.2 8.8 8.6 0.70
0.50 9.5 9,3 9.2 9.3 8.0 6.5 d
0.75 9.0 9.0 9.1 9.2 7.8 5.2 d
1 .0 9.1 8.8 8.8 8.4 6.8 4.3 d
1 .25 7.8 7.5 7.1 6.4 4.2 d d
1 .50 3.2 2 .6 1.8 1 .5 d d d
T a b l e 5.31 I n f l u e n c e o f N i on Cd t o x i c i t y t o w i l d - t y p e Anacystis;
- 1 7
the r e s u l t s based on u n i t s ml x 10 j age o f t h e a l g a
5 days; d = d i e d .
Cd (rag 1 ^ N i (mg r 1 )
0 0.02 0.04 0.08 0.10 0.20 0.24
0 9.6 9.3 9.5 9.4 8.8 7.5 4.2
0.05 9.4 9.2 9.2 9.0 8.8 3.2 1 .2
0.10 9.3 8.8 8.8 7.6 4.5 2.1 d
0.20 8.8 8.6 6.4 5.2 3.1 1 .6 d
0.40 6.8 5.8 3.4 2.2 d d d
0.60 0.1 0.06 d d d ' d d
T a b l e 5.32 I n f l u e n c e o f Cu on Cd t o x i c i t y t o w i l d - t y p e Anacystis;
- 1 7
t h e r e s u l t s based on u n i t s ml x 10 ; age o f t h e a l g a
5 days; d = d i e d .
Cd (mg 1 _ 1 ) Cu (mg r 1 )
0 0.02 0.04 0.06 0.08 0.10 0.16 0.20
0 9.5 9.2 9.5 9.0 8.8 8.6 4.2 2.4
0.05 9.3 9 .1 9.2 9.3 8.5 7.8 1 .8 d
0.10* 9.2 8.6 8.3 8.2 7.8 6.8 d d
0.20 9.0 8.8 6.4 3.2 d d d d
0.40 6.6 3.4 d d d d d d
0.60 0.08 d d d d d d d
T a b l e 5.33 I n f l u e n c e o f Hg on Cd t o x i c i t y t o w i l d - t y p e Anacystis;
-1 7
the r e s u l t s based on u n i t s ml x 10 ; age o f t h e a l g a
5 days; d = d i e d .
Cd (mg 1 1 ) Hg (mg r 1 )
0 0.02 0.04 0.10 0.15 0.20 0.22 0.24
0 9.3 8.7 8.5 7.6 5.8 1 .8 0.2 d
0.05 9.4 8.2 8.2 7.4 5.8 2.1 0.1 d
0.10 8.7 7.4 6.8 5.5 4.2 1 .5 0.08 d
0.20 8.8 7 .6 6.2 5.2 3.1 2.2 d d
0.40 6.8 5.8 5.6 3.4 2.2 1 .5 d d
0.60 0.08 d d d d d d d
T a b l e 5.34 I n f l u e n c e o f Pb on Cd t o x i c i t y t o w i l d - t y p e Anacystis;
-1 7
t h e r e s u l t s based on u n i t s ml x 10 j age o f t h e a l g a
5 days; d = d i e d -
- 1 Cd (mg 1 ) Pb (mg l " 1 )
0 1 2 5 10 20 40
0 9.3 9.4 9.4 9.3 9.3 8.6 1.3
0.05 9.5 9.2 9.0 9.2 8.8 7.2 d
0.10 8.8 8.8 7.8 7.8 6.2 4.4 d
0.20 8.7 8.6 7.8 7.2 5.8 3.5 d
0.40 7.2 6.8 5.2 3.8 2.3 d d
0.60 0.06 d d d d d d
0.80 d d d d d d d
172 Wild-type 0-4 Cd,0-2 Zn 1-8
1-6
p 0-6 Cd,0-4 Zn 1-4
cn 1-2
Ol
•0 0-4 Cd. 0-04 Zn (_>
0-8
0-6
0-4 0-6 Cd,0-04 Zn
0-2
24 48 72 96 120 144 168 192
time h)
F i g 5.15 I n f l u e n c e o f Zn on Cd t o x i c i t y t o w i l d - t y p e Anacystis
Zn-t 5 0 0-6 Cd, 1-0 Zn
1-8
1-6
•4
1-2 0€Cd,0-04 Zn Ol
•0
0-8
1-2 Cd,1-0 Zn 0-6
0-4
/ 0-2
A 1-2 Cd, 0-04 Zn F
24 48 72 96 120 144 168 192
time (h)
F i g 5.16 I n f l u e n c e o f Zn on Cd t o x i c i t y t o Zn-t5.0 Anacystis
5 0
s t r a i n .
10-0 Wild-type pH 9-0 7 3
8-0 10
D 7-0
6-0 \ \ 5-0 \ \
8-0 4-0 \
\
3-0 7-5
2-0 6-5
1-0 7-0 6-0
0 0-5 1-0 1-5 2-0 2-5
Zn (mg M )
5.17 I n f l u e n c e o f pH on Zn t o x i c i t y t o w i l d - t y p e Anacystis.
10 - i
O X
Zn- t 5 0 pH 8 CO
\ \ 8-0
7-5
6-5 7-0
6-0
t 2 U 6 8 10 12
Zn (mg (-1)
F i g . 5.18 I n f l u e n c e o f pH on Zn t o x i c i t y t o Zn-t5.0 Anacystis
s t r a i n .
174.
r i s e i n pH l e d t o i n c r e a s e d Zn t o x i c i t y t o t h e two Z n - t o l e r a n t
s t r a i n s ( F i g s 5.18, 5.19; T a b l e A5.28, A5.29). I n t h e case o f b o t h
w i l d - t y p e and Cu-t0.5 s t r a i n s , pH had no d e t e c t a b l e i n f l u e n c e on t h e
Cu t o x i c i t y . An i n c r e a s i n g pH v a l u e between 6.0 and 8.0 caused a
marked decrease i n t h e t o x i c i t y o f Cd t o t h e w i l d - t y p e ( F i g . 5.20;
T a b l e A5.30).
5.8 I n f l u e n c e o f i n o c u l u m s i z e 4 -1
The i n o c u l u m s i z e was i n c r e a s e d from 2 x 10 u n i t s ml t o 7
2 x 10 u n i t s f o r w i l d - t y p e Anacystis t o see what i s the i n f l u e n c e o f i n o c u l u m s i z e on Zn t o x i c i t y . The r e s u l t s ( F i g . 5.21; T a b l e A5.31 ,
5 -.1
A5.3 2) c o n f i r m t h a t t h e s i z e o f i n o c u l u m chosen (2 x 10 u n i t s ml ' )
i s t h e most s u i t a b l e . Use o f a l a r g e r i n o c u l u m l e d t o reduced t o x i c i t y
and e r r a t i c r e s u l t s .
8 Zn- t 120 pH
E
70 65 85 7-5 & 6 0 80
i
8 12 16
Zn (mg (-1)
5.19 I n f l u e n c e o f pH on Zn t o x i c i t y t o Z n - t l 2 . 0 Anacystis
s t r a i n .
10-0
Wild-type pH
8-0
3 7-0
6-0 8-0
5-0
7-5 A-0
o
\ 3-0
\ 2-0
1-0 6-0 6-5 \
1 0-2 0-6 06 10 •2 Cd (mg 1-1)
P i g . 5.20 I n f l u e n c e o f pH onCd t o x i c i t y t o w i l d - t y p e Anacusti
2U
Wild-type Inoculum size 2 2
y > 2 0 £ Ol 18
O 16
1-4
1 0
0 8
0-6
OA
0 2 2«106 2x10 2«10 2«10
0 0-5 1 0 1 5 2 0 25
Zn (mg H)
F ± g - 5.2i I n f l u e n c e o f i n o c u l u m s i z e on Zn t o x i c i t y t o
w i l d - t y p e Anacystis; c h l a used as g r o w t h c r i t e r i o n .
177.
CHAPTER 6
A c c u m u l a t i o n o f z i n c by Anacystis nidulans
6.1 I n t r o d u c t i o n
An i n v e s t i g a t i o n was c a r r i e d o u t t o compare Zn a c c u m u l a t i o n by
w i l d - t y p e and Z n - t o l e r a n t Anacystis s t r a i n s . These a l g a e were
s u b j e c t e d t o v a r i o u s l e v e l s o f Zn ( 0 . 1 , 1.0 mg 1 ' f o r w i l d - t y p e ;
0.1, 1.0 and 10.0 mg l " 1 f o r Z n - t l 2 . 0 ) f o r p e r i o d s up t o 192 h, b u t
o t h e r w i s e s t a n d a r d g r o w t h c o n d i t i o n s were used ( s e c t i o n 2.33; p. 4 8 ) .
Growth c u r v e s f o r b o t h algae are shown i n F i g s 6.1 and 6.2, w i t h d r y
w e i g h t used as a g r o w t h c r i t e r i o n . These c u r v e s may be compared
w i t h F i g s 4.19 and 3.9 i n which p o p u l a t i o n d e n s i t y was used as a
g r o w t h c r i t e r i o n . The e x p e r i m e n t s were r e p e a t e d t w i c e . Comparisons
were made f i r s t t o s t u d y t h e a b i l i t y o f w i l d - t y p e and Zn-t5.0
Anacystis s t r a i n s t o accumulate e n v i r o n m e n t a l Zn, u s i n g c e n t r i f u g a t i o n
( s e c t i o n 2.513) t o s e p a r a t e t h e a l g a f r o m t h e medium. The second
e x p e r i m e n t was q u i t e s i m i l a r , b u t compared w i l d - t y p e and Z n - t l 2 . 0
s t r a i n s and used the f i l t r a t i o n ( s e c t i o n 2 . 9 ( i i ) ) t o h a r v e s t t h e a l g a .
Because b o t h e x p e r i m e n t s l e d t o s i m i l a r r e s u l t s , o n l y t h e l a t t e r w i l l
be d i s c u s s e d h e r e . I n o c u l a were grown w i t h as low a l e v e l o f Zn as
p o s s i b l e i n t h e medium (0.04 mg 1 S see 2 . 2 ) . The i n o c u l a f o r w i l d -
t y p e and Z n - t l 2 . 0 had 7.5 and 12.5 u.g Zn g 1 d r y w e i g h t , r e s p e c t i v e l y .
There was no s a t i s f a c t o r y t e c h n i q u e t o s e p a r a t e t h e a c t u a l l e v e l o f Zn
a s s o c i a t e d w i t h a l g a f r o m t h a t due t o p r e c i p i t a t i o n . ( I n a d d i t i o n t o
removal o f Zn by t h e a l g a and consequent l o w e r i n g o f t h e o v e r a l l Zn
l e v e l , changes i n CC^ d u r i n g t h e g r o w t h o f a l g a may i n f l u e n c e t h i s
p r e c i p i t a t i o n . ) The r e s u l t s are summarized i n two ways:
( i ) Zn i n a l g a p l u s f i l t e r .
( i i ) As ( i ) l e s s v a l u e o b t a i n e d from f i l t e r used as a c o n t r o l .
Wild-type 0- 1 mg 1-1 Zn 1- 0
24 48 72 96 120 144 168 192
time (h)
6 . 1 I n f l u e n c e o f Z n o n g r o w t h o f w i l d - t y p e Anacystis
d r y w e i g h t w a s u s e d a s a g r o w t h c r i t e r i o n .
Zn-t 120 K ) mg 1-1 Zn
10-0
2U 48 72 96 120 144 168 192
time (h)
6 . 2 I n f l u e n c e o f Z n o n g r o w t h o f Z n
d r y w e i g h t w a s u s e d a s a g r o w t h
- t l 2 . 0 Anacystis ,-
c r i t e r i o n .
179.
The " t r u e " value of Zn associated w i t h alga must l i e between these
two extremes. The l e v e l of Zn associated w i t h a Nuclepore f i l t e r
through which 0.1, 1.0 and 10.0 mg 1 1 Zn, followed by EDTA washes,
had been passed were 0.008, 0.008 and 0.022 mg 1 * Zn, r e s p e c t i v e l y ;
unwashed f i l t e r s were 0.016, 0.026 and 0.89 mg 1 r e s p e c t i v e l y .
The p h y s i c a l adsorption of Zn onto the c e l l surface, was examined
by washing w i t h 40 mg l " 1 EDTA (= 0.138 mM; Table 2.15). At the
beginning of the experiments i t was thought t h a t 3x EDTA washes might
be enough t o release a l l the Zn adsorbed. I t was l a t e r r e a l i z e d t h a t
these were not enough f o r the higher Zn concentrations.
6.2 Influence of c u l t u r e density on removal of Zn from medium
An experiment was i n i t i a t e d to determine whether the i n i t i a l
c u l t u r e d e n s i t y a f f e c t e d the amount of Zn removed from the medium
over a sho r t p e r i o d of time (30 minutes). Cultures i n which i n i t i a l
u n i t s m l " 1 were 0, 4 x 10 6, 6 x 10 &, 8 x 10 6, 10 x 10 6, 20 x 10 6,
30 x 10 6, 40 x 10 6 and 50 x 10 6 were exposed t o 0.1, 0.5, 1.0 and
1.5 mg 1 * Zn. The amounts o f Zn remaining i n the medium are given
i n Table 6.1. The percentage of Zn removed from the medium r i s e s -1
s l i g h t l y w i t h increased p o p u l a t i o n d e n s i t y . For instance a t 1.5 mg 1 Zn, 12% Zn removed by 4 x 10^ u n i t s ml * while 22.6% was removed by
6 -1 50 x 10 u n i t s ml
6.3 Influence o f environmental Zn concentration on Zn
accumulation by Anacystis nidulans
The t o t a l Zn accumulation and accumulation r a t i o by two Anacystis
nidulans s t r a i n s subjected t o various environmental Zn concentrations
i s given i n Tables 6.2, 6.3, 6.4, 6.5, 6.6, A6.1 and A6.2. The growth
of both s t r a i n s i n medium c o n t a i n i n g 0.1 and 1.0 mg 1 * Zn f o r w i l d -
type and 0.1, 1.0 and 10.0 rng 1 1 Zn f o r Zn-tl2.0 are shown i n Figs 6.1
1 8 0 .
Table 6 . 1 I n f l u e n c e o f c u l t u r e d e n s i t y on removal of Zn from
c u l t u r e medium over a sho r t p e r i o d of time ( 3 0 minutes)
n i t i a l Zn (mg l - 1 )
p o p u l a t i o n d e n s i t y u n i t s ml *
Zn l e f t i n medium (mg I " 1 )
0 . 1 0 0 . 0 0 . 10 0 . 5 0 0 . 0 0 . 4 8 1 .0 0 . 0 0 . 8 4 1 .5 0 . 0 1 . 3 2
0 . 1 0 4 X 0 . 0 9 3 0 . 5 0 4 X 0 . 4 8 1 .0 4 X 0 . 8 5 1 ,5 4 X 1 0 6 1 . 3 0
0 . 1 0 6 X 0 . 0 9 0 . 5 0 6 X 0 . 4 6 1 . 0 6 X
1 0 6 0 . 8 8
1 .5 6 X i o 6 1 . 2 6
0 . 1 0 8 X 0 . 0 9 0 . 5 0 8 X 1 0 6 0 . 4 8 1 . 0 8 X 1 0 6 0 . 8 6 1 . 5 8 X i o 6 1 . 2 7
0 . 1 0 10 X 0 . 0 8 3 0 . 5 0 10 X
1 0 6 0 . 4 5
1 . 0 10 X 0 . 8 7 1 .5 10 X i o 6 1 . 2 8
0 . 1 0 20 X 1 0 6
0 . 0 8 0 0 . 5 0 20 X
1 0 6 0 . 4 5
1 . 0 2 0 X 1 0 6
0 . 8 6 1 . 5 2 0 X i o 6 1 . 2 5
0 . 1 0 3 0 X , ~6 1 U 6 0 . 0 8 0
0 . 5 0 30 X 1 0 6
0 . 4 6 1 . 0 3 0 X
1 0 6 0 . 8 4
1 . 5 30 X i o 6 1 . 2 2
0 . 1 0 4 0 X 0 . 0 8 0 0 . 5 0 4 0 X 1 0 6 0 . 4 5 1 . 0 4 0 X
1 0 6 0 . 8 4
1 . 5 4 0 X i o 6 1 . 2 0
0 . 1 0 5 0 X 0 . 0 7 2 0 . 5 0 5 0 X 0 . 4 5 1 . 0 5 0 X
1 0 6 0 . 8 5
1 . 5 50 X i o 6 1 . 1 8
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C N O en o C O o X) LO C O C N C N C N CM CM C O
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CM CO X! r~ X) CO o CM
X! CO cn cn CM r-l CN X) LT) o 00 o CO CO X) O
O T—t T—1 CM CN CN CN CO
O CO CO tn CO CM
CM CM r-l O LO o CM CO CO o CO co
CO CO m X) r-i r—1
X) CO o o r— X) m X> CO CO r> CO GO r-H CM CN LO CM CM co cn X) CO <S"
CM CM CN CO X)
e 4J 3 IP -H CU nj
rH 0) E
C N C
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ID i •H rH P C •H N Cn C E
ro 00 X) i n co CO CO <—1 CN cn CO cn CM CN o O CM CM CO X) o o O o O O o o
m CD o i n m o CN o O
cc co r- XI X) X) X)
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x> X) 00 X) o CO CM o
CM cn CM XI cn o *—< T—(
o o
187 U-0
Cn Wild-type
washed t i l t e r 10 mg 1-1 Zn <= 3-0
.0
0-1 c n
0 8 1
1-0 Inoculum—>
100 200 300 dry weight (mg H )
4-0 Zn-t 12 0
10 mg 1-1 Zn washed CD r i l t a r i t
>o 1-0 fc= 3-0
0 c n
0-1
cn 2-0 .Q«b2 0 cn
inoculum—1> 1-0
100 200 300 dry weight (mg M)
and 6.2, r e s p e c t i v e l y . These show a s i m i l a r growth p a t t e r n f o r
each alga; the maximum growth r a t e occurred during the f i r s t few
days of i n o c u l a t i o n , then growth r a t e l e v e l l e d - o f f w i t h increased
i n c u b a t i o n time. There was a l i n e a r r e l a t i o n s h i p between the
logar i t h m of the Zn composition of the alga and the dry weight of the
c u l t u r e over the whole 192 h periods (Figs 6.3, 6.4). The amount of
Zn i n the alga increases w i t h i n c r e a s i n g Zn i n the medium (Figs 6.3,
6.4). The r a t i o between t h a t taken up at ( i n i t i a l ) 10 mg 1 * Zn
and t h a t a t ( i n i t i a l ) 1 rag 1 ' Zn i s about 5.0 over the whole growth
p e r i o d (Fig. 6.4). The r a t i o between t h a t taken up a t ( i n i t i a l )
1 mg 1 1 Zn and ( i n i t i a l ) 0.1 mg 1 * Zn changes during growth,
presumably a t l e a s t p a r t l y because there i s a greater change i n
environmental Zn during batch c u l t u r e s t a r t i n g w i t h 0.1 mg 1 * Zn
(Tables 6.2, 6.3) .
C o r r e l a t i o n c o e f f i c i e n t s were c a l c u l a t e d f o r both s t r a i n s w i t h
y = logarithm |ig Zn g 1 dry weight; x = dry weight (mg 1 1 ) , the
r e s u l t s are included w i t h Figs 6.3, 6.4, 6.5 and 6.6. Anomalous
c o r r e l a t i o n c o e f f i c i e n t s were obtained when no allowance was made f o r
the presence of Zn i n the f i l t e r .
6.4 Release o f Zn from Anacystis by washing w i t h EDTA
The t o t a l amount of Zn associated w i t h w i l d - t y p e and Zn-tl2.0
Anacystis, w i t h o u t EDTA washes du r i n g the growth i n batch c u l t u r e are
shown i n Figs 6.5 and 6.6. These Figs may be compared w i t h those i n
Figs 6.3 and 6.4, i n which alga were washed w i t h EDTA, and d e t a i l s o f
the r e s u l t s are given i n Tables A6.1 andA6.2. These r e s u l t s suggest
t h a t the i n i t i a l h i gh Zn uptake observed, may be due t o Zn only
l o o s e l y associated w i t h the c e l l s .
W i l d - t y p e 190 Unwashed- f i l t e r
cn 0)
1-0 mg l-i Zn
3-0 0
cn
N 0-1
cn 0-87S
2-0
cn
— > Inoculum 1-0
1 1 , - r -
0 100 200 300
d r y we igh t (mg H )
5-0 Z n - t 5 0 Unwashed - f i l t e r
10-0 mg l"1 Zn
4-0
cn
1-0
3 0
cn
cn r = 0-564
2-0
cn o
Inoculum 1-0
100 200 300
d r y weight (mg (-1)
191 .
CHAPTER 7
STUDIES ON ZINC RESISTANCE OF STRAINS ISOLATED FROM ZINC POLLUTED SITES
7.1 I n t r o d u c t i o n
Blue-green algae are the dominants of some s i t e s w i t h elevated
z i n c , the organisms present u s u a l l y being narrow forms of Plectonema
(section 1.1). The purpose of the present chapter i s t o summarize
the features of examples of species which are t o l e r a n t t o high l e v e l s
of zinc.
7.2 O r i g i n s , i s o l a t i o n and c u l t u r e
Clonal s t r a i n s i s o l a t e d from s i t e s w i t h elevated z i n c , i n
England, France and U.S.A. (Table 2.7 ) were obtained by p l a t i n g .
Two d i f f e r e n t media were re q u i r e d f o r c u l t u r e (see 2.21).
7.3 T o x i c i t y o f Zn and Cd under standard c o n d i t i o n s
The studies reported i n t h i s section were planned t o determine
the l a b o r a t o r y responses t o Zn of i s o l a t e s from s i t e s w i t h elevated
z i n c . S t r a i n s from presumed low zinc s i t e s were included as " c o n t r o l s " .
I s o l a t e s were f i r s t t e s t e d f o r s e n s i t i v i t y t o Zn using the assay whose
r e s u l t s summarized as a Tolerance Index Concentration (T.I.C.See
sect i o n 2.52) . I s o l a t e s from high Zn s i t e s , i n general, t o l e r a t e d
considerably higher l e v e l s o f Zn than the " c o n t r o l " s t r a i n s (Table 7.1).
The s i t u a t i o n i s however not completely c l e a r cut:
( i ) Calothrix D 473 was i s o l a t e d from a dry stream w i t h sediments
r e l a t i v e l y r i c h i n Zn «210 M-m f r a c t i o n : 540 ug g 1 Zn) , y et was
r e l a t i v e l y s e n s i t i v e i n l a b o r a t o r y assay.
( i i ) The r e s u l t s obtained w i t h Synechococcus D 561 were v a r i a b l e but
i n a l l cases the s t r a i n was much more s e n s i t i v e than Gloeothece D 562
192.
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rH X . 0 c 4-> a 4J 0) •H M
A , M
IW rO 0) 0 4-1 en o
+J e G tn a (0 0 a QJ M in •H 0) •H (0 G rH M r4 o O rO 4-1 r4 E-i 04 in •H 6 > 0) 0 4H c X5
O O 0) 4-1
0) ra c c o H -H C
4-1 N (1) (0
U -< 4 J I C rH 0)
oi u CP c e o ~ u
o c ra u
o
•H T) 0) S3
>l rfl in in rd
4->
rrj —
O I G rH •H N CP
6 144 O
Q) rH (J 01 M > 3 Oi O
rH in
o c
x .
3 J - i _
o (Ti LD r- CTl O CO CO ro
O o o T~t O ro ro m
0
s o u -< < 3 x; u
u <
ID O O
I X
o rsi
o
i n 0> rH
0) X) > rfl •H •H > >4
CM in (0 * 3 >
o LO tfl T—( i—. 4-1
C 3 > (0 X)
0 O
2 2 2 2 2 2 2 2 + + 1 + 1 + 1 1 o o O o o O O o X T-H T—I T-H T-H T-H T-t T-H T-H o u 3 3 3 3 3 3 H 3 < <
x ; X! X. x ; X! XI X! x : u U u u CJ u O u
s o
* x ; o
it
Zn
+1 o o o o o
CP. • 4-1 0) • • • • IX) CO C 4-1 co ID LD CO o
me
ro >
o •H 0) » • • T l rH 0
II
as
0)
0) CM ro O •3- ro CN m T-f U ro IT, in co vD <x> 3 m rH in T—t m
•H 0) QJ QJ
m q 0 0 tn tn ro to
•H c; rO q q Ss rrj 4-1 ro
T3 ^H tn V-i 3 • C D ro X, +J
e •H t l 0 6 » • 3 to to to fH •H 0) ra OH to rfl 3 -H 31 R QJ S q 10 to 0 C 0 0 •H Q) e 0 rO tn Q) X 4J rX| 0 3 0 CP <0 •H -c; •H QJ •H •H QJ •H •H u 4J 4J JH •H b -q 1 3 Q 0 QJ tn 0 •3 >q 4J •H "d X!
03 3 l q 4-1 ro 4-1 4J 0 E 6 0 rQ 0 ro 0 c 0 0 0) b >H OJ
C ro <0 ^H - H N r~H 0 0 o q C OH rO • ro rfl r-H x . x ; *C O O X! U t j O a , a , 1/5
CP
lo
hi
OJ rH X) rfl EH
i s o l a t e d from the same sample.
( i i i ) Calothrix D 550 was r e l a t i v e l y Z n - r e s i s t a n t although i t comes
from a s i t e w i t h low Zn l e v e l s . The alga had been i s o l a t e d by
p h y s i c a l means and subcultured i n a medium w i t h 0 . 0 4 mg 1 * Zn u n t i l
the time of assay, so presumably Zn-resistance can not have been
enhanced during i s o l a t i o n of the s t r a i n .
In a l l four instances where the alga was assayed i n the presence
and absence of combined n i t r o g e n , i t was more s e n s i t i v e under the
l a t t e r c o n d i t i o n s . The d i f f e r e n c e i n response was due not only t o a
decreased growth r a t e , but at l e a s t f o r Anabaena cylindrica and
Calothrix membranacea also to the alga being k i l l e d a t lower concen
t r a t i o n s of Zn. The i n f l u e n c e of Zn on the growth r a t e of Calothrix
D 1 8 4 , both i n the presence arid absence of combined n i t r o g e n i s shown
i n F i g . 7.1. The alga was more s e n s i t i v e under the l a t t e r c o n d i t i o n s .
One s t r a i n , Phormidium autumnale D 475, was chosen f o r d e t a i l e d
i n v e s t i g a t i o n . I t was selected because of easy growth i n ACM medium,
the one used f o r a l l major experiments, and because of a s l i g h t l y f a s t e r growth
rates than other s t r a i n s i . e . Calothrix D 184, Calothrix membranacea D179,
Calothrix D 473, and Phormidium sp. D 476. The alga was t e s t e d f o r
i t s s e n s i t i v i t y both t o Zn and Cd, the two metals t o which a l l the a l g a l
populations had been exposed i n the f i e l d t o some ext e n t . The
in f l u e n c e of Zn on the growth of Phormidium D 475 using c h l a as a.
growth c r i t e r i o n i s shown i n Fig. 7 . 2 . The alga t o l e r a t e d up t o
25 mg 1 * Zn. The i n f l u e n c e of Cd on growth i s shown i n Fig . 7 . 3 .
The alga t o l e r a t e d up t o 0 . 8 mg 1 1 Cd, At these concentrations of Zn
and Cd, there was a lag of about 6 days.
10 i
9
8
194 , Calothrix D184
ADM + N
Calothrix D184
contro l
2-0 mg 1-1 Zn
20 24
time (d)
ADM - N
cont ro l 2-0 mg 1-1 Zn
4-0
6-0
8-0 10-0
20 24
time (d)
F i g . 7„1 I n f l u e n c e of Zn on growth o f Calothrix D184, i n the
presence and absence of combined n i t r o g e n .
to
9
8
£ o control
2-5 mg l ' 1 Zn O 6 5 0
10-0
15-0
2 0 0
1
8 12 16 20 24
time (d) p i g . 7.2 Influence of Zn on growth of Phormidi urn autumnale D475
10
8 CD
O control - o -
g jtt mg [-1 Cd O 6
0-6
4 - 0-8
1
time (d) Fig. 7.3 Inf l u e n c e of Cd on growth of Phormidium autumnale D475
196.
7.4 Factors i n f l u e n c i n g t o x i c i t y
A comparison was made of the i n f l u e n c e of environmental f a c t o r s
upon Zn and Cd t o x i c i t y t o a s t r a i n i s o l a t e d from a high Zn s i t e
{Phormidium autumnale D 475) and a " c o n t r o l " s t r a i n (Anacystis nidulans):
( i ) Ca A moderate e f f e c t upon the growth of alga a t d i f f e r e n t Ca
l e v e l s (5 - 200 mg 1 ') was evident i n the metal-free c o n t r o l s . The
in f l u e n c e of Ca on the t o x i c i t y o f Zn i s given i n Table 7.2. A marked
a m e l i o r a t i n g e f f e c t occurred up t o the highest l e v e l (200 mg 1 1 Ca)
i n v e s t i g a t e d . Increasing Ca caused a s i m i l a r increase i n tolerance
t o Cd (Table 7.3).
( i i ) EDTA caused marked decreases i n t o x i c i t y i n a l l cases.
( i i i ) PO -P A s l i g h t decrease upon the growth of alga occurred i n -1
basal medium a t ^ > 56 mg 1 PO^-P. The i n f l u e n c e of PO -P on the
t o x i c i t y of both Zn and Cd i s shown i n Tables 7.4, 7.5. A similar-
decrease on the t o x i c i t y o f Zn and Cd was evident i n a l l cases up t o
-1 28 mg 1 P04~P.
(i v ) pH_ Only very s l i g h t growth of the alga occurred a t pH 7.0
i n c o n t r o l s i n Zn-free medium. A moderate decrease i n the t o x i c i t y
of Zn occurred when the pH was r a i s e d from 6.5 t o 8.0, (Table 7.6),
but C d - t o x i c i t y was s l i g h t l y a f f e c t e d by a s i m i l a r r i s e i n pH (Table
7.7) .
(v) Zn and Cd i n t e r a c t i o n The i n f l u e n c e o f Zn on the t o x i c i t y o f
Cd, using c h l a as a c r i t e r i o n of growth i s shown i n Table 7.8. A
marked increase i n the tolerance of Cd, occurred w i t h i n c r e a s i n g Zn
concentrations. I n c o n t r a s t Cd brought about no detectable change i n
the t o x i c i t y of Zn.
J2 U
w Cn ia B
vO m c i
. o P w 1) m
TS <u
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3 <0 j | E 3
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<D -r-J 0 XJ XJ II
c N 4-J ,^ O i_n >i II
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4-> 1 fl r-l XI CD £ Cn <£> • P B
% ' C <D 0 u a rd P
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OJ r-H •S EH
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• • • CN • o o CO <—<
o +1 +i HH +l
m CI CN CO IX T) T> » • • • O CN m
•3* CN CN in oo T3 O CN ci CO in VO W O O O o o o
o -H + 1 -H +i -H -H ci
in CO IN o IX • • • • • •
O CN CN in r-
t in 00 T—H o CN CO CN c i 1 rH 0) O C o o »-H CP O +i -H +1 +1 + 1 +t E CM -—' 0 CN CO iX) o CO a IX t • • • • •
CN C! in iX>
00 CN in i£> m in vo m cn in
(/) o o o o o o -H +i +1 +i +i + 1
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in in in
. c i ^ > d CTi CO r~- in CO
o W o o O o
o +i -H -H +l -H +1
m co CN d IX *
r- r- co CO
^ ^
i m o m O o o
CP «—i CN in o o a *—H CN
(0
198.
03
Q
(D M 0)
3 + J 3
s O -c; OH
o 4-1 T3 U 4-1 o >1 4-1 •H U •H X o 4-3 <u
+ J
c o o
14-1 o 0> u c d) 3
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a co r-Q)
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s
o • CO CM •a CO CO 00 CO
Cfi T3 t • • • o O o
CM 4H -H + 0) • N
•r4 CO to CO 03 IX T3 T3
CN CO in e a •—i 3 • CM oo in U CM VD in 00 0 c in o O o o o -H o +1 +1 +i -H
s
U
0> 3 m a) E
to > i rd T3
0) S4 3 4->
3 U (D
iw
o • Q)
D i rtj
a) m o •H IX T) • i • • T3 CN m in in
• CN 00 CO GO CO r- < O
. . in 1 o
03 o o o o t-H
II i - l CO 4-1 +1 -H -H +1 +1
S1
T3 U
O IX
i n
CN
CM
CO
CM
m in
o
oo
en e
• "0* CN ro in m v£ >x>
03 o o O o •r-H
+ 1 •H -H +1 -H -H
CN CN CM CO IX
m v£> <£> r-
in CM r- m in
U3 o O t T H
-H -H -H -H •H +1
<£> CO CO O CM o IX
r- oo 00 CO
in O in o O o CN m o o
CO u
199.
0) I rtf rH
>x> r- s • m m C O CM
o ro ro L D • n <v • • • •
Q ro o o o o QJ o + i + i +1 -H
(0 w m [3 •c ro ro 3 o I X •a T3 • • • •
+ J <u O C N ro N
H3 •H w
• O in C O in O 3 E o V£> CM >sr >X>
• H t) rH w o o O O o O • H D
E O o •H +1 -H -H + i +1 U C ro 0 C
•H O C O CM o CM
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•H T3
N - • o *—i CM CM CM II VD m in C O
14-1 1 • • t • • • 0 T3 rH to o O o o o o >i .V CP O -H +1 +1 + i +1 -H 4-> in B CM
•rH u II VO C O C O CM
•H C I X X C to CM ro ro *t in in 0 4->
.—. <U >. • CM O i£> co CM
r C I rti ^ > C O in ro m >£> • P rH T5
tn O o o »—i o O C 0 0 E o + 1 -H + i - H +1 + 1
ft <D 1 <c M o CM m C O »x>
3 I X o rH 4-> in ft r C rH
o 3 4-1 U 0 • CM C O CM in & m
rQ 0) in ro r~- ro ro 0) r C u ID 4-' U) o O O O o o c o Q) U MH » -H -H + l - H + i +i 3 3 O o
r-l w <4-t 03 <D m C O co in c <u CP I X M S IB r-
T-t ft 1 C O rH 1 rH in O O o o X3 Id O Cn t—i C O C O EH ft E CM in
200.
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O ro ro
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CM CM in co o I X • • • • •
m in in in
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t i <X> m 7 3 O CN i£>
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i n r -7 3 o >X) • 7 3 7 3 • •
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+i + 1 +1
c o m I X 7 3 7 3 » • •
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CO V0 CO i n 7 3 O m r o i n • 73 « » f •
CO o o o o +1 + i +1 •H .
CN CN IX) iX> I X •a • » •
m
B i n CN m 7 3 CO CM
7 3 • • » • CO o o o o
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202.
<B
tH .C U
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6 in ro
Q o
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<n S rH 3
••H. u T3 0
0 .c ft 0 4J
T j U
0 >i 4-1 •H U
rH
E-i
T3
T3
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0 > i 4-) 1
rH T J Q) X3 CP 4-1 e C 0) 0 <o
UJT
03 rH 4-> ft r C rH O 3
UH u 0
X3 (U 0) x : o 4-1 c Q) Q) in UH 3 3 0
rH Ul <4H fti 0) C <U CP rH 6
c o CM ro ro
• T3 • • V) O o
CM • + 1 +i
ro T> 73 T J • •
CM ro
CO >X> in
• "O T5 T J • • W O o
o • + 1 •H
IX T3 T3 T J • • in m
{ , CN CM CM
1 •a T ! • • • rH W O o o
CO • +i -H
_a o in CM in
t > IX T3 T> • * • o CM in m
m CM in O m in
• T ) • • • • o W O o o o
• + 1 +i +t -H o
IX) CM ro 1 X TS • • • •
in iX> >X>
CM CM CM in
• •a • » • • W o o *—i o
o +1 +i + 1 +i
CO in CO o IX » • • •
CM r-
o in O in o B ft c o
203.
ni tO r -<*
Q
d) M <0
3
3
n E 3
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a,
5 4-1 O >•.
• P •rH D •H X o •a c o c
0
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a> 3 i—I 14-1 c
co
0)
EH
CP e in ro
tn
(1) N •H Ui
S 3
r-4 3 U o C •H
-a Q)
•H
•a m II c
CP s
u
>1
a>
3 w (0 0) e
>1 -a to
0) n 3
3 U
o Q) CP (0
CP a
a
CP e.
T3 o
• to 00 <3> CN CN T-4 CN
l/l O O o o O o t +1 +1 +1 •H •H *—I
oo O to IX • • • » »
in in m
o CN CO T-H
TJ CN CN r-H
w o o O O O m • + i +1 +1 +1 -H
o CNJ tO CO m
IX tO tO in CO
»-l CO o n ro CN o ro
in O O o o O • +1 +1 -H +1 -H
o O CN CO
IX tO to in CN
in o o TJ CN CN T 1 CN O TJ
o w O o o O o o + l -H +1 +1 -H
IX CN
tO <3-m
o ro CN
o o
to CO o o o d o
204.
7.5 Nitrogen f i x a t i o n
I n view of the f a c t t h a t non-heterocystous forms occurred
widely a t higher l e v e l s of Zn i n the f i e l d ( s e c t i o n 1.2), while
h e t e r o c y s t o u s forms were r a r e , i t seemed p o s s i b l e t h a t Zn might
i n t e r f e r e with n i t r o g e n f i x a t i o n . On the b a s i s of s e n s i t i v i t y of
the algae to growth i n combined n i t r o g e n - f r e e medium a t d i f f e r e n t
l e v e l s of Zn, a l a b o r a t o r y assay was c a r r i e d out u s i n g two
h e t e r o c y s t o u s forms, Anabaena cylindrica and Calothrix D 184 to e s t a b l i s h
the e f f e c t of Zn on ni t r o g e n a s e a c t i v i t y , using the a c e t y l e n e r e d u c t i o n
assay ( s e c t i o n 2.9) as i n d i c a t o r . The comparison i s made on a s t r a i n
from a presumed low Zn s i t e ( Anabaena cylindrica) and from a high Zn
s i t e (Calothrix D 184).
The r e s u l t s are summarized i n Table 7.9. I t can be seen t h a t
while Anabaena cylindrica i s only s l i g h t l y more s e n s i t i v e than Calothrix
D 184 when Zn i s f i r s t added, the e f f e c t i s pronounced 24 h a f t e r the -1
a d d i t i o n of the Zn. For i n s t a n c e , a t 5 mg 1 Zn, the r a t e of a c e t y l e n e -1 -1 r e d u c t i o n was 0.003 and 0.016 nmol C„H ug c h l a min. f o r Anabaena 2 4
and Calothrix r e s p e c t i v e l y .
205.
a rd
- P to
•H co 0) i c
N
>i £1 C O
•H +J rd X
•H in C 0) 81
p •H C
H
rd
in rd
rd W 10 (0 C O
•H P O 3
T3 OJ
0) c 0
rH >1
p 0J u <
c •H s
x : u
tn 3 .
u rH O 6
P*
3 u 0
J3 x>
C 0) Nl 0)
3
4-1 c —
G in rd C O 6 0) N
•H U) H P •H P • P O C to 3 rd 0)
•o Cn O P 0 M SH O X\ X i
.—, P P 0) 10 i n tn •H C 3 c 0) O II 0)
rH •P rH >! 0) c P >1 0) u • 0) u o c rd !H •H 3
0) B P p C P H O 0) o 3 X
X ! CT. 0 0) U X I C n 3 0
-H > CO P N •H >
P o CD •H C 0
O 0) p •H •H C 3 4-1 M
<D 0) 0 3 a U 01 0 C 1 T> M 0) c 0) 3 tsi • H
rH SH 144 T) >H c C rd H rd O
I X
•H C rd
cn
o
CN Stf m r - l o o o o o o o o
CN CO oo ^H o o o o o o o o o o
o o o o
+1
i n m ro T-t < O O O O
O O O O
oo o m t - i CN T - I O O o o o o
o o o o o o o o
o r-< m o
a
o
i n o
CN
o CD m -1 o o o o o o o
<3> CN — < O o o o o o o o o
o o o o o o o o
C N I - ~ . C N C N oo ix> r~- m CN .-i <^ <-l C N ^ H O O o o o o o o o o
o o o o o o o o
o m o o «—< C N
o m o o r-t CN
CN
S
m o •H
q CO •H M 31 Q CJ
rd •H q OJ • q ra 4 J
- q 0 Cd M q rd •< O
206.
CHAPTER 8
DISCUSSION
8.1 Production of r e s i s t a n t s t r a i n s
I t i s evident t h a t f o r Anacystis nidulans the development of
tolerance t o any one p a r t i c u l a r heavy metal takes place e a s i l y , but
t h a t t h i s does not confer general resistance t o a l l metals. Marked
changes i n resistance t o metals other than the one used t o 'develop'
tolerance were few and there were as many examples of a decrease as of
an increase i n resistance (Table 8.1). Only i n the case of the Cd-
t o l e r a n t s t r a i n taken from a Cd-rich medium was there increased
resistance t o a l l the other four metals {Table 8.1). Most f i e l d s i t e s
associated w i t h heavy metal mining a c t i v i t i e s are contaminated
simultaneously by several metals, so any blue-green alga behaving i n a
s i m i l a r manner t o A. nidulans would need t o acquire separate mechanisms
of tolerance f o r each metal. Presumably t h i s makes e v o l u t i o n of
t o l e r a n t populations considerably more d i f f i c u l t than i f only one
metal was present a t elevated l e v e l s . The c o n d i t i o n s r e q u i r e d i n
c u l t u r e of g r a d u a l l y i n c r e a s i n g s t r o n g l y i n h i b i t o r y l e v e l s are however
l i k e l y t o be f u l f i l l e d a t many f i e l d s i t e s , such as the surroundings
of mine wastes. T y p i c a l l y , a wide range of m i c r o - h a b i t a t s w i t h
d i f f e r i n g metal l e v e l s occurs a t such s i t e s . At l e a s t under l a b o r a t o r y
c o n d i t i o n s , s t r a i n s of A. nidulans can grow at comparatively high l e v e l s
of metals w i t h only s l i g h t changes i n l a g , exponential growth r a t e and
f i n a l y i e l d (Table 3.2). The r e s i s t a n t s t r a i n s obtained by t r a i n i n g
w i t h these metals do not lose t h e i r resistance by s u b c u l t u r i n g without
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c u l t u r e s presumably e l i m i n a t e s s e n s i t i v e u n i t s and thus renders the
r e s i s t a n t p o p u l a t i o n s t a b l e . The l e v e l of resistance t o Zn of both
Zn-t5.0 and Zn-tl2.0 t o l e r a n t s t r a i n s d i d not change du r i n g long term
s u b c u l t u r i n g , 72 and 96 generations r e s p e c t i v e l y (Table 3.3) a t a low
concentration of Zn (0.04 mg 1 S. The f a c t t h a t resistance i s not
l o s t even a f t e r several subcultures of the r e s i s t a n t s t r a i n s i n metal-
fre e media s t r o n g l y suggests t h a t these s t r a i n s are mutant s t r a i n s
(s e c t i o n 1.3).
The uses of s t r o n g l y i n h i b i t o r y l e v e l s of metal was suggested
e a r l i e r f o r producing Cu-resistant s t r a i n s of fungi and yeasts by
(Ashida 1965), who concluded t h a t " i n successful l a b o r a t o r y t r a i n i n g
f o r adaptation t o metal t o x i c a n t s f u n g i are u s u a l l y exposed f o r many
passages t o the t o x i c a n t s at concentrations which p a r t i a l l y i n h i b i t
the growth of i n o c u l a n t s " . I n a d d i t i o n DE F i l l i p p i s and Pallaghy
(1976b) were able t o develop Zn and Hg I I resistance i n the Emerson
s t r a i n of Chlorella under l a b o r a t o r y c u l t u r i n g c o n d i t i o n s by m a i n t a i n i n g
c e l l s i n the exponential phase of growth while exposing them t o sub
l e t h a l concentration of e i t h e r Zn or Hg I I . The lack of mutants t o l e r a n t
t o Cu reported p r e v i o u s l y (Sarma 1979) may perhaps have been due t o
c u l t u r e s not being made from s t r o n g l y i n h i b i t o r y l e v e l s of metals.
The reason why such l e v e l s o f metals are needed t o i s o l a t e metal t o l e r a n t
mutants are not c l e a r . As mentioned above f o r Zn, a p o s s i b l e s p e c i f i c
r o l e of the metal cannot be excluded, but a more l i k e l y e xplanation i s
t h a t the mutation r a t e i s g r e a t l y increased i n the slower growing c u l t u r e s
c o n t a i n i n g many f i l a m e n t s or sub-spherical s t r u c t u r e s . Nevertheless there
was apparently no increased r a t e of mutation f o r a n t i b i o t i c r e s i s t a n c e
(Section 3.32). A f u r t h e r explanation may be t h a t u n i t s growing i n a
s t r o n g l y i n h i b i t o r y l e v e l of metal have already undergone phenotypic
adaptation f o r p a r t i a l r e s i s t a n c e t o t h a t metal. I n basal medium any mutant,
which i s p o t e n t i a l l y capable of growing a t a higher l e v e l of metal than t h a t
p o s s i b l e f o r the w i l d - t y p e , w i l l not have undergone such phenotypic
adaptation. Only a mutant a c q u i r i n g i n one step a p a r t i c u l a r l y
large increase i n r e s i s t a n c e w i l l grow on t r a n s f e r from basal medium
t o a p o t e n t i a l l y i n h i b i t o r y l e v e l of metal. This l a s t seems p l a u s i b l e
f o r an element l i k e Zn where there i s a d i s t i n c t d i f f e r e n c e i n the
lag shown by the w i l d - t y p e according t o whether or not the c u l t u r e was
grown p r e v i o u s l y a t a s t r o n g l y i n h i b i t o r y l e v e l .
The present r e s u l t s f i t i n w i t h Ashida's (1965) conclusions f o r
b a c t e r i a , filamentous f u n g i and yeasts t h a t r e s i s t a n c e t o some
organic t o x i c a n t s and a n t i b i o t i c s may develop more r e a d i l y than t o
metal t o x i c a n t s . I n the present study w i t h A. nidulans, i t was found
t h a t none of the m e t a l - r e s i s t a n t s t r a i n s i s o l a t e d a f t e r a t l e a s t 25
subcultures had increased i n tolerance by a f a c t o r greater than 10
times t h a t of the w i l d - t y p e (Table 3.1 ) . I n c o n t r a s t , s t r a i n s of
A. nidulans r e s i s t a n t t o 3 mg 1 * streptomycin (300 x w i l d - t y p e ) , and
1.25 mg 1 * p e n i c i l l i n (125 x w i l d - t y p e ) were i s o l a t e d a f t e r 4 and 7
subcultures i n t o a medium w i t h s t r o n g l y i n h i b i t o r y l e v e l s of streptomycin
and p e n i c i l l i n r e s p e c t i v e l y . These r e s u l t s suggest a s i m i l a r behaviour
t o t h a t recorded by Kumar (1964) who i s o l a t e d A. nidulans t o l e r a n t t o
50 mg 1 * streptomycin (50,000 x w i l d - t y p e ) and 8 mg 1 * p e n i c i l l i n
(400 x w i l d - t y p e ) a f t e r 15 and 10 s e r i a l s u b c u l t u r e s , r e s p e c t i v e l y .
The production of filamentous forms f o l l o w i n g treatment of
A. nidulans w i t h heavy metals, was s i m i l a r t o t h a t reported w i t h
mutagenic or other t o x i c agents ( s e c t i o n 1.32). The metal-induced
r e s i s t a n t s t r a i n s of A. nidulans showed profuse f i l a m e n t a t i o n during
growth i n t h i s metal supplemented medium, no such e f f e c t was
obtained i n the w i l d - t y p e s t r a i n . C y t o l o g i c a l studies were not made t o
e s t a b l i s h the extent t o which these f i l a m e n t s were t r u l y m u l t i c e l l u l a r
or only coenocytic l i k e the mutants described i n Kunisawa and Cohn-
Bazire (1970). Sarma (1979) suggested t h a t formation o f long
filamentous c e l l s by A. nidulans a f t e r exposure t o Cu might be due
to the Cu sorbed t o the c e l l w a l l s i n t e r f e r i n g i n some way w i t h c e l l
d i v i s i o n .
I n the present study an i n i t i a l morphological response t o Cu was
the formation of sub-spherical u n i t s q u i t e d i f f e r e n t i n shape from
the normal rods (Fig. 3 .10 ) - This observation i s s i m i l a r t o t h a t
reported e a r l i e r by Sadler and Trudinger (1967) f o r Pseudomonas. At
s u b - l e t h a l concentrations o f Cu, morphological changes took place by
conversion of b a c t e r i a l rods i n t o s p h e r i c a l forms. I n the case of
A. nidulans, filamentous s t r u c t u r e s d i d e v e n t u a l l y dominate i n
c u l t u r e s of the Cu-resistant s t r a i n .
The p o s s i b i l i t y t h a t metal treatment, i n a d d i t i o n t o leading to
the s e l e c t i o n of mutations of the w i l d - t y p e f o r metal-resistance,
might also have selected f o r other mutations from rods t o f i l a m e n t s
can not be r u l e d out. However the f i l a m e n t a t i o n process was
r e v e r s i b l e , i . e . a l l the m e t a l - t o l e r a n t s t r a i n s showed normal rod
shaped u n i t s when grown i n the absence of metal, although the average
len g t h of the u n i t s of Zn-tl2.0 s t r a i n was gr e a t e r ( F i g . 3 . 1 0 , 3.11).
8.2 Laboratory tolerance and t o x i c i t y
A l l the heavy metals whose i n f l u e n c e was t e s t e d on the growth of
A. nidulans were found t o be more t o x i c than Zn t o a l l the s t r a i n s
t e s t e d ( s e c t i o n 4.1 ) ; Cu was found i n general t o be the most t o x i c t o
both w i l d - t y p e and m e t a l - t o l e r a n t s t r a i n s . These r e s u l t s confirm the
observations on filamentous green algae made by Whitton (1970a) where
Cu was demonstrated t o be more t o x i c than Zn. The r e s u l t s of Sparling
(1968) c o n t r a s t w i t h the observations of other workers i n t h a t Zn and
Cu each had the same e f f e c t on the t o t a l growth of A.nidulans. Up to
5.0 mg 1 1 of both metals s t i m u l a t e d growth. I n a d d i t i o n the present
211.
r e s u l t s f o r Pb agree w i t h those of Whitton (1970a) i n t h a t when Pb i s
added as (Pb(N0 ) ) , i t i s much less t o x i c than Zn; however under these 3 2
circumstances l i t t l e of Pb i s i n s o l u t i o n . Davies et al. (1976)
found t h a t Pb was h i g h l y t o x i c t o rainbow t r o u t and the t o x i c i t y
was more pronounced i n s o f t than hard water.
The tolerance of A. nidulans t o various metals appears i n
general t o be considerably less than e x h i b i t e d by the green algae
(se c t i o n 1.2). Rana and Kumar (1974b) demonstrated t h a t A. nidulans,
Oscillatoria sp. and Arthrospira jenneri were a l l very s e n s i t i v e t o
Zn, w h i l e Chlorella vulgaris and Scenedesmus sp. were h i g h l y t o l e r a n t ,
growing i n 25 and 30 mg 1 1 Zn, r e s p e c t i v e l y . K a t a g i r i (1975) found
t h a t 0.10 mg 1 ^ Cd caused a 50% r e d u c t i o n i n the growth r a t e of
A. nidulans. I n c o n t r a s t , Chlorella sp. r e q u i r e d more than 0.5 i g 1 '
Cd t o i n h i b i t the growth r a t e by a s i m i l a r amount (Hart and Scaife
197 7). The maximum and median tolerance l i m i t s of some blue and green
algae t o Cu and Zn found by Rana and Kumar (1974b) ( c a l c u l a t e d from
copper sulphate quoted by the authors) are given below (Table 8.2) :
Table 8.2 Tolerance t o Cu and Zn of species s t u d i e d by Rana and Kumar (1974).
maximum tolerance median tolerance (mg 1-1) (mg l " 1 )
Cu Zn Cu Zn Anacystis nidulans 0.4 1.5 0.16 1.0
Oscillatoria sp. 0.4 1.0 0.2 0.5
Arthrospira jenneri 0.4 1.5 0.16 0.4
Plectonema boryanum 3.98 30 0.16 10
Nodularia spumigena 0.279 2.0 0.2 0.7
Anabaena doliolum 0.2 1 .0 - -
Fischerella muscicola 0.2 1.0 - -
Chlorella vulgaris 2.0 25 1.0 5.0
Scenedesmus sp. 3.2 30 1 .6 5.0
212.
S p a r l i n g ' s (1968) r e s u l t s are s u r p r i s i n g i n t h a t A. nidulans was
reported to r e q u i r e 5 mg 1 * Zn f o r 50% r e d u c t i o n of the t o t a l growth.
His r e s u l t s c o n t r a s t with the much g r e a t e r s e n s i t i v i t y found i n the
p r e s e n t study (Table 8.1). Rama and Kumar (1974.b) reported t h a t
A. nidulans i s v e r y s e n s i t i v e to high c o n c e n t r a t i o n s of Zn, any
i n c r e a s e i n the c o n c e n t r a t i o n s beyond t h a t of b a s a l medium (presumably
about 0.05 eg 1 Zn) had a growth r e t a r d i n g e f f e c t . T h e i r r e s u l t s are
s i m i l a r to those of K a t a g i r i (1975) who found t h a t A. nidulans c u l t u r e s
with 0.001 and 0.01 mg 1 * Zn had doubling times of 8 h, whereas i n the
presence of 0.1 and 1.0 mg 1 ' Zn, they had doubling times of 10 and
15 h, r e s p e c t i v e l y . I n comparison the p r e s e n t study found 0.04 mg 1 * Zn
l e d to a doubling time of 8.2 h, and 0.5 and 1.0 mg 1 1 Zn to doubling
times of 11.6 h and 15 h, r e s p e c t i v e l y .
Z i n c requirement The f a i l u r e to demonstrate a requirement f o r Zn
i n the growth of e i t h e r w i l d - t y p e or Z n - t o l e r a n t A. nidulans s t r a i n s
may be due to the f a c t t h a t the l e v e l of Zn i n the medium was not
reduced below 0.04 mg 1 the l e v e l d e r i v e d as contaminants from other
chemicals (Table 2.3), beside glassware and d e i o n i z e d water. A survey
of the l i t e r a t u r e ( s e c t i o n 1.21) summarized i n Table 8.3 shows t h a t the
amount of Zn r e q u i r e d by most organisms f o r t h e i r optimum growth i s v e r y
s m a l l . T h i s c o n t r a s t s with the r e s u l t s of Coleman et al. (1971) where
Chlorella vulgaris r e q u i r e d 18.03 mg 1 * Zn f o r optimum growth. The
r e s u l t s of Lange (1971) are anomalous. Although most workers who have
i n v e s t i g a t e d m i c r o n u t r i e n t requirements have found i t i m p o s s i b l e to d e t e c t
l e v e l s of Z n ^ 1 ug 1 Lange reported a d d i t i o n of 0.08 ug 1 * Zn to
f i l t e r e d Lake E r i e water enhanced the growth of 15 c u l t u r e s of Anabaena
cirinalis, and Nostoc muscorum i n a t l e a s t 3 and 5 i n s t a n c e s r e s p e c t i v e l y .
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8.3 Environmental f a c t o r s i n f l u e n c i n g m e t a l - t o x i c i t y to
Anacystis nidulans s t r a i n s
The r e s u l t s presented i n Chapter 5 show c l e a r l y t h a t s e v e r a l
environmental f a c t o r s have a marked i n f l u e n c e on the t o x i c i t y of the
thr e e metals t e s t e d (Zn, Cu, Cd), but i n each case the f a c t o r s d i f f e r
(Table 5.2). In view of the l a r g e l i t e r a t u r e on environmental e f f e c t s
on metal t o x i c i t y ( s e c t i o n 1.4) only a few comparative comments w i l l
be made. Many of the e f f e c t s are s i m i l a r to those reported f o r other
organisms. For i n s t a n c e , the marked i n f l u e n c e of EDTA and PO^-P i n
reducing Z n - t o x i c i t y i s found a l s o i n many e u k a r y o t i c p l a n t s
(Whitton 1980) . Phosphate a l s o reduced the t o x i c i t y of Zn to
Plectonema boryanum (Rana and Kumar 1974b), while n i t r a t e had l i t t l e
e f f e c t on reducing Z n - t o x i c i t y , thus resembling the p r e s e n t r e s u l t s
for A. nidulans. A p o s s i b l e e x p l a n a t i o n of these r e s u l t s may be t h a t
Zn and phosphate, but not n i t r a t e , e n t e r the c e l l s a t the same s i t e
thus i n t e r f e r i n g with each other during uptake by a l g a e . T h i s
i n t e r f e r e n c e of phosphate and Zn with the e n t r y of each other a c r o s s
the c e l l membrane p r e v i o u s l y suggested by Rana and Kumar (1974a) may be
supported by the r e s u l t s i n Table 5.21. When phosphate was introduced
a f t e r i n o c u l a t i o n , i t had much l e s s e f f e c t i n reducing Z n - t o x i c i t y
than i f introduced together w i t h Zn. R e c e n t l y Rigby et al. (1980) found
t h a t Zn i n h i b i t e d phosphate uptake by Synechococcus leopoliensis, while
other c a t i o n s (except Mg) s t i m u l a t e d i t . I t i s t h e r e f o r e p o s s i b l e t h a t
some of the observed e f f e c t s of metals on Zn t o x i c i t y reported i n
Chapter 5 may be due to i n d i r e c t e f f e c t s on phosphate uptake i n a d d i t i o n
to d i r e c t c a t i o n competition. Both Ca and Mg reduced the t o x i c i t y of
Zn and Cd to A. rd dulans in most c a s e s . With the two Z n - t o l e r a n t s t r a i n s ,
the e f f e c t of Mg was g r e a t e r a t s e v e r a l lower c o n c e n t r a t i o n s than t h a t
of Ca, but the i n f l u e n c e of Ca i n c r e a s e d over a much g r e a t e r range of
215.
c o n c e n t r a t i o n s . The g r e a t e r i n f l u e n c e of Mg i n reducing Zn t o x i c i t y
to t o l e r a n t r a t h e r than s e n s i t i v e s t r a i n s has been r e p o r t e d f o r the
green a l g a e , Hormidium rivulare (Say and Whitton 1977) and Stigeoclonum
tenue (Harding and Whitton 1977). The e f f e c t i v e n e s s of Ca a t higher
l e v e l s i n reducing the t o x i c i t y of Zn and Cd to both w i l d - t y p e and
t o l e r a n t s t r a i n s would f i t the hy p o t h e s i s t h a t a mechanism e x i s t s whereby
the Zn i s i n i t i a l l y bound p a s s i v e l y . With such a mechanism, Ca might
compete with Zn f o r these s i t e s . I t i s w e l l documented t h a t the presence
of Ca around roo t s may g r e a t l y reduce heavy metal t o x i c i t y (Wyn-Jones and
Lunt 1967). The e f f e c t of Mg could be by way of the same p r o c e s s ,
p a r t i c u l a r l y s i n c e t h i s element sh a r e s some s i m i l a r i o n i c p r o p e r t i e s
w i t h Zn. The lack, of any i n f l u e n c e of Mg and the only s l i g h t e f f e c t of
Ca on C u - t o x i c i t y i s s u r p r i s i n g . The e x p l a n a t i o n may perhaps l i e i n the
r e l e a s e of a strong Cu complexing agent, such as has been shown f o r
Anabaena flos-aguae (McKnight and Morel 1979). I n c o n t r a s t to Anacystis
nidulans, Mg antagonizes C u - t o x i c i t y to Bacillus licheniforme more than
Zn or C d - t o x i c i t y (Haavik 1976).
Comparison of the r e s u l t s of the t o x i c i t y t e s t s w i t h o b s e r v a t i o n s
on the growth of A. nidulans i n a m e t a l - f r e e medium, but w i t h a s i m i l a r
c o n c e n t r a t i o n of i o n s , showed t h a t Mg, Ca, Pe and PO^-P had q u i t e
a d i f f e r e n t i n f l u e n c e on antagonism than on growth. The d i f f e r e n c e i s
most obvious f o r Mg and Fe, where r a i s i n g the c o n c e n t r a t i o n s from 0.25
to 160 mg 1 ' and 0.5 to 20 mg 1 * r e s p e c t i v e l y , brought about an
i n c r e a s e d antagonism to the t o x i c i t y of Zn, Tables 5.6, 5.17, a t the
same time causing a marked r e d u c t i o n i n t o t a l growth of w i l d - t y p e .
T h i s i s s i m i l a r to the r e s u l t s of Harding and Whitton (1977) f o r
Stigeoclonum tenue i n which Mg had a q u i t e d i f f e r e n t i n f l u e n c e on
antagonism than on growth. The p r e s e n t r e s u l t s on the i n f l u e n c e of Pe
on the t o x i c i t y of Cu to A. nidulans c o n t r a s t with those of Steeman
N i e l s e n and Kamp=Nielsen (1970) on Chlorella pyrenoidosa. I n the
216.
l a t t e r Fe d e t o x i f i e d the, e f f e c t of Cu a t the c o n c e n t r a t i o n s used i n
growth medium (6 jig 1 ' Fe) , but i t had no e f f e c t on Cu t o x i c i t y to
Anacystis nidulans. SteemanoNielsen and Kamp-Nielsen suggested t h a t
t h e i r r e s u l t s might be due to the presence of f e r r o u s c h l o r i d e , which
i n a l k a l i n e s o l u t i o n forms n e g a t i v e l y charged c o l l o i d s capable of
adsorbing c a t i o n s or p o s i t i v e l y charged complexes. Greene e t al. (1975)
suggested t h a t the p r o t e c t i o n of Selenastrum capricornutum from the
t o x i c e f f e c t s of Zn i o n s b y N a , Mg, Ca and P was due l a r g e l y to
i n c r e a s e s i n i o n i c s t r e n g t h . They f u r t h e r suggested t h a t the formation
of an 'ion p a i r ' between Zn and such i o n s might lower the a v a i l a b i l i t y
of Zn to the a l g a . T h i s does not f i t w i t h the p r e s e n t r e s u l t s f o r
Anacystis nidulans (Tables 5.1; A5.2, A5.4, A5.6) or f o r Stigeoclonum
tenue (Harding and Whitton 1977) ; i n both c a s e s some ions (e.g. K +, CI , 2- + SO^ and to some e x t e n t Na ) had no d e t e c t a b l e e f f e c t on t o x i c i t y even
a t very high c o n c e n t r a t i o n s . The behaviour of Anacystis nidulans i n
mixed s o l u t i o n s of Zn and Cd a l s o d i f f e r s markedly from some other organisms.
For Bacillus subtilis s s p . niger, Pseudomonas sp. ( P i c k e t t and Dean 1979)
and Hormidium rivulare (Say and Whitton 1977) Zn and Cd are s y n e r g i s t i c
i n t h e i r t o x i c e f f e c t s . On the other hand Zn reduces markedly Cd
t o x i c i t y to wi l d - t y p e Anacystis nidulans ( F i g . 5.15j Table A5.25)
and probably a l s o the two Z n - t o l e r a n t s t r a i n s ( F i g . 5.16; Table A5.26);
s i m i l a r l y the presence of Cd appears to reduce Zn t o x i c i t y to the
C d - t o l e r a n t mutant (Table 8.1) thus resembling the r e s u l t s of Nakano et al.
(1979) f o r Euglena gracilis and of P i c k e t t and Dean (1976) f o r Klebsiella
(Aerobacter) aerogenes, i n which an a n t a g o n i s t i c i n t e r a c t i o n o c c urred
between Zn and Cd. The remainder of the data i n Table 5.1 i n d i c a t e t h a t
the t o x i c e f f e c t of Zn with other heavy metals was a d d i t i v e or perhaps
sometimes s y n e r g i s t i c (e.g. Cu) i n t h e i r a c t i o n (Tables 5.28, 5.32).
The i n f l u e n c e of pH on Zn t o x i c i t y d i f f e r e d between wild-type
and the two Z n - t o l e r a n t s t r a i n s ( F i g s 5.17, 5.18, 5.19). A r i s e i n
pH however brought about a marked re d u c t i o n i n Zn and Cd t o x i c i t y to
Wild-type ( F i g s 5.17, 5.20) while the same r i s e i n pH caused i n c r e a s e
i n Zn t o x i c i t y to the Z n - t o l e r a n t s t r a i n s ( F i g s 5.16, 5.19). An
i n c r e a s e i n pH over the range 6.5 to 8.0 le a d s to i n c r e a s e d p r e c i p i
t a t i o n , so t h i s alone might perhaps e x p l a i n the decreased t o x i c i t y
found with the w i l d - t y p e ; i t ob v i o u s l y can not e x p l a i n the i n c r e a s e d
t o x i c i t y found with the other two s t r a i n s . The o b s e r v a t i o n t h a t a
r i s e i n pH over the range pH 6.5 to 8.0 l e d to a decrease i n the
t o x i c i t y of Zn c o n t r a s t s w i t h some o b s e r v a t i o n s i n the l i t e r a t u r e
( s e c t i o n 1.4). Mount (1966) showed t h a t a r i s e i n pH l e d to an
i n c r e a s e i n Zn t o x i c i t y to fathead minnow. Hargreaves and Whitton
(1976) demonstrated t h a t r i s e s i n pH above 3.0 l e d to an i n c r e a s e i n
the t o x i c i t y of Zn to a population of Hormidium rivulare i s o l a t e d
from a stream a t pH 3.1. Say and Whitton (1977) confirmed t h a t r i s e s
i n pH from pH 3.0 to pH 8.0 l e d to an i n c r e a s e i n the t o x i c i t y of Zn
to Z n - s e n s i t i v e and Z n - t o l e r a n t p o p u l a t i o n s of Hormidium rivulare.
On the other hand the p r e s e n t r e s u l t s are confirmed by the r e s u l t s of
K a t a g i r i (1975) f o r Anacystis nidulans and Hart and S c a i f e (1977) f o r
Chlorella pyrenoidosa; i n both cases a r i s e i n pH between 7 and 8
reduced the t o x i c i t y o f Cd. R i s e s i n pH a l s o reduced the t o x i c i t y of
Zn to both Z n - s e n s i t i v e and Z n - t o l e r a n t p o p u l a t i o n s of Stigeoclonum
tenue, but t h i s e f f e c t was more marked with the l a t t e r (Harding and
Whitton 1977). There was no i n f l u e n c e of pH on the t o x i c i t y of Cu to
e i t h e r w i l d - t y p e or Cu-t5.0 t o l e r a n t s t r a i n s of A. nidulans.
8.31 Organic compounds i n the medium
A major problem i n any heavy metal t o x i c i t y i n v e s t i g a t i o n i s to
understand the i n f l u e n c e of f a c t o r s e t h e r than the p a r t i c u l a r one
218.
under study. T h i s i s p a r t i c u l a r l y so when the metal l e v e l s are low
and t o x i c e f f e c t s could e a s i l y be masked. Of the i n o r g a n i c s y n t h e t i c
media a v a i l a b l e i n the l i t e r a t u r e ACM has been formulated s p e c i f i c a l l y
f o r Anacystis nidulans. T h i s medium i n c l u d e s EDTA as a c h e l a t i n g
agent. Since EDTA i s known to form complexes with (presumably a l l )
metal i o n s , ion t o x i c i t y i s l i k e l y to be reduced. I t was not p o s s i b l e
to exclude EDTA completely from the medium, but i t s l e v e l was kept as
low as p o s s i b l e .
The lower l e v e l of phosphate i n the medium l e d to i n c r e a s e d
need to b u f f e r the pH. Of the common b u f f e r s used i n freshwater
c u l t u r e s most are known to p o s s e s s some complexing a b i l i t y , or to be
u n s u i t a b l e f o r some other reason such as being t o x i c , p r e c i p i t a t i o n on
a u t o c l a v i n g , or being used up by algae as e s s e n t i a l n u t r i e n t s
(Smith and Foy 1974). HEPES was chosen as a b u f f e r i n g agent before i t
was r e a l i z e d t h a t t h i s too can a c t as a c h e l a t i n g agent ( s e c t i o n 2.22).
The p r e s e n t work showed s l i g h t changes i n the t o x i c i t y of Zn to w i l d -
type A. nidulans w i t h i n c r e a s i n g l e v e l s of HEPES (Table 5.26)
which can not be ex p l a i n e d by the poor b u f f e r i n g w i t h the lower l e v e l s
of HEPES; they are presumably a d i r e c t e f f e c t of c h e l a t i n g by the
HEPES molecule. The s i t u a t i o n i s s l i g h t l y c omplicated because both
EDTA and HEPES were p r e s e n t s i m u l t a n e o u s l y i n the medium,- with EDTA
known to a c t as a strong c h e l a t i n g agent. The i n f l u e n c e of HEPES i n
the absence of EDTA has not so f a r been s t u d i e d . However the l e v e l s
of EDTA and HEPES used f o r v a r i o u s experiments were given i n Table 2.4.
8.4 Accumulation of Zn by Anacystis nidulans
Anacystis nidulans c u l t u r e s are capable of accumulating high
c o n c e n t r a t i o n s of Zn and there i s l i t t l e i n d i c a t i o n t h a t r e s i s t a n t
s t r a i n s accumulate d i f f e r e n t amounts of z i n c compared with the w i l d - t y p e .
219.
For i n s t a n c e w i l d - t y p e and Zn-tl2.0 s t r a i n s grown i n batch c u l t u r e
with 1.0 mg 1 * Zn e v e n t u a l l y accumulate 1605 and 1792 ug Zn g *
dry weight, r e s p e c t i v e l y (Tables 6.2 and 6.4); r e s p e c t i v e accumulation
r a t i o s a r e 2866 and 4074. The i n i t i a l high Zn uptake i s mostly
removed by EDTA washes ( s e c t i o n 6.3), suggesting t h a t t h i s Zn i s only
l o o s e l y a s s o c i a t e d with the c e l l s . At l a t e r s t a g e s i n growth only a
s m a l l percentage of the Zn i s removable with EDTA, i n d i c a t i n g t h a t
i t s accumulation i s a s s o c i a t e d with growth of the a l g a . With i n c r e a s i n g
Zn i n the medium, i n c r e a s e d Zn i s accumulated by the a l g a . With the
Zn-tl2.0 s t r a i n the p r o p o r t i o n a l i n c r e a s e i n o l d c u l t u r e s i s about the
same between 0.1 and 1 mg 1 * Zn as between 1 and 10 mg 1 * Zn. For
i n s t a n c e a t a growth stage with 300 mg 1 1 dry weight ( F i g . 6.4) there
i s a 4.8-fold i n c r e a s e i n a l g a l Zn f o r a 10-fold i n c r e a s e i n e n v i r o n
mental Zn.
No i n v e s t i g a t i o n was c a r r i e d out to study the e f f e c t of other
f a c t o r s on Zn accumulation by A. nidulans, but K a t a g i r i (1975)
found t h a t Cd accumulation i s pH dependent, more accumulation o c c u r r i n g
a t n e u t r a l pH than a t more a l k a l i n e v a l u e s . A s i m i l a r o b s e r v a t i o n
has been made with Chlorella (Hart and S c a i f e 1977). T h i s phenomenon
may be r e l a t e d to the f a c t t h a t s o l u b i l i t y of metal i s reduced a t
more a l k a l i n e c o n d i t i o n s ; the Cd may be p r e s e n t i n a form which
cannot be t r a n s p o r t e d by the c e l l .
8.5 Tol e r a n c e and adaptation shown by f i e l d m a t e r i a l
I t i s c l e a r t h a t some blue-green algae are capable of r e s i s t i n g
very high l e v e l s of Zn. At l e a s t i n streams these are u s u a l l y very
narrow forms which can o f t e n be r e f e r r e d to Plectonema ( s e c t i o n 1.1).
220.
Members of C h l o r o c o c c a l e s may a l s o be p r e s e n t , and, although u s u a l l y
s m a l l - c e l l e d , l a r g e r forms may occur. Laboratory a s s a y s ( T a b l e 7.4 )
have shown t h a t most i s o l a t e s from high Zn s i t e s are adapted forms,
being more t o l e r a n t of high Zn l e v e l s than most i s o l a t e s from s i t e s
l a c k i n g Zn-enrichment, thus resembling o b s e r v a t i o n s f o r Stigeoclonum
teuue (Harding and Whitton 1977) and Hormidium rivulare (Say and
Whitton 1977) . However the r e s u l t s of Calothrix D 473 and Calothrix
parietina D 550 d i d not f i t w i t h the other r e s u l t s . Although
Calothrix D 473 was i s o l a t e d from sediments r e l a t i v e l y r i c h i n Zn, i t
was r e l a t i v e l y s e n s i t i v e i n the l a b o r a t o r y a s s a y . A p o s s i b l e
e x p l a n a t i o n i s t h a t the s i t e i s h i g h l y c a l c a r e o u s , a f a c t o r reducing
the t o x i c i t y of Zn to most organisms ( s e c t i o n 1.4). C. parietina D 550
was r e l a t i v e l y Z n - r e s i s t a n t although i t comes from a s i t e w i t h low Zn
l e v e l s (Table 2.7 ) . I t i s d i f f i c u l t to suggest any e x p l a n a t i o n . The
a l g a was i s o l a t e d by p h y s i c a l means and s u b c u l t u r e d i n a medium with
0.04 mg 1 1 Zn u n t i l the time of a s s a y . A study was c a r r i e d out by
Stokes e t al. (1973) on'the heavy metal t o l e r a n c e of algae from contaminated
l a k e s i n the Sudbury mining a r e a of O n t a r i o . W h i l s t not g i v i n g c l e a r
evidence f o r g e n e t i c adaptation of s p e c i e s of Chlorella vulgaris and
Scenedesmus acuminatus to the t o x i c i t y of Cu and Ni , i t d i d show t h a t
s t r a i n s i s o l a t e d from contaminated l a k e s were more t o l e r a n t than c l o s e l y
r e l a t e d l a b o r a t o r y s t r a i n s of s p e c i e s i n the same genera. I n d i s c u s s i n g
t h e i r r e s u l t s , Stokes et al. p o s t u l a t e t h a t the two genera may be
i n h e r e n t l y more adaptable to the t o x i c e f f e c t of the metals than most
other a l g a e .
I n s p i t e of o b s e r v a t i o n s such as th e s e , Whitton and Say (1975)
conclude t h a t i t seems probable t h a t not a l l algae growing i n high
c o n c e n t r a t i o n s of heavy metals i n the f i e l d have developed s p e c i a l
221 .
t o l e r a n c e to the p a r t i c u l a r m e t a l ( s ) . As the number of s p e c i e s of
algae which can grow i n non-polluted streams i s so l a r g e , s e v e r a l
s p e c i e s may e x i s t which may p o s s e s s the c a p a c i t y to grow i n e l e v a t e d
c o n c e n t r a t i o n s of metal 'by a c c i d e n t ' and t h a t might be what happened
with Calothrix parietina D 550.
8.51 Nitrogen f i x a t i o n
There are two p o s s i b l e e x p l a n a t i o n s f o r the p o s s i b l e absence of
n i t r o g e n f i x a t i o n i n streams with l e v e l s of Zn above 10 mg 1 \ and
i t s r a r i t y (judged by frequency of h e t e r o c y s t o u s organisms (Whitton
1980)) a t s l i g h t l y lower l e v e l s . I t may be due simply to these
environments c a r r y i n g s u f f i c i e n t combined n i t r o g e n t h a t p o t e n t i a l
n i t r o g e n f i x e r s have no s e l e c t i v e advantage. On the other hand i t may
be due to the p a r t i c u l a r s e n s i t i v i t y to Zn of n i t r o g e n a s e or some other
f e a t u r e of the n i t r o g e n f i x e r s . The g r e a t e r s e n s i t i v i t y of s t r a i n s
when grown i n the absence of combined n i t r o g e n r a t h e r than i t s
presence (Table 7.1 ) suggests t h a t the second e x p l a n a t i o n may be
important. N e v e r t h e l e s s i t can not be the s o l e reason because
Calothrix D 184 i s capable of growing i n a medium f r e e of combined
n i t r o g e n ( F i g . 7.1 ) , y e t w i t h a Zn l e v e l h i g h e r than y e t recorded i n
the f i e l d f o r a n i t r o g e n - f i x i n g blue-green a l g a .
8.6 Concluding remarks
The occurrence of blue-green algae a t s i t e s p o l l u t e d by heavy
metals was reviewed a t the beginning of t h i s t h e s i s . During the p r e s e n t
study i t has been shown t h a t Anacystis nidulans can adapt to the heavy
metals Co, Ni, Cu, Zn and Cd, a f t e r repeated s u b c u l t u r e i n the presence
of these r e s p e c t i v e metals.A h i g h l y - i n h i b i t o r y l e v e l of metals was e s s e n t i a l
f o r o b t a i n i n g these a d a p t a t i o n s . I t thus seems l i k e l y t h a t such adaptation
can take p l a c e q u i t e e a s i l y i n nature where a wide range of metal l e v e l s
222.
are l i k e l y to occur w i t h i n one region such as the v i c i n i t y of a mine.
I t i s to be hoped t h a t f u r t h e r work, u s i n g a range of l a b o r a t o r y
s t r a i n s and both l i q u i d and s o l i d media, may l e a d to a more complete
understanding of a d a p t a t i o n f o r heavy metal r e s i s t a n c e by blue-green
'algae.
The i n d i c a t i o n t h a t f a c t o r s such as Ca, Mg and PO^-P p l a y a r o l e
i n reducing metal t o x i c i t y to A. nidulans f i t s the h y p o t h e s i s
t h a t more than one t o l e r a n c e mechanism may be p r e s e n t and shows how
complicated the s i t u a t i o n may be i n the f i e l d . A p a s s i v e binding of
Zn to exchange s i t e s i n the r e g i o n of the c e l l w a l l might be
s u b j e c t e d to competition from other ions such as Ca and Mg. From the
morphological o b s e r v a t i o n s on A. nidulans s t r a i n s i t was
p o s s i b l e only to s p e c u l a t e how the Zn sorbed to the c e l l w a l l s may
i n t e r f e r e i n some way, such as with c e l l d i v i s i o n , l e a d i n g to the
production of filamentous or s u b - s p h e r i c a l forms, i t i s c l e a r t h a t more
d e t a i l e d study of the morphology of A. nidulans would h e l p
c l a r i f y the p o s s i b l e r o l e of Zn i n producing morphological changes.
Other s t u d i e s which would h e l p understanding of the i n f l u e n c e of Zn
are ones u s i n g ^ Z n to f o l l o w up the accumulation work reported here.
While most of the experiments r e p o r t e d here were performed with
l a b o r a t o r y c u l t u r e s , some t o x i c i t y t e s t s were c a r r i e d out on algae
i s o l a t e d from high Zn s i t e s . These algae s t i l l showed high Zn
t o l e r a n c e i n the l a b o r a t o r y a f t e r i s o l a t i o n . The f a c t t h a t non-
h e t e r o c y s t o u s forms of blue-green algae occurred a t higher l e v e l s of
Zn i n the f i e l d than d i d h e t e r o c y s t o u s forms a l s o r e q u i r e s f u r t h e r
study to see i f Zn i n h i b i t s n i t r o g e n f i x a t i o n d i r e c t l y .
223.
SUMMARY
( i ) R e l a t i v e l y low c o n c e n t r a t i o n s of heavy metals i n h i b i t e d the
growth of Anacystis nidulans. The maximum c o n c e n t r a t i o n s of Co, Ni,
Cu, Zn and Cd t o l e r a t e d by w i l d - t y p e a l g a , a s judged by the l e v e l s of
metals proving s t r o n g l y i n h i b i t o r y , were 0.32, 0.16, 0.15, 1.5 and 0.55 -1 -1 mg 1 , r e s p e c t i v e l y . I n c o n c e n t r a t i o n s < 0.5 mg 1 Zn, growth was
almost the same as i n c o n t r o l , both as regards the length of the l a g
phase and the f i n a l p o p u l a t i o n d e n s i t y .
( i i ) Wild-type Anacystis nidulans was found a l s o to be v e r y s e n s i t i v e
to low c o n c e n t r a t i o n s of a n t i b i o t i c s , i n h i b i t i o n of growth o c c u r r i n g -1
a t 0.01 ug ml of p e n i c i l l i n , polymyxin or streptomycin, as judged
by the c o n c e n t r a t i o n l e a d i n g to a 50% re d u c t i o n i n growth r a t e .
( i i i ) Mutants of Anacystis nidulans t o l e r a n t to high l e v e l s of Co, Ni,
Cu, Zn or Cd were obtained by repeated s u b c u l t u r i n g a t s t r o n g l y i n
h i b i t o r y l e v e l s of metals. For i n s t a n c e the l e v e l of Zn a t which strong
i n h i b i t i o n o c c u r r e d was r a i s e d from 1.45 to 16.5 mg 1 1 Zn a f t e r 75
s u b c u l t u r e s . For Co, Ni, Cu or Cd the l e v e l s were r a i s e d to 2.45, 1.30,
0.55 and 2.5 mg 1 * , r e s p e c t i v e l y , a f t e r 25 s u b c u l t u r e s . The comparative
response of v a r i o u s s t r a i n s to a p a r t i c u l a r metal was i n g e n e r a l q u i t e
s i m i l a r whether judged by growth r a t e , l a g or y i e l d
( i v ) Colony formation on agar w i t h d i f f e r e n t l e v e l s of Zn showed t h a t
the a c q u i s i t i o n of i n c r e a s e d r e s i s t a n c e was due to the production of
mutants.
The mutants correspond to
those regarded as spontaneous elsewhere i n the blue-green a l g a l
l i t e r a t u r e , but c r i t i c a l experiments were not made to r u l e out the
p o s s i b i l i t y t h a t Zn i t s e l f has a r o l e as a mutagenic agent.
224.
(v) I t proved i m p o s s i b l e to demonstrate tle_presence of mutants
when making s u b c u l t u r e s from medium l a c k i n g Zn-enrichment. On the
other hand the presence of s u b - i n h i b i t o r y Zn d i d not l e a d to any
i n c r e a s e i n the r a t e of 'spontaneous' mutation f o r p e n i c i l l i n or
streptomycin t o l e r a n c e . The use of NTG (N-methyl-N'-nitro-N-
n i t r o s o g u a n i d i n e ) as a mutagenic agent f a i l e d to l e a d to any d e t e c t a b l e
i n c r e a s e i n the r a t e of mutation f o r Z n - t o l e r a n c e . NTG a l s o f a i l e d to g
l e a d to d e t e c t a b l e mutation (1 i n 5 x 10 u n i t s ) i n c u l t u r e s l a c k i n g
Zn-enrichment.
( v i ) A l l the metal t o l e r a n t s t r a i n s s t i l l grew w e l l i n b a s a l medium.
There was no i n d i c a t i o n t h a t they now r e q u i r e d higher l e v e l s of these
metals f o r optimum growth; only very s l i g h t changes were d e t e c t a b l e i n
the l a g , e x p o n e n t i a l growth r a t e and y i e l d as compared w i t h the w i l d -
type .
( v i i ) Two of the mutants w i t h high t o l e r a n c e f o r Zn and one f o r each of
the other metals were chosen f o r comparative s t u d i e s . Assays of c r o s s -
r e s i s t a n c e of each of the f i v e types of mutant were made to other four
m e t a l s . I n most c a s e s changes i n c r o s s - r e s i s t a n c e were only s l i g h t ,
w ith about equal numbers of examples of i n c r e a s e d and decreased
r e s i s t a n c e . Examples of marked changes were i n c r e a s e d C o - r e s i s t a n c e
of a C d - t o l e r a n t s t r a i n , and decreased C d - r e s i s t a n c e of a N i - t o l e r a n t
s t r a i n .
( v i i i ) The i n f l u e n c e was t e s t e d of a range of f a c t o r s on the t o x i c i t y of
Cu, Zn and Cd to the w i l d - t y p e , of Zn to two Z n - t o l e r a n t s t r a i n s , and of
Cu to a C u - t o l e r a n t s t r a i n . K (5 - 500 mg l " 1 ) and C I (10 - 200 mg l " 1 ) had
no d e t e c t a b l e e f f e c t . HEPES N-2 hydroxyethylpiperazine-N'-2-ethanesulphonic
a c i d (0.3 - 1.2 mg 1 l e d to s l i g h t r e d u c t i o n i n Zn t o x i c i t y to w i l d - t y p e ;
not s u r p r i s i n g l y , EDTA caused a marked r e d u c t i o n i n t o x i c i t y i n a l l c a s e s .
225.
I n c r e a s i n g l e v e l s of Cu, Cd and Hg a l l l e d to an i n c r e a s e d t o x i c i t y ,
the e f f e c t s of c o n c e n t r a t i o n s of the i n d i v i d u a l metals apparently
being a d d i t i v e r a t h e r than s y n e r g i s t i c . I n c r e a s e s i n Ca, Mn, Fe,
Mg and P reduced Zn t o x i c i t y to both wild-type and Z n - t o l e r a n t s t r a i n s ,
but the s t r a i n s d i f f e r e d i n t h e i r response to pH. With the wild-type
a r i s e i n pH (6.0 - 8.0) brought about major r e d u c t i o n s i n t o x i c i t y ,
w h i l e t h a t r i s e brought about an i n c r e a s e i n the t o x i c i t y to
Z n - t o l e r a n t s t r a i n s .
( i x ) The responses of Zn-t5.0 and Zn-tl2.0 s t r a i n s to p e n i c i l l i n ,
polymyxin and streptomycin were a l s o t e s t e d . I n every case there
was a s l i g h t i n c r e a s e i n r e s i s t a n c e to about 6x the l e v e l t o l e r a t e d
by w i l d - t y p e .
(x) Morphological changes were noted a t the higher l e v e l s of
metals and i n some ca s e s there was c o n s i d e r a b l e d i v e r s i t y w i t h i n a
s i n g l e f l a s k . At the l e v e l of metals s u b - i n h i b i t o r y f o r each of
these s t r a i n s f i l a m e n t s were produced. The morphological response
to Cu was d i f f e r e n t . At i n h i b i t o r y l e v e l s of Cu, w i l d - t y p e ,
Cu-t0.5 and Zn-t5.0 a l l formed many s u b - s p h e r i c a l u n i t s . A f u r t h e r
marked change took p l a c e with Cu-t0.5 during s u b c u l t u r e s 22 to 25.
Although there was no d e t e c t a b l e change i n the Cu-tolerance of the
p o p u l a t i o n as a whole, the s u b s p h e r i c a l s t r u c t u r e became r e p l a c e d
completely by f i l a m e n t s . S u b s p h e r i c a l s t r u c t u r e s s i m i l a r to those
found a t high l e v e l s of Cu were a l s o noted w i t h C o - t l . 8 grown a t high
Co l e v e l s , but here these u n i t s formed l e s s than 1% of the p o p u l a t i o n .
( x i ) No b i g s h i f t s i n absorption maxima of pigments between mutants
and wild-type were observed, but p a r t i c u l a r l y a t i n t e r m e d i a t e Cd-
c o n c e n t r a t i o n s , there was a marked i n c r e a s e i n the r e l a t i v e phycocyanin
226.
content of wi l d - t y p e Anacystis nidulans. Phycocyanin was not
observed a t any c o n c e n t r a t i o n s of Cd i n the growth of C d - t o l e r a n t
s t r a i n s .
( x i i ) The Zn c o n c e n t r a t i o n s in the a l g a (ug g * dry weight) i n c r e a s e d
w i t h i n c r e a s e d Zn i n the medium and g e n e r a l l y a l s o w i t h the age of
the c u l t u r e .
( x i i i ) A r e s i s t a n t s t r a i n of Anacystis (Zn-tl2.0) took up approximately
the same amount of Zn as wild-type from environmental c o n c e n t r a t i o n s a t
which they both grew (0.1 and 1.0 mg 1 * Zn).
(x i v ) Algae i s o l a t e d from high c o n c e n t r a t i o n s of Zn i n the f i e l d
had i n g e n e r a l a much g r e a t e r r e s i s t a n c e to the metal than Ones
growing i n lower c o n c e n t r a t i o n s or obtained from a l g a l c u l t u r e c o l l e c t i o n s .
The s i t u a t i o n i s however not completely c l e a r - c u t , because one i s o l a t e
of Calothrix (C. parietina D 550) from a low Zn s i t e showed c o n s i d e r a b l e
Zn r e s i s t a n c e .
(xv) A l a b o r a t o r y comparison was made of the i n f l u e n c e of Zn on n i t r o g e n
f i x a t i o n from a s t r a i n from a presumed low Zn s i t e (Anabaena cylindrica)
and one from a high Zn s i t e {Calothrix D 184) shows t h a t the former was
only s l i g h t l y more s e n s i t i v e than Calothrix when Zn was f i r s t added.
The e f f e c t was however pronounced 24 h l a t e r .
2 2 7 .
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239.
APPENDIX
240.
C o n t e n t l i s t A3 page
T a b l e A3.1 I n f l u e n c e o f Co on t h e g r o w t h o f C o - t l . 8 . 244
A3.2 I n f l u e n c e o f Ni on t h e g r o w t h o f N i - t l . O . 244
A3.3 I n f l u e n c e o f Cu on t h e g r o w t h o f Cu-t0.5. 245
A3.4 I n f l u e n c e o f Zn on t h e g r o w t h o f Zn-t5.0. 245
A3.5 I n f l u e n c e o f Zn on t h e g r o w t h o f Z n - t l 2 . 0 . 246
A3.6 I n f l u e n c e o f Cd on t h e g r o w t h o f Cd-t2.0. • ••• 246
A3.7 I n f l u e n c e o f Zn and t i m e o f h a r v e s t i n g i n 247
b a t c h c u l t u r e on l e n g t h o f w i l d - t y p e Anacystis.
A3.8 I n f l u e n c e o f Zn and t i m e o f h a r v e s t i n g i n 248
b a t c h c u l t u r e on l e n g t h o f Z n - t 5 . 0 ; i n o c u l u m
f r o m b a s a l medium.
A. 3.9 I n f l u e n c e o f Zn and t i m e o f h a r v e s t i n g i n 249
b a t c h c u l t u r e on l e n g t h o f Z n - t 5 . 0 ; i n o c u l u m
f r o m s t r o n g l y i n h i b i t o r y l e v e l o f Zn.
Content l i s t A4.0 page 241
Table A4.1 I n f l u e n c e of Co on the growth of wild-type 250
A4.2 I n f l u e n c e of Co on the growth of N i - t l . 0 250
A4.3 I n f l u e n c e of Co on the growth of Cu-t0.5 251
A4.4 I n f l u e n c e of Co on the growth of Zn-t5.0 251
A4.5 I n f l u e n c e of Co on the growth of Z n - t l 2 . 0 252
A4.6 I n f l u e n c e of Co on the growth of Cd-t2.0 252
A4.7 I n f l u e n c e of Ni on the growth of wi l d - t y p e 253
A4.8 I n f l u e n c e of Ni on the growth of C o - t l . 8 253
A4.9 I n f l u e n c e of Ni on the growth of Cu-t0.5 254
A4.10 I n f l u e n c e of Ni on the growth of Zn-t5.0 254
A 4 . l l I n f l u e n c e of Ni on the growth of Z n - t l 2 . 0 255
A4.12 I n f l u e n c e of Ni on t h e growth of Cd-t2.0 255
A4.13 I n f l u e n c e of Cu on the growth of wi l d - t y p e 256
A4.14 I n f l u e n c e of Cu on the growth of C o - t l . 8 256
A4.15 I n f l u e n c e of Cu on the growth of N i - t l . 0 257
A4.16 I n f l u e n c e of Cu on the growth of Zn-t5.0. 257
A4.17 I n f l u e n c e of Cu on the growth of Z n - t l 2 . 0 . 258
A4.18 I n f l u e n c e of Cu on the growth of Cd-t2.0 258
A4.19 I n f l u e n c e of Zn on the growth of w i l d - t y p e . 259
A4.20 I n f l u e n c e of Zn on the growth of C o - t l . 8 . 259
A4.21 I n f l u e n c e of Zn on the growth of N i - t l . 0 260
A4.22 I n f l u e n c e of Zn on the growth of Cu-t0.5. 2 6 0
A4.23 I n f l u e n c e of Zn on the growth of Cd-t2.0. 261
A4.24 I n f l u e n c e of Cd on the growth of w i l d - t y p e . 261
A4.25 I n f l u e n c e of Cd on the growth of C o - t l . 8 . 262
A4.26 I n f l u e n c e of Cd on the growth of N i - t l . 0 . 262
A4.2 7 I n f l u e n c e of Cd on t h e growth of Cu-t0.5. 263
A4.28 I n f l u e n c e of Cd on the growtn of Zn-t5.0 263
A4.29 I n f l u e n c e of Cd on the growth of Z n - t l 2 . 0 264
A4.30 I n f l u e n c e of Hg on the growth of wi l d - t y p e 264
A4.31 I n f l u e n c e of Pb on the growth of wi l d - t y p e 265
A4.32 Pigment changes during growth of wi l d - t y p e , 266
Anacystis a t d i f f e r e n t Cd c o n c e n t r a t i o n s .
242.
page Content l i s t A5.0
Table A5.1 I n f l u e n c e of Na on Cd t o x i c i t y to wi l d - t y p e Anacystis. ... 267
A5.2 I n f l u e n c e of K on Zn t o x i c i t y to wild-type Anacystis 267
A5.3 I n f l u e n c e of K on Cd t o x i c i t y to wi l d - t y p e Anacystis 268
A5.4 I n f l u e n c e of Ca on Zn t o x i c i t y to wi l d - t y p e Anacystis. ... 268
A5.5 I n f l u e n c e of Ca on Zn t o x i c i t y to Zn-t5.0 Anacystis 269
A5.6 I n f l u e n c e of Ca on Zn t o x i c i t y to Zn-tl2.0 Anacystis 269
A5.7 I n f l u e n c e of Ca on Cd t o x i c i t y to wi l d - t y p e Anacystis. ... 270
A5.8 I n f l u e n c e of Mn on Zn t o x i c i t y to Zn-t5.0 Anacystis 270
A5.9 I n f l u e n c e of Mn on Zn t o x i c i t y to Zn-12.0 Anacystis 271
A5.10 I n f l u e n c e of Fe on Zn t o x i c i t y to Zn-t5.0 Anacystis 271
A5.11 I n f l u e n c e of Fe on Zn t o x i c i t y to Zn-tl2.0 Anacystis 272
A5.12 I n f l u e n c e of Fe on Cu t o x i c i t y to wild-type Anacystis. ... 272
A5.13 I n f l u e n c e of Fe on Cu t o x i c i t y to Cu-t0.5 Anacystis 273
A5.14 I n f l u e n c e of PO^-P on Zn t o x i t i t y to w i l d - t y p e Anacystis- 273
A5.15 I n f l u e n c e of PO^-P on Zn t o x i c i t y to Zn-t5.0 Anacystis. .. 274
A5.16 I n f l u e n c e of PO^-P on Zn t o x i c i t y to Zn-tl2.0 Anacystis. . 274
A5.17 I n f l u e n c e of PO^-P on Cd t o x i c i t y to w i l d - t y p e Anacystis. 275
A5.18 I n f l u e n c e of NO^-N on Zn t o x i c i t y to w i l d - t y p e Anacystis. 275
A5.19 I n f l u e n c e of EDTA on Zn t o x i c i t y to wi l d - t y p e Anacystis. • 276
A5.20 I n f l u e n c e of EDTA on Zn t o x i c i t y to Zn-t5.0 A n a c y s t i s . . . . 276
A5.21 I n f l u e n c e of EDTA on Zn t o x i c i t y to Zn-tl2.0 Anacystis... 277
A5.22 I n f l u e n c e of EDTA on Cd t o x i c i t y to wi l d - t y p e Anacystis. . 277
A5.23 I n f l u e n c e of Zn on Cd t o x i c i t y to w i l d - t y p e A n a c y s t i s . . . . 278
u n i t s ml was used as a growth c r i t e r i o n .
243.
page
T a b l e A5.24 I n f l u e n c e o f Zn on Cd t o x i c i t y t o w i l d - t y p e Anacystis; ... 278
c h l a was used as a g r o w t h c r i t e r i o n .
A5.25 I n f l u e n c e o f low Zn l e v e l s on Cd t o x i c i t y t o Zn-t5.0 .... 279
Anacystis.
A5.26 I n f l u e n c e o f h i g h Zn l e v e l s on Cd t o x i c i t y t o Zn-t5.0 .... 279
Anacystis.
A5.27 I n f l u e n c e o f pH on Zn t o x i c i t y t o w i l d - t y p e Anacystis 280
A5.28 I n f l u e n c e o f pH on Zn t o x i c i t y t o Zn-t5.0 Anacystis 280
A5.29 I n f l u e n c e o f pH on Zn t o x i c i t y t o Zn-tl2.0 Anacystis 281
A5.30 I n f l u e n c e o f pH on Cd t o x i c i t y t o w i l d - t y p e Anacystis 281
A5.31 I n f l u e n c e o f i n o c u l u m s i z e on Zn t o x i c i t y t o 282
w i l d - t y p e Anacystis; u n i t s ml * was used as a
g r o w t h c r i t e r i o n .
A5.32 I n f l u e n c e o f i n o c u l u m s i z e on Zn t o x i c i t y t o 282
w i l d - t y p e Anacystis; c h l a was used as a g r o w t h
c r i t e r i o n .
C o n t e n t l i s t A6.0
6.1 A c c u m u l a t i o n o f Zn d u r i n g b a t c h c u l t u r e g r o w t h 283
o f w i l d - t y p e Anacystis. R e s u l t s show t o t a l
v a l u e s f o r a l g a + f i l t e r .
6.2 A c c u m u l a t i o n o f Zn d u r i n g b a t c h c u l t u r e g r o w t h 284
o f Z n - t l 2 . 0 Anacystis. R e s u l t s show t o t a l
v a l u e s f o r a l g a + f i l t e r .
T a b l e A 3 . 1 I n f l u e n c e o f C o o n t h e g r o w t h o f C o - c l . 8
t i m e C h )
S o u r c e o f i n o c u l u m
t i m e C h )
f r o m s t r o n g l y i n h i b i t o r y l e y e l o f m e t a l t i m e C h )
C o ( m g l " 1 )
t i m e C h )
0 0 . 3 0 . 6 1 . 2 1 . 8
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
2 4 7. 5 x l 0 3 6 . 3 x l 0 5 4 x l 0 5 3 . 2 x l 0 5 2 . 5 x l 0 5
4 8 3 . 2 x l 0 6
6 2 . 8 x 1 0
6 1 . 8 x 1 0 8 x l 0 5 4 x l 0 5
7 2 2 . 5 x l 0 ? 2 . 2 x l 0 7 1 . 3 x l 0 ? 2 . 5 x l 0 6 1 . 3 x l 0 6
9 6
1 l x l O 8 8 x l 0 7 5 x l 0 7
6 8 x 1 0 3 . 2 x l 0 6
1 1 2 0 1 . 3 x l 0 8 l x l O 8 8 x l 0 7 1 . 8 x l 0 ? 5 . 6 x l 0 6
1 4 4 !
a 1 . 6 x 1 0 1 . 4 x l 0 8 l x l O 8 3 . 2 x l 0 7 1 . 4 x l 0 7
l g
1 6 8 1 . 8 x 1 0 1 . 6 x l 0 8 l . l x l O 8 !
i
7 5 x 1 0 2 x l 0 7
T a b l e A 3 . 2 I n f l u e n c e o f N i o n t h e g r o w t h o f N i - t t . O
t i m e ' h )
S o u r c e o f i n o c u l u m
t i m e ' h )
f r o m c t r o r g l y i n h i b i t o r y l e v e l o f m e t a l t i m e ' h )
N i ( m g l " 1 )
t i m e ' h )
0 0 . 2 0 . 4 0 . 8 1 . 0
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
2 4 8 x l 0 5 6 . 3 x l 0 5 5 x l 0 5 3 . 6 x l 0 5 2 . 8 x l 0 5
4 8 5 . 6 x l 0 6 4 . 5 x l 0 6 2 . 5 x l 0 6 1 . 4 x l 0 6 6 . 3 x l 0 5
7 2 3 . 2 x l 0 ?
7 2 . 2 x 1 0 l x l O 7
6 5 . 6 x 1 0
6 2 . 5 x 1 0
9 6 H
1 x 1 0 5 . 6 x l 0 ?
7 3 . 2 x 1 0
7 1 . 8 x 1 0
6 8 x 1 0
1 2 0 8
1 . 4 x 1 0 l x l O 8 5 x l 0 7
7 3 . 2 x 1 0 l . S x l O 7
1 4 4 1 . 6 x l 0 8 1 . l x l O 8 6 . 3 x l 0 ? 4 . 5 x l 0 7 2 . 8 x l 0 7
1 6 8 1 . 8 x l 0 8 1 . 3 x l 0 8 8 x l 0 ? 5 . 6 x l 0 7 4 x l 0 7
TabLe A3.3 I n f l u e n c e of Cu on growth of Cu-t0.5 245.
time (h)
Source of inoculum
time (h)
from s t r o n g l y i n h i b i t o r y l e v e l o f metal time (h)
Cu 'mg 1 ^)
time (h)
0 0.1 0.2 0.4 0.5
0 2 x l 0 5 2 x l 0 5 5
2x10 2K10 5 2 x l 0 5
24 8 x l 0 5 7 . l x l O 5 5 x l 0 5 3 . 2 x l 0 5 2 . 5 x l 0 5
48 6 . 3 x l 0 6 5 . 6 x l 0 6 3 . 2 x l 0 6 1 . 4 x l 0 6 6
1x10
72 3 . 2 x l 0 ? 2 . 5 x l 0 ? 1 . 6 x l 0 ? 6 . 3 x l 0 6 2 . 8 x l 0 6
96 U 1 0 8 8 x l 0 ? 5. 6 x l 0 ? 2 x l 0 ? 8 x l 0 6
120 , - , 8
l . b x l O 8 1.3x10
7 8x10 3.2xlO ? 1 . 6 x l 0 7
144 1 . 6 x l 0 8 1 . 3 x l 0 8 l x l O 8 5 x l 0 ? 2 . 5 x l 0 ?
168 1 . 8 x l 0 8 1 . 4 x l 0 8 I x l O 8 5 . 6 x l 0 7 3 . 2 x l 0 ?
Table A3.4 I n f l u e n c e of Zn on the growth of Zn-t5.0
time Ch)
Source of inoculum
time Ch) bas a l medium s t r o n g l y i n h i b i t o r y l e v e l of metal a t which
adapted time Ch)
Zn 'mg 1 l ) Zn mg 1 1 )
time Ch)
0.04 2.0 4.0 5.0 0.04 2.0 4.0 5.0 6.0
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 2 x l 0 6 8 x l 0 5 2 . 5 x l 0 5 1. l x l O 5 2 x l 0 6 1 . 8 x l 0 6 3 . 2 x l 0 5 2 x l 0 5 2 x l 0 5
48 1. l x l O 7 6
6x10 4 x l 0 5 1 . 4 x l 0 5 7
1.8x10 1 . 6 x l 0 7 6
5.6x10 6
1x10 2 x l 0 5
72 8 1x10 4.2xlO ?
6 3.2x10
6 1.1x10
7 8x10
7 6.3x10 3.6xlO ?
6 8x10 2 . 5 x l 0 5
96 8
4x10 8
1.1x10 2.2xlO ? 5x10° 2 . 2 x l 0 8 2 x l 0 8 8
1x10 3.2xlO ? 3 . 6 x l 0 5
120 5 x l 0 8 3 x l 0 8 7 . l x l O 7 I x l O 7 3 . 6 x l 0 8 8
3.2x10 8
1.6x10 5.6xlO ? 5 . 6 x l 0 5
144 5 . 3 x l 0 8 8
4x10 8
1.6x10 4 x l 0 7 5 . l x l O 8 8
4x10 1 . 8 x l 0 8 6 . 3 x l 0 7 8 x l 0 5
168 5 . 6 x l 0 8 , 8 4.5x10
8 1.5x10 & x l 0 7
8 5.6x10 4 . 5 x l 0 8
8 2x10
7 8x10 1 . I x l O 6
24^.
T a b l e A3.5 I n f l u e n c e o f Zn on t h e g r o w t h o f Z n - t l 2 . 0
t i m e ( h )
Source o f i n o c u l u m
t i m e ( h ) b a s a l medium s t r o n g l y I n h i b i t o r y l e v e l o f m e t a l a t w h i c h a d a p t e d t i m e ( h )
Zn frag 1 S Zn 'mg 1 l )
t i m e ( h )
0.04 4 8 10 11 12 0.04 4 8 10 U 12
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 3 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 l x l O 6 5 . 6 x l 0 5 3. 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 1 . 4 x l 0 6 4 . 2 x l 0 5 1 . 8 x l 0 5 1 . 4 x l 0 5 1 . 4 x l 0 5 l . l x l O 5
48 7 . 1 x l 0 6 3 . 2 x l 0 6 1 . 6 x l 0 6 5 . 6 x l 0 5 3 . 6 x l 0 5 2 . 5 x l 0 5 5 . U 1 0 6 2 x l 0 6 4 . 2 x l 0 5 3 . 2 x l 0 5 1 . 4 x l 0 5 l . l x l O 5
72 1 . 8 x l 0 ? 7
1x10 6
4x10 6
1.4x10 6
1.4x10 4 x l 0 5 2 . 2 x l O ? 2 x l 0 7 S . l x l O 6 l . b x l o 6 1 . 4 x l 0 6 5 . 1 x l 0 5
96 7
2.8x10 1.8x10' 8 x l 0 6 3 . 2 x l 0 6 6
1.8x10 7 . 1 x l 0 5 5. I x l O 7 4 x l 0 7 2 . 5 x l 0 7 1 . 6 x l 0 ? 6
5.6x10 1 . 3 x l 0 5
120 6.3 x 1 0 7 7
4x10 l . S x l O 7 5 . 6 x l 0 6 3 . 6 x l 0 6 l . B x l O 6 8 x l 0 7 8 x l 0 7 5 . 6 x l O ? 4 . 2 x l 0 7 1 . 6 x ) 0 7 2 x l 0 6
144 7
8x10 b . 3 x l 0 7 7
3.2x10 1 . 4 x l 0 ? 8 x l 0 6 6
4x10 8
1.6x10 1 . 4 x l 0 8 8
1x10 7 . 1 x l 0 7 3 . 2 x l O ? 6
6.3x10
168 8
1.2x10 l . l x l O 8 8 . 8 x l o ' 5 . 2 x l o ' 3 . 2 x l o ' 7 1.0x10 1 . 6 x l 0 8 1 . 5 x t 0 8 1 . U 1 0 8 I x l O 8 7 . U 1 0 7
7 2.3x10
T a b l e A3.6 I n f l u e n c e o f Cd on g r o w t h o f C d - t 2 . 0
Source o f i n o c u l u m
t i m e ( h )
f r o r . s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l t i m e ( h )
Cd (mg l " 1 )
0.0 0.5 1.0 1.5 2.5
0 2 x l 0 5 2 x l 0 5 2x10 5 2 x l 0 5 2 x l 0 5
24 l x l O 6 7 . l x l O 5 5 x l 0 5 3.bxlOD 2 . S x l O 3
48 1 . 3 x l 0 7 S x l O 6 5 . 6 x l 0 6 2 . 5 x l 0 6 8 x l 0 5
I 72 1 3 . 6 x l 0 7
1 2 . 5 x l 0 7 1 . 4 x l 0 ? 5 . 6 x l 0 6 l . S x l O 6
| 96 | l x l O 8 7 . U 1 0 7 3 . 6 x l 0 7 1 . 3 x l 0 ? 3 . 2 x l 0 6
120 1 . 3 x l 0 8 8 x l 0 7 5 x l 0 ? 2 . 2 x l O ? 8 x l 0 6
144 1 . 4 x l 0 8 l x l O 8 6 . 3 x l 0 7 3 . 2 x l 0 7 1 . 4 x l 0 7
168 1 . 8 x l 0 8 l . l x l O 8 7 . 1 x l 0 7 4 x l 0 ? 2 . 5 x l 0 7
Table A3 . I I n f l u e n c e o f Zn and t i m e o f h a r v e s t i n g i n b a t c h c u l t u r e
on l e n g t h o f w i l d - t y p e Anacystis.
t i m e Zn ^ l e n g t h c l a s s e s o f c e l l s (h) (mg 1
3 < 6 . 0 6.0<9.0 9.0<12.0 12 .< 18.0 s *C "*>
24 0 .04 90 + 8 .2 10 + 2 .6 0 .5 88 + 8 .5 12 + 3 .0 0 .75 61 + 6 .6 38 4 + 6 . 1 1 .0 45 4 ± 8 . 1 53 6 ± 1 1
48 0 .04 85 ± 5 .7 15 ± 3 .2 0 .5 76 + 10 . 1 24 ± 4 .5 0 .75 52 3 ± 8 . 1 47 7 + 7 .5 1 .0 39 5 + 7 .5 60 5 ± 1 3 .4 1 .25 27 4 + 1 .3 72 6 ± 9 .8
0. 04 62 + 6 38 ± 9 4 0. 5 50 i 8 .5 50 ± 8 .9 0. 75 24 + 3 .6 76 ± 10 7 1 . 0 16 + 1 5 84 ± 9 1 1 . 25 0 92 . 8 ± 7 9 7 2 + 0 .8
0. 04 47 4 ± 5 42. 6 ± 7 0 0 0. 5 29 7 ± 5 4 70. 3 ± 7 .2 0 0. 75 9 6 ± 2 .2 90. 4 + 6 3 0 1. 0 0 69. 2 ± 7 5 30 8 ± 5 .6 1. 25 0 29. 4 4 8 70 6 ± 11 .5
248.
Table A3.8 I n f l u e n c e o f Zn a-d t i m e o f h a r v e s t i n g i n b a t c h c u l t u r e
on l e n g t h o f Z n - t 5 . 0 ; i n o c u l u m was t a k e n f r o m b a s a l medium.
t i m e Z n _ i l e n g t h c l a s s e s o f c e l l s (h) (mg 1 _ )
9^12.0 12^: 24 2 4 ^ 3 6 3 6 ^ 4 8
0 .04 84 .6 + 10 15 .4 + 3 .2 1 .0 83 .6 ± 6. 6 16 .4 + 3 .3 2 .0 78 ± 9. 2 22 ± 4 .8 3 .0 66 .7 ± 14 33 .3 ± 7 .8 4 .0 27 .4 ± 6 72 .6 + 16 .9 5 .0 16 .2 + 4. 5 83 .8 ± 13 .6
0 .04 79 .3 ± 8. 5 20 .7 ± 4 .3 1 .0 71 .2 ± 10. 4 28 .8 ± 5 .4 2 .0 64 + 13. 1 36 ± 8 .0 3 .0 52 .7 + 8. 2 47 .3 + 12 .8 4 .0 32 .3 + 9. 3 67 .7 + 15 . 1 5 .0 25 .6 ± 6. 4 74 .4 + 12 .2
0.04 65 .4 + 10 34.6 ± 10 0 1.0 42 .7 ± 9 57.3 + 9. 8 0 2.0 25 .6 ± 6.2 74.4 ± 12. 4 0 3.0 24 .7 ± 6.8 75.3 + 14. 8 0 4.0 21 .7 ± 5.5 59.7 + 12. 5 18 6 ± 3 .2 5.0 11 .7 ± 3.8 63.1 + 10. 8 25 2 + 3 .5
0.04 61 .6 + 7 .2 38.4 + 8 .4 0 1.0 33 .6 + 6 .5 46 + 12 .5 20.4 + 3 .2 2.0 17 .4 + 3 .6 52 + 17 .2 30.6 + 6 .6 3.0 5 .7 + 1 .4 48 + 11 .8 46.3 + 1 .5 4.0 0 45 + 14 . 1 42.5 + 3 .4 13 .5 ± 4 .2 5.0 0 40 + 13 .7 47 .6 + 5 .4 12 .4 + 3 .7
249.
T a b l e A3.9 I n f l u e n c e o f Zn and t i m e o f h a r v e s t i n g i n b a t c h c u l t u r e
on l e n g t h o f Z n - t 5 . 0 ; i n o c u l u m was taken f r o m s t r o n g l y -
i n h i b i t o r y l e v e l .
t i m e Zn l e n g t h c l a s s e s o f c e l l s (h) (mg l " )
6 < 9 . 0 9 < 12 12< 24 24< 36
0 .04 21 .9 + 3 78. 1 + 6 .7 0 1 .0 20 .30 64 . 5 + 2 .6 15 .2 2 .0 16 .6 + 4 .3 65. 1 + 6 .8 18 • 4 ± 2. 5 3 .0 13 .5 ± 5 .4 43. 2 ± 5 .8 29 .7 ± 4. 2 4 .0 10 + 3 .6 45. 2 + 7 .5 44 .8 + 8. 5 5 .0 8 -9 ± 2 .5 29. 8 + 8 .3 61 .3 ± 3. 3
48 0.04 16.8 83.8 1.0 0 80.9 2.0 0 70.4 3.0 0 61.2 4.0 0 42.9 5.0 31.8
96 0.04 86.2 1.0 71.4 2.0 58.3 3.0 28.4 4.0 13 5.0
192 0-04 67.3 1.0 48.4 2.0 21.6 3.0 16.6 4.0 13.4 5.0
+ 9 .5 0 + 10 19 .1 + 6.8 + 7 .8 29 .6 ± 3.9 + 11 .2 38 .8 ± 7.2 + 10 .6 57 .1 ± 4.3 ± 6 .6 68 .2 + 6.5
+ 6 .8 13 .8 + 1.8 0 + 7 .5 28 .6 ± 6.9 0 ± 11 41 .7 + 4.1 0 + 3 .7 38 .9 ± 7.2 32 .7 + 6. 3 ± 3 .8 42 + 5.6 45 + 8. 5 - 4 7 .2 + 5.4 52.8 ± 5. 3
+ 7 . 1 32 .7 ± 4.8 0 ± 5 .6 51 .6 + 8.2 0 + 3 .8 78 .4 + 7 .5 0 ± 4 .0 52 .4 + 5.7 31 + 5. 7 + 3 .8 45 .6 ± 6.6 41 ± 4. 8 0 36 .2 + 5.4 63 .8 ± 8. 1
2 50.
Table A4.1 I n f l u e n c e of Co on the g r o w o I v i l d - t v p p
Source of Inoculum
t i n e (h>
b a o e l medium s t r o n g l y I n h i b i t o r y l e v e l ot m e t a l
Co (Qg r l ) Co tag l " 1 )
0 0.02 0.04 0.08 0. 1 0.16 0.2 0.32 0 0.02 0.04 0.08 0.10 0.16 0.2 0.4
0 2x10* 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l O S 2 x l 0 5 2 x l 0 3 2 x l 0 5 2 x l 0 5 2 x l 0 5 2x10* 2x10*
24 1 . 3 x l 0 S 7 . 1 x l 0 5 6.3X10 5 4.5xlO S 3 . 6 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 l x l O 6 7 . I x l O 5 5.6X10 5 4 x l 0 5 3 . 2 x l 0 5 J . 2 x l 0 5 3 . 2 x l 0 5 3 . 2 x l 0 5
48 8x10* 5 . I x l O 6 6
3.6x10 l . S x l O 6 l . l x l O 6 5 . 6 x l 0 5 3 . 6 x l 0 5 2 . 5 x l 0 5 7 . U 1 0 6 5 x l 0 6 2.5x10* 1 . 6 x l 0 6 8 x l 0 5 5 . I x l O 5 4x10* 3.2x10*
72 3 . 2 x l 0 ? 2 x l 0 7 8 x l 0 6 6 . 3 x l 0 6 4 x l O S l x l O 6 6 . 3 x l 0 5 4 x l 0 5 2 . 5 x l 0 7 l . S x l O 6 I x l O 7 5 . 6 x l 0 6 2.8x10* l x l O 6 7 . 1 x l 0 5 5 . l x l O 5
96 9 x l 0 ? 6 . 3 x l 0 7 2.5X10 7 2 x l 0 ? l . l x l O 7 2.5X10 6 l x t O 6 5 . U 1 0 5 7 . l x l O 7 5 . U 1 0 7 3 . 2 x l 0 7 l . S x l O 7 8x10* 3.2x10* 1.6x10* 7 . l x l O 3
120 1 . 3 x l 0 8 8 x l O ? 6 . 3 x l 0 7 3.6X10 7 2 x l 0 7 4«10 6 3.6x10* 6 . 3 x l 0 5 1.6xlO S l x l O 8 5 . 1 x l 0 7 3 . 2 x l 0 7 1 . 6 x l 0 7 7.1x10* 4.10 6 l . S x l O 6
144 8
1.6x10 1.3X10 8 Sxl'O 7 5 . 6 x l 0 7 3 . 2 x l 0 7 6 . 3 x l 0 6 l . S x l O 6 8 x l 0 6 2 x l 0 8 1 . 3 x l 0 8 8 x 1 0 7 5 . 1 x l 0 7 2 . 5 x l 0 ? I x l O 7 3 . 6 x l 0 S 2 . 5 x l O S
168 2 . 5 x l 0 8 1 . 6 x l 0 8 1x10* 6 . 3 x l 0 7 4 x l 0 7 I x l O 7 3.2X10 6 1.3.10 6 2 . 5 x l 0 8 l . S x l O 8 1.3xlO S 8 x l 0 7 4 . 5 x l O ? l . S x l O 7 9 x l O S 3 . 6 x l O S
Table kk, 2 I n f l u e n c e of Co on the g r o w t h of N i - c l . O
t i m e (h)
Source o f i n o c u l u m
t i m e (h) b l H l medium e t r o n g l y I n h i b i t o r y l e v e l of met a l a t which
adapted t i m e (h)
Co Cmg l'S Co (mg t " 1 )
t i m e (h)
0 0.05 0.1 0.2 0.4 0.6 0.8 0 0.05 0.1 0.2 0.4 0.6
0 2x10 5 2«10 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2x10 5 2x10 5 2x10 5 2x10 5 2x10 5 2x10 3 2 x l 0 5 2x10 5
24 1.3xlO S B x l O 5 6 . 3 x l 0 5 5 . 6 x l 0 5 4 x l 0 5 2x10 5 1 . 8 x l 0 5 l . l s l o 6 l x l O 6 7 . 1 x l 0 5 4x10 5 2 . 5 x l 0 5 1 . 8 x l 0 5
48 l . l x l O 7 8 x l O S 4 x l 0 6 3 . 2 x l O S 1.3x10* 3. 2 x l O S 1 . 6 x l 0 5 l . l x l O 7 8 x l O S 3 . 6 x l O S 1x10* 4 . 5 x l O S l . S x l O 5
72 5 x l 0 7 4 . 5 x l 0 7 1 . 6 x l 0 7 I x l O 7 2.5X10 6 3 . 6 x l 0 5 l x l O 3 j.esio 7 l . S x l O 7 I x l O 7 3 . 2 x l 0 S 8 x l 0 5 2x10*
96 8x10 7 7 . 1 x l 0 7 4x10 7 2 . 5 x l 0 7 4 s l 0 S 6 . 3 x l 0 5 1 . 3 x l o ' l x l O 8 6 . 3 x l 0 7 2 . 5 x l 0 7 6 . 3 x l O S 1.6xlO S 3 . 6 x l 0 5
120 I.61IO 8 1 . 3 x l 0 8 8x10 7 6 . 3 x l 0 7 8 x l O S I x l O 6 1.8x10* 1 . 6 x l 0 8 8x10 7 3 . 6 x l 0 7 1 . 8 x l 0 7 6
2.5x10 5x10 5
144 2 . 5 x l 0 e 1 . 6 x l 0 S l . l x l O 8 8x10 7 l . S x l O 7 2 . 5 x l 0 6 2 x l 0 5 2.5X10 8 1.3X10 8 6.3x10 7 3 . 6 x l 0 7 5 . 6 x l O S 7 . U 1 0 5
168 3 . 2 x l O S 2 . 5 x l 0 8 1 j 4 x l 0 8 1 . 3 x l 0 8 2 . 8 x l 0 7 5 . 6 x l 0 6 4x10 5 2 . 8 x l 0 5 1.8x10* 8x10 7 4 . 5 x l 0 7 1 . 3 x l o ' l x l O 6
25!
Table . J I n t l u e n c e at O on the uro w t h o f Cu-tO. 4)
time ' h)
Source o f Inoculum
time ' h) baaa l medium s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l t o which adapted time ' h)
Co (mg I ') Co (tug 1
time ' h)
0 0.05 0. 1 0.2 0.4 0.6 0 0.05 0. 1 0.2 0.4 0.6
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l C 5 5
2x10 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 1x10° 7 . I x l O 5 4 . 5 x l 0 5 3 . 2 x l o 5 2 . 5 x l 0 5 2 . 3 x l 0 5 l x l O 6 8 x l 0 5 4 x l 0 5 2 . 5 x l 0 5 2 x l 0 5 1 . 4 x l 0 5
48 b . 3 x l O b 3 . 2 x l 0 6 i x l 0 b 5 . 6 x l 0 5 4 x l 0 5 2 . 5 x l 0 5 6
6.3x10 3 . 2 x l 0 6 l x l O 6 4 x l 0 5 2 . 5 x l 0 5 1 . 4 x l 0 5
72 ? . 2 x l 0 7 7
1.5x10 4 x l 0 b 6
2x10 8 x l 0 3 3 , b x l 0 5 3 . 2 x l O ? l . S x l O 7 6
3.2x10 8 x l 0 5 3 . 2 x l 0 5 1 . 4 x l 0 !
96 8 1.8x10 1 . 3 x l 0 8
7 1.3x10 4.5xlO t > 1 . 6 x l 0 6 5 . l x l O 5 I x l O 8 5 . 1 x l 0 7 6 . 3 x l 0 6 1.4x10° 5 x l 0 5 d
120 2 . 5 x l 0 8 n
7x10" 2 .5x10'' 7
1.3x10 6
2.5x10 7 . 1 x l 0 5 1 . 6 x l 0 9 l x l O 8 1 . 8 x l 0 7 6
2.5x10 8 x l 0 J d
144 3 . 2 x l 0 8 2 . 5 x l 0 8 5 x l o ' 2 x l 0 7 6 . 3 x l 0 6 1.3X10 6 1 . 8 x l o 8 8
1.4x10 7
3.2x10 6
5x10 1.4x10* d
168 8
3.6x10 2 . 5 x l 0 8 8 x l 0 7 4 x l o ' I x l O 7 2 x l 0 6 8
1.8x10 1.6X10 8 3 . 6 x l 0 7 8 x l 0 6 2.5x10* d
Table A£«. U I n f l u e n c e of Co on t h e g r o w t h of Zn-t5.0
time ( h )
Source of ino c u l u m
time ( h ) basal medium s t r o n g l y i o h l b i t o r y l s w l o f m e t a l a t which adapted time ( h )
Co (rog l " S Co (mg l " 1 )
time ( h )
0 0.05 0. 1 0.2 0.3 0.4 0 0.05 0.1 0.2 0.3 0.4
0 1.8xlo' i 1 . 8 x l 0 5 1 . 8 x l 0 5 1 . 8 x l 0 5 1 . 8 x l 0 5 1 . 8 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 l x l O 6 5 x l 0 5 4 x l 0 3 3 . 6 x l 0 5 2 x l 0 5 l . S x l O 5 l x l O 6 5 . 6 x l 0 5 4 x l 0 5 3 . 2 x l 0 5 2 . 5 x l 0 5 2 x l 0 5
48 8 x l 0 6 2.8x10° 1 . 3 x l 0 6 8 x l 0 5 2 . 5 x l 0 5 l . S x l O 5 8 x l 0 6 2.5.10 6 1.3x10° S x l O 5 3 . 6 x l 0 5 2 . 5 x l 0 S
72 3.3xlO ? 1 . 3 x l 0 ? 6.3X10 6 3 . 2 x l 0 6 l x l O 6 2 x l 0 5 2 . 8 x l 0 7 1 . 2 x l 0 7 6 . 3 x l 0 6 2 . 8 x l 0 6 1x10° 5 x l 0 5
96 1 . l x l O 8 7
4x10 1 . 8 x l 0 7 l x l O 7 2 . 5 x l 0 6 4 x l 0 5 l x l O 8 6 . 3 x l 0 ? 2 . 5 x l o 7 l . l x l o 7 2.5x10* 1 . 3 x l 0 6
120 1 . 8 x l 0 8 7
8x10 4 . 0 x l 0 7 2 . 5 x l 0 7 6 . 3 x l o 6 I x l O 6 1.6X10 8 l . i o 8 4 . 5 x l 0 7 3 . 1 l l 0 7 8x10* 2.SxlO 6
144 2 x l 0 8 8
1.3x10 8 x l 0 7 4 x l 0 ? l . l x l O 7 1 . 6 x l 0 6 2 x l 0 8 1 . 6 x l 0 8 8 x l 0 ? 4 . 5 x l O ? l . S x l O 7 4.5x10*
166 8 3.1x10 l . b x l o 8 I x l O 8 9 x l 0 7 1 . 6 x l 0 7 2 . 5 x l 0 6 2 . 8 x l 0 8 l . S x l O 8 U 1 0 8 6.3x10 7 2 . 5 x l 0 7 7 . I x l O 6
252 .
Table Ai.*) I n f l u e n t of Co the growth of ;'. n- t 12 .0
ti m e ( h i
Source of inoculum
t i m e ( h i basal medium s t r o n g l y i n h i b i t o r y l e v e l of mecal a t w h i c h adapted t i m e ( h i
Co (mg 1~S Co (m* 1 ' ')
t i m e ( h i
7
0 0.C5 0. 10 0.20 0.30 0.40 0 0.05 0. 10 0. 20 0.30 0.40
0 2«10 5 2 x l 0 5 2 x l 0 5 2 x . 0 5 2 x l 0 5 2 x l 0 3 2 x l 0 5 2 x l n 5 2* l o 5 2 x l 0 5 2 * l o 5 2 x l 0 5
24 l . O x l O 6 8 x l 0 5 4 . 5 x l o 5 4 x l 0 5 2.SxlO 5 2 x l o 5 1.OxlO 6 5 . 6 x l 0 5 3 . 2 x l 0 5 2 . 5 x l o 5 2 x l o 5 1 .SxlO 5
48 1 . 3 x l 0 7 2 . 5 x l 0 6 1 . 8 x l 0 6 9 x l 0 5 2 . 5 x l 0 5 2 x l 0 5 1 . l x l O 7 1 . 3 x l o 6 l x l o 6 4 . 5 x l 0 5 2 . 8 x l 0 5 l . s x i o 5
72 4 x l 0 ? 1 . 3 x l o ' 6
6.3x10 2 x l 0 6 4 x l 0 5 2 x l 0 5 4 x l 0 7 b
9x10 2.5x10" 6. 3 x l o 5 4 . l x l O 5 2 . 5 x l p 3
96 8 1x10 4 x l 0 ?
7 2.5x10
6 4x10 6 . 3 x l 0 5 2 . 8 x l 0 5
8 1x10 3 . 2 x l o ? 6. I x l O 6 l . S x l o 6 I x l o " 2.8x10 5
120 1 . 8 x l o " 6 . 3 x l 0 ? 4 x l o ' 8 x l 0 6 l x l O 6 3 . 6 x l 0 5 1 . 8 x l 0 B
7 5x10 1 . I x l o ' 2 . 8 x l o < > 1.4xlO b 2 . 8 x l 0 5
144 2 . 5 i c l 0 8 9 x l 0 ? 6 . 3 x l 0 7 7
1.3x10 6
2x10 4 x l 0 3 2 . 6 x l O B 6 . 3 x l o 7 2 x l o ' s x l o " 2.8x10" 0 . J x U l 5
168 8 2.8x10 I x l O 8 8 x l o 7 2 x l 0 ? 2 . 6 x l 0 6 6 . 3 x l 0 5 3 . 2 x l 0 8
8 1x10 4 x l o ' I . 3xi.. ' 4xlC ' 1.4.10°
Table Ai*. b I n f l u e n c e of Co on Che growth o f C d - t 2 . i l
time ( h )
Source of i n o c u l u m
time ( h ) basal medium s t r o n g l y i n h i b i t o r y l e v e l o l m e t a l a t which adapted time ( h )
Co (mg l " 1 ) Co (mg l " 1 )
time ( h )
0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 1.0 1. 5
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 6 1.3x10 9 x l 0 5 5 . 6 x l 0 5 3 . 6 x l 0 5 1 . 8 x l o 5 l x l O 6 I x l O 6 8 x l 0 5 6. 3 x l 0 5 4 x l 0 5 2.SxlO 5 I x l O 3
48 1 . 3 x l 0 7 8 . 8 x l 0 6 6
2.5x10 8 x l 0 5 2 . 5 x l 0 5 l . l x l O 7 l x l O 7 4 . 5 x l 0 6 3 . 2 x l 0 6 9 x l 0 5 2 . 5 x l o 5 d
72 3 . 2 x l 0 7 2.5xlO ? S x l O 6 1 . 3 x l 0 6 3 . 2 x l 0 5 3.2xlO ? 7
4x10 1 . 8 x l 0 7 S x l O 6 1 . 6 x l 0 6 4 x l o 5 d
96 8 x l 0 ? 5 x l 0 ? 2 . 5 x l 0 7 2 . 8 x l 0 6 5 . 6 x l 0 5 9 x l 0 7 7
5.6x10 4 x l 0 7 2 . 5 x l 0 7 3.2xL0 6 7 . I x l o 5 d
120 1 . 3 x l 0 8 7 . l x l O 7 4 x l o 7 6 . 3 x l 0 6 l . 3 x l 0 6 8
1.6x10 8 x l o 7 5 . 6 x l 0 ? 4 x l 0 7 5 x l 0 6 1 . 3 x l o 6 d
144 l . B x l O 8 1 . U 1 0 8 6 . 3 x l o 7 1 . 3 x l 0 ? 1 . 6 x l 0 6 8
1.8x10 8
1x10 8 x l 0 7 5.SxlO 7 1 . 3 x l 0 7 I . S x l O 6 d
168 2 . 8 x l 0 8 1 . 3 x l 0 8 S x l O 7 1 . 8 x l o ' 2 x l 0 6 2 . 5 x l 0 8 1 . 6 x l 0 8 8 x l 0 ? 7
6.3x10 2 . 5 x l o 7 2. b x l O 6 d
,01»' 9 0 , M 9 0 , » o C ^01>"p
9 °""'' 8 0 , " Z 90T « r c 891
8 o , . 8 Z (01»8'J (0T»6 9 0 , . 9 I 9 o i » r c VVI
, O W I 0W\ (0I»<7 g o i * 9 i S0I«8 toi*z e 0 , . f , 9 o i x e - , 0Z1
^om £0I»8 ^OIXB'Z
; 0 I « f 9 9o,»,-, 96
, w t ,01*8 {01*S , o i x S - z tom ^ i * ?
{01«6 ,01»€-T {01«<7 OI«T 9
,OIX» , 0 1 « f 9 O i x c i I
81
s01»4'I , w s0!*8 , 0 1 X 1 9 o i » r i
{ 0 m t01*7 {01»Z 5
S0TX2 ; 0 i x z 0
« 1 0 I'O 50-0 0 Z'O SI-0 I -o so'o 0
(H) 3IUT3
( ^ 1 «»> 111 < 1 *«) IN
(H) 3IUT3 P A ] D » P 6
<]:>?<1A ] v i n s a j o |»ABI Xjo3T9Tq uT *l8no;l}« M N J P A O I Y E S E Q (H) 3IUT3
N M J N S O U I J O A O J N O < ;
(H) 3IUT3
p ,01*9 « ^01*1 j O t x j ' 2 ^01*5 9 oi . i 9 0 I . 9 - 1 80,«8-Z p ;0T*8 t 0 i x 8 ' I ; 0 I X < ) C ( 0 W ? ;0IXZ'8 90I«9-I 891
p ,01*9'C ,01*9-5 ^ O W l ^ o i x e z ^01*C9 9 0 I X , 9 0 , x j p J01«'7 901XS-Z z o i » r i ; 0 1 X S Z ; 0 I X 8 8 0 i x , - l
p 9omz ,01X8 i O T X 9 l ^ O T x z t £ 0 1 X 9 - 5 9 o,«c-i p 50IX-? 9 0 | X 9 - I 9 0 1 X 8 ; 0 1 X 8 - 1 ^0IX9-£ ^OIXC'9 90,X, o z i
p ,0T»C-1 ,01*8-1 ,01*9'C ,0TX» 0 1 * 9 - 1 0 I * r € ^ O i x j p j O W Z ? 0 i x f q 90ix„ toi»i (01X;'Z ( 0 i x s •>, ^01*8 96
p { 0 i x f 9 jOTXg ,01X9-1 , 0 i x 9 1 ,01X9 5 ^ 0 1 * 1 ^ O i x z - t p 501«8-I 9 0 1 X F L , 0 1 X 5 - 2 9 0 1 X B ^ 0 1 X 8 - I ; 0 ] X Z - t It
p 501X9'E S 0 I X 5 S01X9-C S 0 ] X 8 ,01«9-I 9OI«5-Z ,OIXJ-; p { 0 I X [ ' l o i x s - ; s
5 0 i x f 5 5 0 i x f 9 9 o i x B - z 9 o i x s - i 9 0 I X 9 8t>
p jOTXJ-Z 5 0 T x e - j 50IXJ'C 5 0 i x ^ {01X5 s o t * r z , o i x t - i p 01*C'l ;0TXZ ?oi«z-z 5 o i x r 5 S 0 1 X C 9 q 0 i x , 1Z
q0\'Z {01*Z ?0TXJ 501*Z sOIX2 { 0 1 * Z S 0 1 X J S 0 1 X J s o i * z S 0 1 X I 5 o i * t S 0 1 X J 5 0 i x z ; 0 I X Z 5 o m 0
« ' 0 z-o 91 '0 T O 80 "0 W O ZO'O 0 SZ'O Z'O 91'0 i r o 1
80-0 w o zo-o j o l
( 4 ) 3 0 1 1 3
( i a»> m *») IN
( 4 ) 3 0 1 1 3 \*13ni J O < < J 0 3 1 < i m u i AlSuo-UG umipam J B S E Q ( 4 ) 3 0 1 1 3
INNFLNAOUT J O S D J N O S
( 4 ) 3 0 1 1 3
254
Source of i n o c u um
t ime 1 h ) basa l medium s t r o n g l y i n h i b i t o r y l e v e l o
adapted me c a 1 a t w h i c h
Nt mg r ' ) N . mg 1 1 )
0 0.05 0. 1 0.15 0.2 0 0.05 0 1 0.15 0.2
o 2 x l o 3 5
2x10 2 x l o ' 2X10 3 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2x1.) 5
24 1. l x U l ' ' 6 . I x l O 5 4x10 1 . 2 x l 0 5 2 x l 0 5 1x10° 7 . I x l O 5 4 x l l l 5 2.5X1..5 2 x l 0 5
48 Ul . > ' 4* 10° t>
U K ) 1 . l x l ( . 5 2 . 5 x l 0 5 7
1x10 5 . l x l o 6 2,10° 8 x l 0 5 2 . 5 x l o 5
72 ;
5.6x1(1 u i o ' 3.2x1.." 6
L . ox 10 4 x l 0 5 3 . 4 x l O ? 2 . 5 x l o ' . . 6 6.JxlO 2.5x10° 3.2x10*
9 b I x l O 8 J . 2 x l o ' 5.6x10° 6
J.2x10 7 . l x l O 5 l x l o " 5 x l n 7 1 . 4 x l 0 7 5 x l 0 6 4 x l 0 5
12C 8
1.8x10 li. J x l O 7 1 . 4 x l o ' 6.3x10* 6
1x10 1 . 8 x l o " b x l o 7 2 . 5 x l 0 7 l x l O 7 4. 2 x l 0 5
144 8
2.5x10 7
8x10 2 . 5 x l 0 ? I x l o ' 6
1.bxlO 3 . 2 x l 0 8 8
1.6x10 7
4x10 1 . l x l o ' 6 . i x l o 5
168 3 . 2 x l O B H
1x10 7
3.6x10 7
1.8x10 2 . 5 x l 0 6 3 . 2 x l 0 8 1.SxlO 8 6 . 3 x l 0 7 7
2.5x10 1 . I x l O 6
Tab le A V t O I n f l u e n c e of Ni on the g r o w t h of Zn-15.0
ti m e ' h i
Source o f in o c u l u m
t i m e ' h i b a s a l medium s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t which adapted t i m e ' h i
N i (mg l " 1 ) N i (mg l " 1 )
t i m e ' h i
0 0.05 0.1 0.2 0.22 0.25 0 { 0.05 0.1 0.2 0.22 0.25
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2x10 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 1x10° 5 . 6 x l 0 5 4 x l 0 5 2 . 5 x l 0 5 1 . 8 x l 0 5 1 . 8 x l 0 5 l x l O 6 8 x l 0 5 4 x l 0 5 2 . 5 x l 0 5 l . B x l o 5 l . B x l o 5
48 S x l O 6 2.8x10° 7 . l x l O 5 3 . 2 x l 0 5 2 . 5 x l 0 5 1 . 8 x l 0 5 l x i o 7 3 . 6 x l 0 6 8 x l 0 5 3 . 6 x l 0 5 l . S x l O 5 d
72 3 . 6 x l O ? 1 . 4 x l 0 7 2 . 8 x l 0 6 6 . 3 x l 0 5 4 . 5 x l 0 5 2 x l 0 5 4 x l 0 7 1 . 3 x l 0 7 2 x l 0 6 5.BxlO 5 2 . 5 x l 0 5 d
96 l x l O 8 2 . 8 x l 0 7 5 x l 0 6 1 . 3 x l 0 6 8 x l 0 5 2 . 5 x l 0 5 l x l O 8 2 x l 0 7 3 . l x l O 6 S x l O 5 3 . 2 x l 0 5 d
120 1.8K10 8 5 x l 0 7 1 . 3 x l 0 7 2.5x10° 1.3x10° 3 . 6 x l 0 5 1 . 8 x l 0 8 4 . 5 x l 0 ? 7.2x10° 1.6x10° 4 x l 0 5 d
144 2 x l 0 8 8 x l 0 7 l . S x l O 7 3 . 6 x l 0 6 l . S x l O 6 4 . 5 x l 0 5 8
2.6x10 6 . 3 x l 0 7 1 . 8 x l 0 7 2.5x10° 7 . U 1 0 5 d
168 3 . 2 x l 0 8 1 . 3 x l 0 8 3 . 6 x l 0 7 6 . 3 x l 0 6 6
2.5x10 7 . l x l O 5 3 . 1 x l 0 8 l x l O 8 3 . 2 x l 0 7 5.1x10° S x l O 5 d
Table A^ . I ! I n f l u e n c e of Ni. on the gr o w t h o f Z n - t l 2 . 0
Source of I n o c u l u m
t i m e ( h ) basal medium s t r o n g l y i n h i b i t o r y l e v e l w h i c h Bdapted
of metal a t
N 1 (mg l " 1 ) N i (mg l " 1 )
0 0.05 0. 10 0.15 0.2 0 0.05 0. 10 0.15 0.2
0 2 n l 0 5 2 x l 0 5 2 x l 0 5 2X10 3 2 x l 0 5 2 x l o ' 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 l . S x l O 6 6 . 3 x l 0 5 3 . 6 x l 0 5 2 . 5 x l 0 5 l . S x l O 5 1 . 8 x l 0 6 5 . 5 x l 0 5 2 . 5 x l 0 5 l . S x l O 5 l . l x l O 3
48 1.3X10 7 4 . 5 x l 0 6 8 x l 0 5 4 x l 0 5 1 . 8 x l 0 5 I . I x l o ' 4 x l 0 6 5 . 6 x l 0 5 2 . 5 x l O S 1.0xlO S
72 4 x l o ' 8 x l 0 6 2 . 5 x l 0 6 B x l O 5 2 x l 0 5 4 x l 0 ? 7 . U 1 0 6 1 . 3 x l 0 6 3.2xlO S - d
96 8 1x10 2 . 5 x l 0 7 4 . 5 x l 0 6 l . S x l O 6 2 x l 0 5 I x l O 8 1 . 8 x l 0 7 2 x l 0 6 4 x l 0 5 d
120 l . S x l O 8 4 x l o ' 7 1 .3x10 2 . 8 x l 0 6 3 . 6 x l 0 5 1 . 6 x l o " 3 . 6 x l o ' 4 x l 0 6 5 . 6 x l 0 5 d
144 2 . 8 x l 0 8 6.3x10' L.fix 10' 4 . I x l O 6 5 . 6 x l 0 5 2 . 8 x l 0 8 »xlO ? 5. 6x 1 0 6 6
1x10 d
168 3 . 2 x l 0 8 l x l O 8 J - 2 x l 0 7 b . i x l O 6 l x l O 6 2 . 6 x l 0 8 • , , 8 i . I x l o 1 . 3 x l o ' 1 . 6 x l 0 6 d
T a ble AA . ! 2 I n f l u e n c e " f Ni on the grower, uf Cd-c2-0
t i m e ( h )
Source ot inoculum
t i m e ( h ) b a s a l medium s t r o n g l y i n h i b i t o r y l e v e l ot m e t a l at w h i c h adapted t i m e ( h )
Ni (mg -1
Ni (mg 1 )
t i m e ( h )
0 0.05 0. 10 0.15 0.2 0.25 1
0 j 0.05 0.1 0.2 0.25 0. 3
c 2 x l 0 5 2 x l 0 5 2 x l o 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l o ' 2 x l o ' 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 1.3X10 6 9 x l 0 5 b . 3 x l 0 S 4 x l 0 5 2 . 5 x l 0 5 l x l o ' 1 .JxlO 6 «xlO J 5
5.6x10 5 . 6 x l 0 5 2.SxlO 5 1 . 8 x l 0 5
48 l x l O 7 S.bxlO 6 4 x U 6 1 . 3 x l 0 6 2 . 5 x l 0 5 1 . 2 x l o 5 1 . 8 x l 0 ? 6 . 3 x l 0 6 4x10 2x10*' 5 x l 0 5 1 . 8 x l 0 5
72 3.2xlO ? 1 . 6 x l 0 ? 8 . 8 x l 0 6 2.5x10° 5
3.2x10 d 3 . 6 x l o ' 2 . 8 x l 0 7 l . 8 x l O ? l x l o ' 9 x l o 5 2 . 5 x l O S
96 8
1x10 4 x l 0 ? 1 . 6 x l o ' 7 . I x l O 6 7 . l x l O 5 d 8
1x10 i . b x l o ' 4x10 7 l . b x l O 7 1 . 6 x l 0 6 4 x l 0 5
120 1 . 8 x l 0 8 S x l O 7 4 x l o ' 1 . 6 x l 0 ? 1 . 3 x l 0 6 d l . S x l O 8 1 . l x l O 8 S x l O 7 2.SxlO 7 2.5.10 6 5 x l 0 5
144 2 . 5 x l o 8 1 . 6 x l 0 8 8 x l 0 7 3 . 6 x l 0 7 2 . 5 x l 0 6 A 2 . 5 x l 0 8 1 . 4 x l 0 8 I x l O 8 4x10 b 4x10 7. U 1 0 5
16H 3 . 2 x i o 8 l . S x l O 8 I x l O 8 5 x l o ' 3 . 6 x l 0 6 d 1 . 2 x l 0 8 1 . 3 x l 0 8 1 . I x l O 8 S.bxlO 7
6. 3 x l 0 6
I x i O 6
p ,01»v ^ O i x f z ^0I«f9 got*z- c p 9 o i x 9 - e ^01»C'9 9 o,«ri e O , « 9 E 891
p 9 0 T » 9 C £0T»9'T ^ O W E 901«Z'C p ,01X9'f ;0I«9 E »,'"» p ,OT»S-Z
9 oi«e zOT»Z 9 ot«e-i p ^ O i x g ' t 90T«9-1 OZI
p , 0 i x t l 9owi ,01X8 g01«I p 901X£'I 90I»8 01»Z 8 0 , x T 96
p , 0 i x z ,01*9'C ^ O W v p S 0 1 X I V 9 0 1 X Z C ,,01X8 £ 0 i x c z;
p S01»9-C {OT«8 , 0 W 1 £01XT p 50T«S , 0 W I 90I«9-c 0!«ri
p {0TX<7 S0TXC'9 p S01XS'Z s 0 t X 5 ^01X6 ,01X9 1 11
soi*z ?01*Z s a i * z s 0 i x z s 0 i x z s 0 i x z s o i * z ?01«Z s0I«Z 0
Z'O SVO f O so-o 0 Z'O SI'O r o SO'O 0
( t *>) "3 ( 5 _ 1 8mj "3
peldvpp ajnTpam Teseq
amTfiaoai j o e s j n o g
,01*., ^Otxz ^ o i x e - E ^oixrs ^0ix« g 0 1 » V l e o i x z
{ O W S 9 o i « z ^OTXl ^ O i x s z Z 0 1 X T - S ^ O i x r ^ 8 o , x r i
s 0 ™ 8 9 1
, o w z , 0 1 * f 9 0 W 1 z oixzz ^OIXS ^01x9 9 0 1 » £ 1 { 0 1 X 9 £ , 0 1 - E - l ^OIXE'Z z oi»s ^ O l x f - 9 9 o , x r i goixe-i •ni
, ° w i ,01X 8-Z , 0 1 * 9 ! £ 0 i x i , - l .01X8Z I
^Oixs f 0 l x l ' 2 g O l x f ! s o w z
S 0 T X E 9 ,ot«v t 0 i x r l 01«» ^01X9-5 g 0 i x , 9 0 , x E - l OZI
9 0 i x 8 - z 01x9-{ 9
^ O W l Oixs t I
^01*5 f W l s 0 i x z S 0 1 X 9 E ,oi * z ^0I»1 £ 0 i x z
£01»9-E ^OIXE'9 ,OT«T 96
JOIXS-Z ,01X1 , 0 ' X V I ,01XZ-Z , 0 i x ? ^01X9-1 ^ O W l £01X 9-T j O l x z - E J O I X E - 9 ,01x9-1 ,01 «V ,01«E-9 £ 0 I X 9 - T ^oixe-z li
EOTX8'l {0TXZ-Z J O I X J Z s 0 1 x E v J O I X S ' V S01X8 ,oi*<, 9 0 1 « 9 - S s 0 i x f l 5oi«s-z s0ixc - z S01»V S01X9 'S , 0 1 X E - I ,01X S-Z ,0!XE-9 8V
jOlxg'T g 0 T X f Z 5 0 i x f z j O I x s ' Z J O I X J E s 0 i x i ^ ,01»1 s 0 i x z 5 0 i x j s o i x z s 0 i x z s 0 i x z - c s 0 i x v s 0 1 x t - 9 ,01»1 tl
soixz { 0 I » Z { 0 i x z {01XZ { 0 i x z s o i » z goixz 5 0 i x z soixz S01XZ 5 0 i x z s 0 i x z
soi«z s 0 i x z s 0 i x z s 0 i x z 0
91'0 i r o T O 80-0 W O ZO'O lO'O 0 9 1 0 zro r o 80-0 w o zo-o IO-O 0
8m) no ( j 80) no
CM) acrj.3 TOAST A.i03TqTqui ATSUDJ30 amjpom fDooq CM) acrj.3
o d A q - p i ^ f t j o q i n o j S »q3 uo no j o a o u a n j j u i CI *<7V ' l ^ i
257
Table A 4.15 I n f l u e n c e o f Cu on t h e g r o w t h o f N l - t l . O
t i m e (h>
Source of i n o c u l u m
t i m e (h> b a s a l medium s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t wh i c h
adopted t i m e (h>
Cu (mg 1 S Cu (mg I " 1 )
t i m e (h>
0 0.05 0. 10 0. 15 0.20 0 0.05 0.10 0.15 0.20
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 " 2 x l 0 5 2 x l 0 5
24 I . U I O 6 ' 9 x l 0 5 S x l O 5 2 . 2 x l 0 5 2 . 2 x l 0 5 1.4x10° 6 . J x l O 5 3 . 6 x l 0 5 2.SxlO 5 d
48 u i o 7 3.6x10° I x l O 6 2 . 5 x l 0 5 l . S x l o 5 1. u i o 7 2 . 8 x l 0 6 4 . 5 x l 0 5 1 . 8 x l 0 5 d
72 4 x l 0 ? 1 . 3 x l 0 7 2 . 5 x l 0 6 4 x l 0 5 1 . 8 x l 0 5 3 . 6 x l 0 ? 6.3x10* 8 x l 0 5 l . 3 x l 0 5 d
96 I x l O 8 2 . 5 x l 0 ? 5.6x10* 3 . 2 x l 0 5 1 . 6 x l 0 5 S x l O 7 l x l o 7 l . O x l O 6 1 . 3 x l 0 5 d
120 1 . 8 x l o 8 5 x l o ? 1 . l x l O 7 8 x l 0 5 3 . 2 x l 0 5 1 . 3 x l 0 8 l . 4 x l 0 ? 1 . 6 x l 0 6 1 . 8 x l 0 5 d
144 2 x l 0 8 8 x l 0 ? 2 . 5 x l 0 7 1 . 6 x l 0 6 4 x l 0 5 2 . 8 x l 0 8 3 . 6 x l 0 ? 2 . 5 x l 0 6 2 . 5 x l 0 5 d
168 3 . 6 x l o 8 l . l x l O 8 3 . 6 x l 0 7 3 . 2 x l 0 6 6 . 3 x l 0 5 3 . 2 x l 0 8 6 . 3 x l 0 ? 4 x l 0 6 2 . 5 x l 0 5 d
T a ble A4.16 I n f l u e n c e o f Cu on t h e gr o w t h o f Zn-t5.0
t i m e ( h )
Source o f i n o c u l u m
t i m e ( h ) b a s a l medium s t r o n g l y I n h i b i t o r y l e v e l o f m e t a l a t wh i c h adapted t i m e ( h )
Cu (mg I * 1 ) Cu (mg l " 1 )
t i m e ( h )
0 0.05 0.1 0.2 0.25 0.3 0 0.05 0. 1 0.2 0.25 • 0.3
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 6 1.3x10 7 x l o 5 5 . 6 x l 0 5 2 . 5 x l 0 5 1 . 7 x l 0 5 1 . 7 x l 0 5 l x l O 6 6 . 5 x l 0 5 2 . 5 x l 0 5 2 x l 0 5 2 x l 0 5 1.5X10 5
48 l . O x l O 7 6
7.9x10 6
2x10 3 . 2 x l 0 5 1 . 7 x l 0 5 l . O x l O 5 S x l O 6 3 x l 0 6 4 . 5 x l 0 5 5
3.1x10 2 x l 0 5 d
72 4x10' 2 x l 0 7 6 x l 0 6 4 . 6 x l 0 5 2.SxlO 5 d 2 . 8 x l O ? 1 . 6 x l o ' 1.3x10° S x l O 5 2 . 2 x l 0 5 d
96 2 x l 0 8 l x l O 8 2 x l 0 ? l x l O 6 5 . 6 x l 0 5 d U I O 8 4 x l 0 ? 4 . S x l O 6 6
1x10 2.8xL0 5 d
120 2 . 5 x l 0 8 , 2 x l 0 B 7 x l 0 7 2 . 8 x l 0 6 1 . 6 x l 0 b d 1 .6xlO B 8 x l 0 ? l x l O 7 1.7.10 6 6 . 3 x l 0 5 d
144 3 . 2 x l 0 8 2 . 5 x l 0 8 8
1.2x10 6
8x10 3 x l 0 6 d 2 x l 0 8 l x l O 8 2 . 5 x l 0 7 4x10* 8x10 5 d
168 8 3.2x10 3 . 2 x l 0 8 1 . 6 x l 0 8 2 x l 0 ? 5.6x10° d 2.SxlO 8 1 . 6 x l 0 8 4.SxlO 7 7. U I O 6 1 . U 1 0 6 d
258 .
Tab I e \l* . 1 7 I n f l u e n c e u t Cu en i he $ rowt li of Zn - t I 2 0
t i m e (hi
Source of I n o c u l u m
t i m e (hi ba s a l medium s t r o n g l y i n h i b i t o r y l e v e l of metal at which
adapted t i m e (hi
Cu < mg l " ' ) Cu Img 1 ^ 1
t i m e (hi
0 0.05 0. 10 0.20 0.25 0 0.05 0. 1Q 0. 20 0.25
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x k > 5 2 x l 0 5 2 x l 0 5 2 x l 0 S 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 b 1.0x10 8 x l 0 5 4 x l 0 5 2 . 5 x l o 5 1.8x10* 8 x l 0 5 5 x l 0 S 2 . 5 x l o 5 l x l l > S l x l O 5
48 l x l O 7 5.6X10 6 1 . 3 x l 0 b 2 . 8 x l 0 S 2 x l 0 5 I x l O 7 3 . 2 x l 0 6 b . 3 x l O S I x l O 5 d
7 2 2 . 5 x l 0 7 9 x l 0 6 3 . 2 x l 0 6 4 x l 0 5 2 . 5 x l 0 5 2 x l o ' 8 x 1 0 6 1 . J x l o " 1 . 3 x l ( l 5 d
96 l x l O 8 2 x l 0 ? Hxlo'' H s U ) 5 4 x l o ' S x l ( / 1 . 4 x l < l 7 J . 6 X I 0 6 2 . S x l d 5 d
120 1 , 6 x l 0 8 3.6xlO ? 1 . 3 x l o ' 2 . 8 x l 0 6 B x l O 5 i . i o 8 J . b x l o ' S x l l J 6 4 x l ( | 5 d
144 2 . t x l 0 8 6 . 3 x l 0 ? 2 . 5 x l 0 7 B x l O 6 1 . 4 x l 0 6 1.SxlO 8 6.3xlO ? 1 . 4 x l o ' S x l O 5 d
168 1 1
2 . 8 x l 0 8 9 x l 0 7 4 . 5 x l 0 ? 1.4x10' SxlO 6 2 x l 0 8 8 x l o ' 2 x i o ' I . 3 x l 0 6 d
Table A4 . 18 I n f l u e n c e of Cu on t h e g r o w t h of Cd-t20
Source o f i n o c u l u m
time ( h ) b a s a l medium s t r o n g l y i n h i b i t o r y l e v e l o m e t a l a t wh i c h adapted
Cu (mg l " 1 ) Cu (mg I ^!
0 0.05 0.1 0. 15 0.2 0.25 0.05 1
0. 1 0.15 0.2 0.25 0. 1
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l O S 2 x I 0 5 2 x l O S
24 9 x l 0 5 5 x l C 5 3 . 2 x l 0 5 2 . 5 x l 0 5 l . S x l O 5 1 . 2 x l 0 5 9 x l 0 5 5 . 6 x l 0 5 4 x l 0 5 2 . 8 x l 0 5 2 x l 0 5 2 x l 0 5
48 l x l O 7 6 . 3 x l 0 6 1 . 3 x l 0 6 4 x l 0 5 1 . 3 x l 0 5 d 7
1x10 4 x l 0 6 2 x l 0 6 9 x l 0 5 4. 5 x l 0 5 2 . 5 x l 0 5
72 3 . 2 x l 0 7 2 x l 0 7 4 x l 0 6 S x l O 5 2.SxlO 5 d 3 . 2 x l 0 7 7
2x10 6.3X10 6 2 . 5 x l 0 6 1 . 3 x l 0 6 l . B x l O 5
96 8 . 8 x l 0 7 6 . 3 x l O ? 9 x l 0 6 2 . 2 x l 0 6 4 x l 0 5 d I x l O 8 S x l O 7 2 x l 0 7 5 . l x l O 6 6
3.2x10 3 . 2 x l 0 5
120 1 . 8 x l 0 8 1 . 3 x l 0 8 2 . 8 x l 0 ? 4 x l 0 6 8 x l O S d 1 . 8 x l 0 8 9 x l 0 7 4 x l 0 7 9 x l 0 6 4 . 5 x l 0 6 3 . 6 x l 0 5
144 3 . 2 x l 0 8 2»10 8 4 x l 0 7 S x l O 6 6
1 . I x l O d 2 . 8 x l 0 8 8
1.6x10 6 . 3 x l 0 7 1 6 x l 0 7 8.SxlO 6 b . 3 x l o 5
168 3 . 2 x l 0 8 2 . 5 x l 0 8 9x10 7 2 x l o 7 1 . 4 x l 0 6 d 3 . 2 x l 0 8 2 x l 0 8 l x l O 8 2 . 8 x l 0 7 I . 3 x l 0 ? b
1.3x10
259.
Table I n f l u e n c e of Zn on the gr o w t h of w i l d - t y p e
t i m e (h)
Source o f i n o c u l u m
t i m e (h) b a s a l medium s t r o n g l y i n h i b i t o r y l e v e l of me t a l
t i m e (h)
Zn 'mg 1 S Zn 'mg 1 ^)
t i m e (h)
0.04 0.5 1.0 1.25 1.5 1.75 0.04 0.5 1.0 1.25 1.5 1.75
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l o 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 B x l O 6 7 . 5 x l 0 6 4 x l 0 6 5 x l 0 5 1 . 5 x l 0 5 1.5X10 5 1.6xlO b 9 x l 0 5 6 . 3 x l 0 5 5 . I x l O 5 3 . 2 x l 0 5 l . B x l O 5
48 8 x l 0 ? 6 x l o ? 4 x l 0 ? 7 . 1 x l 0 6 3 . 2 x l 0 5 d I x l O 7 6 . 3 x l 0 6 3 . 2 x l 0 6 l . S x l O 6 b x l O 5 2 . 2 x l 0 5
72 4 x l 0 8 1 . 2 x l 0 8 U l f . 8 1 . 8 x l 0 7 7. I x l O 5 d 5 . I x l O 7 2 . 2 x l 0 7 1 . 6 x l 0 7 8 x l 0 6 l . B x l O 6 5 . 1 x l 0 5
96 5x10° 2 x l 0 8 1 . I x l O 8 2 . 8 x l 0 7 2 . 2 x l 0 6 d 2 x l 0 8 B x l O 7 5 . 6 x l o 7 3 . 2 x l o 7 4 x l 0 6 B x l O 5
120 5 . 6 x l 0 8 2 . 5 x l 0 8 1 . 3 x l 0 8 4 . 5 x l 0 7 5.6x10* d 4,10 8 S
1.Sxlr 1 . 3 x l 0 8 7
8x10 8 x l 0 6 1 . 6 x l 0 6
144 6.3x10® 2 . 8 x l 0 8 1 . 6 x l 0 8 7
6.3x10 l x l O 7 d 5.1x10* 2.5x10* 8
1.6x10' l x l O 8 1 . 6 x l 0 7 6
2 . I x l o
168 7 . 9 x l 0 8 8
3.2x10 l . S x l O 8 S x l O 7 1 . 8 x l 0 7 d 7. l x l O 8 3 . 2 x l 0 8 l . S x i o 8 l . l x l O 8 2 . 5 x l 0 7 4«10 6
Table A i . 2 0 I n f l u e n c e of Zn on the gr o w t h of C o - t l f
time ( h )
Source of in o c u l u m
time ( h ) ba s a l medium atr.my.ly i n h i b i t o r y l e v e l uf m e t a l a t w h i c h adapted time ( h )
Zn (mg l ' 1 ) Zn 'mg 1 ^)
time ( h )
0.04 0.5 1.0 1 . 5 2.0 0.04 0. 5 1.0 1 . 25 1.5
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 1 . I x l O 6 l . l x l O 6 5 x l 0 5 2 . 5 x l 0 5 l x l O 5 l . O x l O 6 l x l O 6 4 x l 0 5 2 . 5 x l 0 5 I x l O 5
48 1 . 3 x l 0 7 5 x l 0 6 l x l O 6 3 . 2 x l 0 5 4
8x10 l . l x l O 7 4.5X10 6 8 x l 0 5 2 . 5 x l o 5 I x l O 5
72 4 . 5 x l O ? 2 x l 0 7 3 . 6 x l 0 6 5 x l o 5 d 3 . 6 x l 0 7 1 . 6 x l 0 ? l . l x l O 6 3 . 2 x l o 5 d
96 1 . l x l O 8 5. l x l O 7 7. I x l O 6 8 x l 0 5 d I x l O 8 3 . 6 x l 0 7 2 . 5 x l 0 6 4 x l o 5 d
120 1 . 8 x l 0 8 8 x l 0 7 1 . 8 x l 0 7 t . 6 x l 0 6 d l . S x l O 8 8 x l O ? 3 . 6 x l o 6 a x l o 5 d
144 3 . 2 x l 0 8 1 . 3 x l 0 8 7
3.6x10 6
2.5x10 d 3 . 2 x l o " l . b x l O 8 6. 3 x l O b I x l O 6 d
168 3 . 4 x l 0 8 l . b x l O 8 4 x l 0 7 3 . 2 x l 0 6 d 3 . 6 x l 0 8 2 . 8 x l o " I x l o 7 ' . i x l n 6 d
260.
Table A4.21 I n f l u e n c e of Zn on the growth of N i - t l . O
Sour ce of i n o c u l u m
Iime 1h) basal mediui s t r o n g l y i n h i b i t o r y l e v e l o f met a l
a t which adapted
V. n (mK 1 ) Zn mg l " 1 )
0 . 04 1
o. 5 1.0 1.5 0.04 0.5 1.0 1.5
0 2 x l l > 3 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 1 . 2 x l 0 6 8 x l 0 3 4 x l 0 5 2 . 5 x l 0 3 1 . 3 x l 0 6 5 . 6 x l 0 5 4 x l 0 5 2 . 5 x l 0 5
48 l x l O 7 Sxlo" 6
1.3x10 4 x l 0 5 1 . l x l O 7 3 . 2 x l 0 6 9 x l 0 5 2 x l 0 5
72 4.bxlO 7 2 x l t ) ? 2 . 5 x l 0 6 b . 3 x l 0 5 4 . b x l 0 ? 6
9x10 2 x l 0 6 l . B x l O 5
9b u i o 8 5 x l 0 ? 5 . b x l o b 8 x l 0 5 , , 8 l x l o 1.bxlO 7 3 . 6 x l 0 6 2 . 5 x l 0 5
120 I . a x l n " V x l o ' 1 . I x l O 7 1 . 3 x l 0 6 1 . 8 x l 0 8 2 . 8 x l 0 7 7 . l x l O 6 4 x l 0 3
144 2 . 5 x l 0 8 1 . 3 x l 0 8 7
2x10 1.8xlO b 2 . 5 x l 0 8 4 x l 0 ? 2 . 5 x l 0 ? 8 x l 0 5
168 8 3.2x10 2 x l 0 8 3 . 6 x l 0 ? 2 . 5 x l 0 6 3 . 6 x l 0 8 8 x l 0 ? 4 . 5 x l 0 7 1 . l x l O 6
Table A4.22 I n f l u e n c e of Zn on che growth of Cu-t0.3
time (h)
Source o f inoculum
time (h) basal medium s t r o n g l y i n h i b i t o r y l e v e l of m e t a l t o which
adapted time (h)
Zn fmg I ^) Zn fmg 1
time (h)
0.04 0.5 1.0 1.5 2.0 0.04 0. 5 1.0 1.2 1.5
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 3 2 x l 0 5 2 x l 0 3 2 x l 0 5 2 x l 0 5
24 l x l O 6 7 . I x l O 3 4 . 5 x l 0 5 2 . 5 x l 0 5 1 . 6 x l 0 3 l x l O 6 5 . 6 x l 0 5 2 . 5 x l 0 5 1 . 8 x l 0 5 l . 6 x l 0 5
48 6 . 3 x l 0 6 5 . l x l O 5 l x l O 6 3 . 6 x l 0 5 1 . 8 x l 0 5 6 . 3 x l 0 6 3 . 2 x l 0 6 5 . l x l O 3 2 . 5 x l 0 3 1 . 8 x l 0 5
72 3.2xlO ? 2 x l 0 ? 4 . 5 x l 0 6 6 . 3 x l 0 5 2 . 5 x l 0 3 3 . 2 x l 0 7 1 . 3 x l 0 ? l x l O 6 3 . 6 x l 0 5 2 x l 0 5
96 1x10 5.6xlO ? 1 . 3 x l 0 7 l x l O 6 3 . 6 x l 0 5 l x l O 8 3 . 6 x l 0 7 2 x l 0 6 5 . 6 x l 0 5 2 . 5 x l 0 5
120 8 1.3x10
7 8x10 8 x l 0 7 2 x l 0 6 6 . 3 x l 0 5 1 . 6 x l 0 8 6 . 3 x l 0 ? 3 . 6 x l 0 6 7 . I x l O 5 3 . 2 x l 0 5
144 1 . 6 x l 0 8 l x l O 8 l x l O 8 3 . 6 x l 0 6 8 x l 0 5 1 . 8 x l 0 8 7 . 1 x l 0 ? 6 . 3 x l 0 6 l x l O 6 3 . 6 x l 0 5
168 1 . 8 x l 0 8 ; 1 . 3 x l 0 8 1 . t x l O 8 6 . 3 x l 0 6 1 . 6 x l 0 6 1.8x10* l x l O 8 I x l O 7 1 . 6 x l 0 6 5 . 1 x l 0 5
261 .
Table A V 23 I n f l u e n c e of Zn on the g r o w t h of Cd-t2.0
time (h)
Source of i n o c u l u m
time (h) b a s a l medium s t r o n g l y I n h i b i t o r y l e v e l of m e t a l a t which adapted time (h)
Zn (mg 1 S Zn (mg I ' )
time (h)
0.04 0.5 1.0 1.5 2.0 0.04 1.0 1.5 2.0 2.5 3.0 J. 5
0 2 x l 0 5 2 x l 0 5 2 x t 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 6 1x10 6 . 3 x l 0 5 3 . 6 x l 0 5 2 . 5 x l 0 5 l . 8 x l 0 5 8 x l 0 5 5 . 6 x l 0 5 4 x l 0 5 4 x l 0 5 2 . 8 x l 0 5 2 . 5 x l 0 5 l . B x l O 5
48 1 . 3 x l 0 ? 5 x l 0 6 5 x l 0 5 2 . 5 x l 0 5 1 . 8 x l 0 5 l x l O 7 6 . 3 x l 0 6 3 . 2 x l 0 6 2 . 5 x l O b 8 x l 0 5 4 x l 0 5 3 . 2 x l 0 5
72 3 . 2 x l 0 ? 2 x l 0 ? l x l O 6 4 . 5 x l 0 5 1 . 8 x l 0 5 2 . 8 x l 0 7 2x10 7 8 x l 0 6 6
4x10 1 . 4 x l 0 6 8 x l 0 5 3 . 2 x l 0 5
96 8 x 1 0 1 5 x l 0 7 3 . 2 x l 0 6 6 . 3 x l o 3 d 8 x l 0 7 4 . 5 x l 0 7 2 . 5 x l 0 7 1 . 3 x l 0 7 3.6X10 6 1 . 8 x l 0 6 4 . 5 x l 0 5
120 1 . 3 x l 0 8 8 x l 0 7 5 . 6 x l 0 6 1 . 3 x l 0 6 d 1 . 4 x l 0 8 l x l O 8 4 . 5 x l 0 7 2 . 8 x l 0 7 l x l O 7 4,10" 8 x l 0 5
144 1 . 6 x l 0 8 1 . 3 x l 0 8 l x l O 7 2 . 5 x l 0 6 d 2 x l 0 8 1 . 4 x l 0 8 8 x 1 0 7 5.6xlO ? 1 . 8 x l 0 7 8 . 8 x l 0 6 1 . 3 x l 0 6
168 2 . 8 x l 0 8 1 . 5 x l 0 8 1 , 8 x l 0 7 4,10 6 d 2 . 8 x l 0 8 1 . 4 x l 0 8 I x l O 8 7 . I x l O 7 2 . 6 x l 0 7 1 . 3 x l 0 7 1 . 8 x l 0 6
T a b l e Ai.24 I n f l u e n c e of Cd on the g r o w t h of w i l d - t y p e
time 'h)
Source o f Inoculum
time 'h) 1
b a s a l medium j s t r o n g l y i n h i b i t o r y l e v e l of metal time 'h)
Cd fog l " 1 ) cd (mg r l (
time 'h)
0 0.15 0.3 0.35 r
0.4 0.5 0.6 0 0. 15 0.3 0.35 1 0.4 1
0. 5 0.6
0 2 x l 0 5 2 x l 0 5 2x10 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l O S 2 x l 0 5 2 x l 0 5 2 x l o 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 2x10* 1.6x10* 6.3xl O S 2 x l 0 5 1 . 8 x l 0 5 1.6X10 5 1 . 6 x l 0 5 l . S x l O 6 6
1.3x10 8 x t 0 5 d 3 . 2 x l 0 5 2 . 5 x l 0 5 2 . 5 x l O S
48 3.6xlO ? 2 . 1 x l 0 7 1.10 6 2 . 5 x l 0 5 2. l x l O 5 2 x l 0 5 1 . 8 x l 0 5 l . S x l O 7 1 . 3 x l 0 7 1.8xlO b d 8 x l 0 5 5 . I x l O 5 3 . 2 x l O S
72 1 . 6 x l 0 8 1 . 4 x l 0 8 2 . 8 x l 0 6 5 . 6 x l 0 5 4 x l 0 5 2 . 6 x l 0 5 2 . 5 x l 0 S 7 . 1 x l 0 7 4 . 5 x l 0 7 5 . l x l o 6 d 1 8 x l 0 6 l x l O 6 5 . 6 x l O S
96 2 . 8 x l 0 8 2 . 2 x l 0 8 1 . 4 x l 0 7 4. 5 x l 0 6 3. 2 x l 0 6 l . S x l O 6 1.10 6 1 . 6 x l 0 8 l x l O 8 1 . 4 x l 0 7 d 3 . 6 x l o 6 2 x l 0 6 9 x l 0 3
120 3 . 6 x l 0 8 2 . 5 x l 0 8 2 . 5 x l 0 7 l . l x l O 7 6. 3 x l 0 6 4«10 6 2 x l 0 6 2 . 2 x l 0 8 1 . 6 x l 0 8 3 . 6 x l 0 7 d 5 . 6 x l 0 6 3 . 2 x l 0 6 1 . 6 x l 0 6
144 4 x l 0 8 2 . 8 x l 0 8 4 . 5 x l 0 7 1 . 6 x l 0 7 8 x l 0 6 5 . 6 x l 0 6 3 . 2 x l 0 6 2 . 8 x l 0 8 2 x l 0 8 7 . l x l O 7 d 1. I x l O 7 6 . 3 x l 0 6 2.5.10 6
168 5 . I x l O 8 2 . 8 x l 0 8 7 . l x l O 7 2 . 8 x l 0 7 1 , 4 x l 0 7 8.10 6 4 x l 0 6 4 . 2 x l 0 8 2 . 5 x l 0 8 8. 2 x l 0 7 d 2 . 2 x l 0 7 1 . 3 x l 0 7 6
5.6x10
262.
Table A4.25 I n f l u e n c e of Cd on t h e g r o w t h o f C o - t l . 8
t i m e ( h )
Source o f i n o c u l u m
t i m e ( h ) b a a a l medium s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l a t w h i c h adapted t i m e ( h )
Cd (mg l " 1 ) Cd (mg 1"')
t i m e ( h )
0 0.05 0.10 0.2 0.3 0.4 0 0.05 0.1 0.15 0.2 0.3
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x L 0 5 2 x l 0 5 2 x l 0 5
24 l . l x l O 6 7 . 1 x l 0 5 4 . 5 x l 0 5 3 . 2 x l 0 5 2 . 5 x l 0 5 2 . 5 x l 0 5 8 x l 0 5 4 x l 0 5 2 . 5 x l 0 5 2 . 2 x l 0 5 2 . 2 x l 0 5 d
48 6 8x10 5x10* 1 . 8 x l 0 6 l x l o 6 5 . U 1 0 5 2 . 5 x l 0 5 8 x l 0 6 2 x l 0 6 6 . 3 x l 0 5 2 . 5 x l 0 5 2 . 5 x l 0 5 d
72 4 . 2 x t O ? 1 . 3 x l 0 7 6 . 3 x l 0 6 6
2.5x10 8 x l 0 5 2 . 5 x l 0 5 2 . 8 x l O ? 7 . U 1 0 6 1 . 8 x l 0 6 6 . 3 x l 0 5 2 . 5 x l 0 5 d
96 l x l O 8 2 . 5 x l 0 ? 1 . 3 x l 0 7 6
5.6x10 2 x l 0 6 4 x l 0 5 1x10° 2 x l 0 ? 3 . 6 x l 0 6 1.8x10* 4 x l 0 5 d
120 8
1.6x10 6 . 3 x l 0 ? 2 . 5 x l 0 7 1 . 3 x l 0 7 4x10* 6 . 3 x l 0 5 8
1.4x10 3 . 6 x 1 0 7 6 . 3 x l 0 6 2.5x10* 8x10 5 d
144 2 x l 0 8 I x l O 8 4 . 5 x l 0 7 1.8xlO ? 6 . 3 x l 0 6 1x10* 1 . 8 x l 0 8 5 . I x l O 7 1 . 3 x l 0 ? S . l x l O 6 6
1.4x10 d
168 2.1x10* 1 . 3 x l 0 8 6 , 3 x l O ? 2 . 8 x l 0 ? 1 . 4 x l 0 ? 1 . 4 x l 0 6 8
2x10 7
6.3x10 2 x l 0 7 7.1x10* 3.2x10* d
Table I n f l u e n c e o f Cd on the gr o w t h o f N i - t l . O
t i m e ( h )
Source of I n o c u l u m
t i m e ( h ) b a g a l medium s t r o n g l y i n h i b i t o r y l e v e l of m e t a l a t w h i c h adapted t i m e ( h )
Cd (mg l " 1 ) Cd (mg l " 1 )
t i m e ( h )
0 0.05 0. 10 0. 15 0.20 0.25 0 0.05 0.10 0.15 0.20 0.25
0 2 x l 0 5 2 x l o 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2»10 5 2 x l 0 5
24 1x10* 5 . l x l O 5 3 . 2 x l 0 5 3 . 2 x l 0 5 3 . 2 x l 0 5 d 8 x l 0 5 6 . 3 x l 0 5 3.2x10* 2 . 5 x l 0 5 2 x l 0 5 d
48 6.3x10* 2 . 8 x l 0 6 l x l O 6 4 x l 0 5 3 . 6 x l 0 5 d 6.3x10* 1.6x10* 8 x l 0 5 6 . 3 x l 0 5 2 . 8 x l 0 5 d
72 3 . 6 x l O ? l x l o 7 6
3.5x10 8 x l 0 5 4 x l 0 5 d 3.2x10 7 6
6.3x10 2.5x10* 8 x l 0 S 4 . 5 x l 0 5 d
96 8 x l 0 7 3 . 2 x l 0 7 l x l O 7 1.8x10* 6 . 3 x l 0 5 d l x l O 8 1 . 6 x l 0 7 6.3x10* 1.3x10* 8 x l 0 5 d
120 1 . 3 x l 0 8 6 . 3 x l 0 7 2x10 7 2.8x10* l x l O 6 i 1 . 4 x l 0 8 4 x l 0 7 1 . 4 x l 0 7 2.5x10* 1.6x10* d
144 1 . 6 x l 0 8 S x l o ' 3.2xlO ? 5 . 6 x l 0 6 1.8x10* i 2 x l 0 8 5 . 6 x l 0 7 2 . 5 x l 0 7 5.6x10* 2.5x10* d
168 2 x l o " l x l o 8 4 x l 0 ? l x l o 7 6
3.2x10 d 2 . 5 x l 0 8 7 . l x l O 7 3 . 6 x l 0 7 1 . 4 x l o 7 3.2x10* d
26 ? .
T a b l e A4.27 I n f l u e n c e o f Cd on t h e g r o w t h o f C u - t 0 . 5
t i m e ( h )
S o u r c e o f i n o c u l u m
t i m e ( h ) b a s a l medium s t r o n g l y i n h i b i t o r y l e v e l o f m e t a l t o w h i c h
a d a p t e d t i m e ( h )
Cd (rag l " 1 ) Cd (mg l " 1 )
t i m e ( h )
0 0.05 0. 10 0.20 0.40 0. 60 0 0.05 0. 10 0. 15 0.20
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 l x l O 6 6 . 3 x l 0 5 5 . I x l O 5 3 . 6 x l 0 5 2 . 5 x l 0 5 2 . 5 x l 0 5 I x l O 6 4 x l 0 5 2 . 5 x l 0 5 2 x l 0 5 1 . 6 x l 0 5
48 6 . 3 x l 0 6 J . 6 x l 0 6 2 x l 0 6 l x l O 6 5 . 6 x l 0 5 2 . 8 x l 0 5 6 . 3 x l 0 6 2 x l 0 6 7. l x l O 5 3 . 6 x l 0 5 2 . 0 x l 0 5
96 l x l O 8 2 x l 0 ? 8 x 1 0 * 5 . l x l O h 6
1.6x10 6 . 3 x l 0 5 I x l O 8 2 x l 0 ? 4 x l 0 6 I x l O 6 3 . 2 x l 0 5
120 1 . 3 x l 0 8 4 x l 0 ? 1 . 3 x l 0 ? l x l O 7 4 . 5 x l 0 6 l x l O 6 l . 6 x l 0 8 4 x l 0 ? 6 . 3 x l 0 6 2 . 5 x 1 0 * 4 x l 0 5
144 8
1.6x10 8 x l 0 7 2 . 5 x l 0 7 1 , 6 x l 0 7 6
8x10 1 . 6 x l 0 6 1 . 8 x l 0 8 6 . 3 x l 0 ? 8 x l 0 6 3 . 6 x l 0 6 7 . 1 x l 0 5
168 1 . 8 x l 0 8 l x l O 8 3 . 6 x l O ? 2 x l O ? 4 . 3 x l 0 6 2 x l 0 6 1 , 8 x l 0 8 7 . U 1 0 7 2 x l 0 ? 6 . 3 x l 0 6 l x l O 6
T a b l e A4.28 I n f l u e n c e o f Cd on t h e g r o w t h o f Z n - t 5 . 0
time ( h )
Source of inoculum
time ( h ) b a s a l medium s t r o n g l y I n h i b i t o r y l e v e l of metel a t v h i c h adapted time ( h )
Cd (mg t " 1 ) Cd (mg l " 1 )
time ( h )
0 0.2 0.3 0.4 0.5 0 0.2 0.3 0.4 0.5 0.6 0.8 1.0
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2x10 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2x10 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 6 1x10 4 x l 0 5 l . S x l O 5 1.8xl0 5 1 . 8 s l 0 5 9 x l 0 5 7.U10 5 4x10 5 3 . 2 x l 0 5 2 . 8 x l 0 5 2 . 5 x l 0 5 2»10 5 1.8xl0 5
48 8x10 6 1.3xl0 6 2 . 5 x l 0 5 1.8xl0 5 1.8xl0 5 5 . 1 x l 0 6 2.8x10* 1x10* 5 x l 0 5 4x10 5 4 x l 0 5 3 . 2 x l 0 5 1.8xl0 5
72 4 . 5 x l 0 7 3 . 6 x l 0 6 3 . 2 x l 0 5 1.8xl0 5 1.8xl0 5 3 . 6 x l 0 7 1.6sl0 7 4 x l 0 6 1.3xl0 6 8 x l 0 5 5 . 6 x l 0 5 4x10 5 2 x l 0 5
96 l x l O 8 6
8x10 8x10 5 2 . 5 x l 0 5 1.8xl0 5 l x l O 8 5 . 6 x l 0 ? 1 . 6 x l 0 ? 3.6x10* 2 . 5 x l 0 6 1x10* 5 x l 0 5 3 . 6 x l 0 5
120 8 1.6x10 1.8xl0 7 1.3xl0 6 4 x l 0 5 2 . 4 x l 0 5 1 . 8 x l 0 8 l x l O 8 4 x l 0 7 8x10* 5.6x10* 3.2x10* 1.3x10* 5 x l 0 5
144 2 x l 0 8 4 x l 0 ? 1.6xl0 6 6.3xl0 5 2 . 8 x l 0 5 2 . 0 x l 0 8 1.6xl0 8 8x10 7 2 . 5 x l 0 7 1.4xl0 ? 5.6x10* 2.8x10* 8 x l 0 5
168 3.2xlO R 6 . 3 x l 0 7 3 . 6 x l 0 6 l . l x l O 6 3.6«105 3 . 2 x l 0 8 8
1.6x10 I x l O 8 3 . 6 x l 0 7 2 . 5 x l 0 7 l . S x l O 7 5.6x10* 1.4x10*
264 .
Table A4.29 I n f l u e n c e of Cd on the growth of Z n - t l 2 . 0
time ( h )
Source of inoculum
time ( h ) basal medium s t r o n g l y i n h i b i t o r y l e v e l o f metal a t which adapted time ( h )
Cd (mg l " 1 ) Cd (mg l " 1 )
t ime ( h )
0 0.2 0.3 0.4 0.5 0 0.2 0.3 0.4 0.5 0.6 0.8 1.0
0 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2x10 5 2x10 5 2 x l 0 5
24 l.OxlO 6 6 . 3 x l 0 5 3 . 2 x l 0 5 1 . 8 x l 0 5 1 . 8 x l 0 5 l.OxlO 6 6 . 3 x l 0 5 4x10 5 3 . 6 x l 0 5 3 . 2 x l 0 5 2 . 5 x l 0 5 2 x l 0 5 1 . 8 x l 0 5
48 1.3x10 7 1 . 8 x l 0 6 4x10 5 1 . 8 x l 0 5 1 . 8 x l 0 5 l . l x l O 7 2 . 5 x l 0 6 1 . 3 x l 0 6 8 x l 0 5 4 . 5 x l 0 5 4 x l 0 5 2 . 8 x l 0 5 1 . 8 x l 0 5
72 4x10 7 6 . 3 x l 6 6 5 . 6 x l 0 5 1 . 8 x l 0 5 1 . 8 x l 0 5 4 x l 0 7 8 x l 0 6 6
3.6x10 6
2.5x10 8x10 5 5.6x10 5 4 x l 0 5 2 . 5 x l 0 5
96 l x l O 8 1.3x10 7 1 . 3 x l 0 6 3 . 6 x l 0 5 1 . 8 x l 0 5 l x l O 8 2 . 8 x l 0 7 l x l O 7 6 . 3 x l 0 6 2 . 5 x l 0 6 1 . 4 x l 0 6 8x10 5 3 . 2 x l 0 5
120 1 . 8 x l 0 8 3.2xlO ? 2 , 5 x l 0 6 l x l O 6 2 . 5 x l 0 5 1 . 8 x l 0 8 8 x l 0 7 2 . 8 x l 0 7 1 . 3 x l 0 7 6 . 3 x l 0 6 3 . 2 x l 0 6 1 . 8 x l 0 6 4 x l 0 5
144 2 . 8 x l 0 8 7 . l x l O 7 3 . 2 x l 0 6 l . 6 x l 0 6 2 . 8 x l 0 5 3 . 2 x l 0 8 1 . 4 x l 0 8 5x10 7 2 . 8 x l 0 7 1.3xl0 7' 5 . 6 x l 0 6 2 . 8 x l 0 6 6 . 3 x l 0 5
1 168 3 . 2 x l 0 8 ! l x l O 8 8 x l 0 6 2 . 8 x l 0 6 4 . 5 x l 0 5 3 . 2 x l 0 8 2 x l 0 8 8 x l 0 7 7 7 5x10 j2.8x10 8 x l 0 6 4 x l 0 6 l x l O 6
Table A4.30 I n f l u e n c e of Hg on the growth of w i l d - t y p e
time ( h )
Source of inoculum
time ( h ) basal medium time ( h )
Hg (mg l " 1 )
time ( h )
0 0.01 0.02 0.03 0.04 0.05 0.06
0 2 x l 0 5 2 x l 0 5 2x10 5 2 x l 0 5 2 x l 0 5 2 x l 0 5 2 x l 0 5
24 8 x l 0 5 6 . 3 x l 0 5 4 . 5 x l 0 5 3 . 2 x l 0 5 2 x l 0 5 1 . 6 x l 0 5 d
48 6 6.3x10 3 . 6 x l 0 6 1 . 8 x l 0 6 3 . 6 x l 0 5 2 . 5 x l 0 5 1 . 3 x l 0 5 d
72 3.2xlO ? 2 x l 0 7 4 x l 0 6 5. l x l O 5 2 . 6 x l 0 5 l x l O 5 d
96 l x l O 8 5 . l x l O 7 l x l O 7 1 . 3 x l 0 6 3 . 2 x l 0 5 1 . 3 x l 0 5 d
120 1 . 4 x l 0 8 7 . 2 x l 0 ? 1.4xlO ? 2 . 5 x l 0 6 6 . 3 x l 0 5 3 . 6 x l 0 5 d
144 1 . 6 x l 0 8 9 x l 0 7 2 x l 0 7 4 . 5 x l 0 6 l x l O 6 6 . 3 x l 0 5 d
168 2 x l 0 8 l x l O 8 2 . 5 x l 0 7 7 . l x l o 6 2 x l 0 6 1 . 3 x l 0 6 d
265 .
in
CM
m X m m m co X CM CM CM m 00 CM
m in
in oo m co co CM CM eg CM
00 in 6 m
CM CM CO m CM CM co oo
42 oo 6 lw 4-1
m m 00 CO in
in CM CM 01 CO
4-1
m CM CO co X CO CM 00 co co in oo
oo eo m oo oo X (0 00 CM CM CO CM
00 vO CM 00 CU CM OS CM
266.
T a b l e A4.3 2 Pigment changes d u r i n g g r o w t h o f w i l d - t y p e
Anacystis a t d i f f e r e n t Cd c o n c e n t r a t i o n s .
t i m e (days)
Cd mg 1 d r y wt (mg l " " 1 )
c h l a (mg I " 1 )
phyco. % c h l a (mg 1 -1) d r y wt
% phyco. phyco: c h l a d r y wt (by d r y wt.)
0 0.0 20 0.20 ND 1 .0 ND ND 0.05 20 0. 20 ND 1 .0 ND ND 0.15 20 0.20 ND 1.0 ND ND 0.25 20 0. 20 ND 1 .0 ND ND
2 0 80 0.83 0.33 1 .04 0.42 0.40 0.05 80 0.70 0.67 0.88 0.84 0.96 0.15 25 0.24 ND 0.96 ND ND 0.25 20 0.18 ND 0.96 ND ND
0.0 0.05 0.15 0.25
130 116 60 55
1 .26 1 .39 0.28 0.28
0.91 2 .25 0.17 0.15
0.97 1 .20 0.47 0.51
0.70 1 .93 0.28 0.27
0.72 1 .60 0.61 0.53
0.0 0.05 0.15 0.25
180 182 120 130
1 .81 0.83 0.83 0.70
1 .25 4.1 1.0 0.91
1 .0 0.46 0.70 0.54
0.70 2.23 0.84
0.70 5.0 1.2 1.15
0.0 0.05 0.15 0.25
216 222 142 136
2.22 0. 56 1 .39 1.11
1.75 4.3 2 .57 2.45
1 .03 0.25 0.98 0.82
0.81 1 .95 1 .80 1.80
0.78 7.68 1.85 2.21
10 0.0 0.05 0.15 0.25
224 200 180 180
2.36 1 .53 1 .67 1 .53
2.5 2.22 4.10 3.76
1 .05 0.77 0.93 0.85
1.11 1.11 2.23 2.10
1 .06 1.45 2.46 2 .47
12 0.0 0.05 0.15 0.25
240 228 200 196
2.10 1 .95 1 .95 1 .67
2.67 2.80 5.57 5 .45
0.88 0.85 0.98 0.85
1.11 1 .23 .78 .78
.27
.43
.96
.26
14 0.0 0.05 0.15 0.25
256 248 225 225
2 .26 1 .98 1 .82 1 .76
2.60 2.73 6.0 6.10
0.88 0.80 0.81 0.78
1.0 1.1 2.67 2.70
15 38
3.36 3.47
267.
T a b l e A5.1 I n f l u e n c e c f Na on Cd t o x i c i t y t o w i l d - t y p e Anacystis.
Cd (mg 1 1 ) Na (mg r 1 )
2.5 5 10 20 40 50 100 200 30 0
0 8.0 8.6 8.6 8.6 8.6 8.8 8.2 8.5 8.6
0.15 6.8 6.8 6.4 6.4 6.6 6.4 6.2 6.6 6.5
0.30 5.4 5.4 5.5 5.2 5.6 5.1 5.4 5.5 5.4
0.45 4.3 4.2 <4 . 4 4.3 4.6 4.3 4.6 4.6 4.4
0.6 1.3 1 .5 1 .3 1 .4 2.8 1.6 1.6 1.5 1 .6
0.9 d d d d d d d d d
Table A5.2 I n f l u e n c e o f K on Zn t o x i c i t y t o w i l d - t y p e Anacystis.
Zn (mg 1 ) K (mg r 1 )
5 10 20 40 80 100 200 300 400 500
0.04 8.8 8.6 8.5 8.4 8.5 8.2 8.1 8.2 8.2 8.1
0.10 8.8 8.7 8.4 8.4 8.6 8.2 8.0 8.2 8.1 8.0
0.20 8.8 8.7 8.4 8.5 8.4 8.0 8.0 8.2 8.0 8.1
0.40 8.6 8.7 8.6 8.2 8.4 8.4 8.2 7.8 7.4 7.5
0.60 8.5 8.6 8.3 8.0 8.5 8.0 8.1 7.6 7.6 7.1
0.70 8.4 8.2 7.8 7.7 7.8 7.5 7.4 7.4 7.6 7.2
0.80 8.4 7 .6 7.6 7.5 7.5 7.2 7.4 7.6 7.0 7.2
1 .0 8.2 7.4 7.2 7.6 7.5 7.2 7.2 7.5 7.2 7.0
1 .25 7.8 7.1 7.0 7.5 7.5 6.8 6.8 6.8 7.0 6.9
1 .50 0.3 0.3 0.24 0.06 0.07 0.05 0.06 0.03 0.06 0.04
1.75 d d d d d d d d d d
268.
Table A5„3 I n f l u e n c e o f K on Cd t o x i c i t y t o w i l d - t y p e Anacystis.
Cd (mg 1 ) K (mg 1 )
5 10 20 40 80 100 200 300 400 500
0 -04 9.2 8.8 8.8 8.7 8.6 8.8 8.8 8.8 8.8 8.8
0.15 8.4 8.2 8.2 8.0 7.8 7.8 7.6 7.4 7.5 7.5
0.30 5.4 5.2 5.5 5.6 5.4 5.4 5.2 5.0 5.1 5.2
0.45 A A H . H 4.5 4.8 A n *-* . 4.2 3.8 3 .6 T O 3.2 J . z
0.60 1 . 5 1.8 1 .8"- 1 .4- 1 .6 1 .4 1 .5 1 . 4-- 1 A'- 1 .4-
0.90 d d d d d d d d d d
Table A5.4 I n f l u e n c e o f Ca on Zn t o x i c i t y t o w i l d - t y p e Anacystis.
-1 Zn (rng 1 )
0.04
0.25
0.50
0.75
1.0
1 .25
1 .50
2.0
7 ,
Ca (rag 1 )
2.5 5
7.5 7.9
7.8 7.9
7
7.5 7.6
7.4 7.5
6.0 7.0
0.05 0.4
d d
10 20 40 80 100 200
8.4 8.6
8.4 8.5
7.8 8.2
7.7 8.0
7.4 7.5
0.66 4.3
0.05 3.7
9.4
9.2
8.9
8.9
8.5
8.3
6.2
5 .6
10.6
10.6
10.2
9.3
9.3
9.4
7.7
6.7
10.7
10.8
10.4
10.2
9.6
9.6
7.8
6.8
11.6
11.2
10.6
10.4
10.4
9.8
8.2
7.1
269.
T a b l e A5.5 I n f l u e n c e o f Ca on Zn t o x i c i t y t o Zn-t5.0 Anacystis.
Zn (mg 1 ) I Ca (mg 1 )
2.5 5 10 20 40 80 100 200
0.04 8.2 8.3 8.4 8.6 8.8 9.0 9.2 9.4
2 7.8 8.0 8.1 8.2 8.5 8.4 8.6 8.7
4 4.6 5.8 7.5 7.8 8.2 8.0 8.5 8.5
5 2.8 3.5 4.0 6.8 7.5 8.2 8.2 8.4
6 d d d 4.8 5.2 8.0 8.0 8.3
8 d d d 0.05 2.3 7.5 7.5 8.0
10 d d d d d 6.8 6.8 8.0
12 d d d d d 5.6 5.6 7.8
Table A5.6 I n f l u e n c e of Ca on Zn t o x i c i t y to Zn-tl2.0 Anacystis.
Zn (mg l " ) [ Ca (mg l " )
2.5 5 10 20 40 80 160 320
0.04 8.2 8.3 8.4 8.6 8.8 9.0 9.2 9.4
4 7.8 8.0 8.0 8.2 8.5 8.4 8.6 8.8
8 4.6 5.8 7.5 7.8 8.2 8.0 8.5 8.5
10 3.8 4.8 5.0 6.8 7.5 8.2 8.2 8.4
12 3.2 3.6 4.2 4.8 5.4 8.0 8.0 8.3
16 d d d 0.05 2.3 7.5 7.5 8.0
20 d d d d d 6.8 6.8 8.0
24 d d d d d 5.6 5.6 7.8
T a b l e A5.7 I n f l u e n c e o f Ca on Cd t o x i c i t y t o w i l d - t y p e Anacystis.
Cd (mg 1 l ) Ca (mg r 1 )
2.5 5 10 20 40 80 100 200
0 8.0 8.8 8.8 8.8 8.4 8.8 8.8 9.4
0.3 4.8 5.4 6.8 8.4 8.0 8.8 9.0 9.6
0.45 4.0 4.2 4.0 5.4 5.4 5.8 6.0 6.4
0.6 1.8 3.0 4.6 4.6 4.8 4.8 4.8 5.4
0.9 d 2.0 3.0 3.2 3 .6 4.2 4.4 4.8
1.2 d d d 1.8 2.1 4.0 4.2 4.5
Table A5.8 I n f l u e n c e o f Mn on Zn t o x i c i t y t o Zn-t5.0 Anacystis.
Zn (mg r 1 ) Mn (mg r 1 )
0.05 0.10 0.50 1.0 2.5 5 10 20
0. 04 8.3 8.3 8.4 8.5 8.6 8.5 8.0 7.2
2 8.4 8.3 8.4 8.5 8.6 7.0 6.2 3.0
4 5.8 5.8 5.7 7.0 8.0 6.8 4.3 d
5 3.4 3.5 3.4 4.8 7.2 6.2 d d
6 d d d 3.4 6.8 5.6 d d
8 d d d 3.4 6.0 3.8 d d
10 d d d 3.2 4.2 3.7 d d
12 d d d 1.8 2.1 1 .6 d d
Table A5.9 I n f l u e n c e of Mn on Zn t o x i c i t y to Z n - t l 2 . 0 Anacystis»
Zn (mg l " 1 ) Mn (mg l " 1 )
0.05 0.1 0.5 1.0 2.5 5 10 20
0.04 8.3 8.3 8.4 8.5 8.6 8.5 7.8 7.0
4 8.4 8.3 8.4 8.5 8.6 7.0 6.2 3.0
8 5.8 5.8 5.7 7.0 8.0 6.8 4.5 d
10 3.4 3.5 3.4 4.8 7.2 6.4 d d
12 d 2.4 3.2 3.4 6.4 5.6 d d
16 d d d 3.4 6.0 3.6 d d
20 d d d 3.2 4.2 3.7 d d
24 d d d 1.8 2.1 1.6 d d
T a b l e A5.10 I n f l u e n c e of Fe on Zn t o x i c i t y to Zn-t5.0 Anacystis
r e s u l t s based on u n i t s ml ^ x 10^; age of the a l g a 5 days-
d = died.
Zn (mg l " 1 ) Fe (mg 1 1 )
0.05 0.1 0.5 1.0 2.5 5 10 20
0.04 5.4 7.5 7.6 7.6 7.8 7.0 6.5 5.0
2 5.8 7.4 7.3 7.2 7.1 7.0 6.2 4.8
4 5.6 7.2 7.0 7.2 7.0 6.8 6.0 4.5
5 5.2 6.4 7.3 7.2 6.8 6.2 5.1 4.0
6 d d d d 3.1 4.1 3.8 3.6
8 d d d d d 2.6 3.4 3.2
10 d d d d d d 2.2 1.8
12 d d d d d d d d
272
Table A5 11 I n f l u e n c e of Fe on Zn t o x i c i t y to Zn-tl2„0 AnacystiS'
r e s u l t s based on u n i t s m l - 1 x 10 7; age of the a l g a 5 days;
d = die d .
Zn (rag l " 1 ) Fe (mg l " 1 )
0.05 0.1 0.5 1.0 2.5 5 10 20
0.04 5.4 7.5 7.6 7.6 7.8 7.0 6.2 5.1
4 5.8 7.4 7.5 7.5 7.3 6.8 5.8 4.6
8 5.6 7.3 7.3 7.4 7.0 6.4 5.4 4.5
10 5.2 7.2 7.2 7.2 6.8 4.1 3.8 3.2
12 5.0 6.8 7.0 7 .0 3.2 2.6 3.4 3.2
16 d d d d d d 2.2 1.8
20 d d d d d d 1.2 d
24 d d d d d d d d
Table A5.12 I n f l u e n c e of Fe on Cu t o x i c i t y to w i l d - t y p e Anacystis
the r e s u l t s based on u n i t s ml x 10 ; age of the
a l g a 5 days; d = died.
Cu 'mg l " 1 ) Fe (mg i - 1 )
0.05 0.10 0.50 1.0 2.5 5 10 20
0.0 5.5 7.6 7.8 7.8 7.8 6.9 6.5 4.8
0.02 5.8 7.6 8.0 7.8 7.8 6.8 6.3 5.2
0.04 4.8 5.0 5.2 5.6 5.8 5.2 5.0 5.0
0.08 2.5 2.5 2.8 3.8 4.0 5.0 5.1 4.8
0.10 0.85 0.85 1.3 1.8 3.6 4.0 4.5 4.2
0.16 0.06 0.06 1.0 1.2 1.2 1.4 2.3 3.8
0.20 d d d d d d d d
0.24 d d d d d d d d
273 .
Table A5.13 I n f l u e n c e of Fe on Cu t o x i c i t y to Cu-tO.5 Anacystis.
r e s u l t s based on u n i t s ml ^ x 10 7; age of the a l g a 5
days; d = died
Cu (mg l " 1 ) Fe (mg r 1 )
0.05 0.10 0.50 1.0 2.5 5 10 20
0.0 5.6 7.8 7.8 7.6 7.8 6.9 6.5 5.0
0.2 6.0 5.6 6.2 6.5 6.2 6.0 6.3 5.0
' 0.4 0.8 1.0 3.6 3.2 4.2 4.6 4.0 3.8
0.5 0.07 0.85 2.1 3.4 3.6 3.0 3.2 3.4
0.6 d d 0.05 0.38 1.8 1.2 0.45 0.5
0.8 d d d 0.02 0.66 0.8 d d
1.0 d d d d 0.52 0.6 d d
1.5 d d d d 0.45 d d d
T a b l e A5. 14 I n f l u e n c e o f PO -P on Zn t o x i c i t y t o w i l d - t y p e Anacystis.
Zn (mg 1 ) P 0 / T p ( m <? 1 5
0.88 1 .75 3.5 7 14 28 56 112
0.04 6.5 7.8 8.5 8 .6 9.4 9.5 10.0 10.2
0.5 5.9 7.6 8.6 8 .6 9.6 9.6 10.0 10.0
1.0 5.7 7.4 8 .4 8 . 5 9.3 9.2 9.6 9.8
1.5 3.6 7.4 7.2 7.8 8.2 8.6 9.2 9.4
2.0 0.4 7.4 7.6 7.8 7.9 8.2 8.8 9.1
2.5 d 6.8 7.2 7.5 7.8 7.8 8.5 8.6
3.0 d 6.8 7.2 7.2 7.5 7.4 8.4 8.4
3.5 d d 4.5 5.8 7.2 7.2 7.8 7.8
4.0 d d 3.8 4.5 6.8 7.0 7.6 7.6
274.
T a b l e A5 .15 I n f l u e n c e o f PO -P on Zn t o x i c i t y t o Z n - t 5 . 0 Anacystis.
Zn (mg 1 1 ) PO 4 -P (mg r 1 )
1 .75 3 .5 7 14 28 56 112
0.04 8.8 8.8 9.6 9.5 9.8 10.0 10.2
2 8.8 8.8 9.6 9.5 9.8 10.0 10.2
4 5.8 6 .2 8.6 9.0 9.4 9.5 9.2
5 4.2 4.8 6.4 8.1 9.0 9.4 9.4
6 d 3.2 5.5 8.0 8.8 9.4 9.5
8 d d 4.5 8.0 8.6 9.2 9.2
10 d d 2.3 7.8 8.0 8.4 8.6
12 d d^ r
d 4.3 7.8 8.0 8.4
Table A5.16 I n f l u e n c e of - P on Zn t o x i c i t y to Zn-tl2.0 Anacystis.
Zn (mg l " 1 ) 1 P ° 4 - P (mg l " 1 )
1.75 3.5 7.0 14 28 56 112
0.04 8.8 8.8 9.6 9. 8 9. 8 10. 0 10 0
4 8.8 8.8 9.6 9. 5 9. 8 10. 0 10 2
8 5.8 5.2 8.6 9. 0 9. 0 9. 5 9 6
10 4.2 4.8 6.4 8. 0 9. 0 9. 4 9 6
12 3.5 4.2 5.5 8. 0 8. 5 9. 4 9 5
16 d d 4.5 8. 0 8. 6 9. 2 9. 2
20 d d 2.6 7. 5 8. 0 8. 4 8. 6
24 d d d 4. 0 6. 8 7. 8 8. 0
275.
T a b l e A5.17 I n f l u e n c e o f PO -P on Cd t o x i c i t y t o w i l d - t y p e Anacystis.
:d (mg 1 1 ) PO -P 4
(mg r 1 )
1 .75 3.5 7 14 28 56 112
0 8.8 8.9 8.8 8.9 9.4 9.8 9.4
0.03 8.8 8.8 8.6 8.8 8.9 9.2 9.6
0.06 8.6 8.6 8.8 8.9 9.1 9.0 9.4
0.12 8.8 8.8 8.8 9.0 8.8 8.8 9.0
0.24 7 .6 7.8 8.8 8.8 8.8 8.9 8.8
0.36 5.5 5.4 6.8 8.4 8.5 8.6 8.8
0.48 4.4 4.2 4.5 5.4 6.4 7.0 7.8
0.60 3 .1 3.0 4.6 4.6 5.8 6.8 7.4
0.90 d d 3.2 3.8 4.6 5.4 7.0
1 .20 d d d 2.2 3.1* 5.2 6.8
T a b l e A5.18 I n f l u e n c e o f NO -N on Zn t o x i c i t y t o w i l d - t y p e Anacystis.
Zn (mg r 1 ) NC -N 3
(mg 1 )
1 2.5 5 10 20 40 50 100 200 400
0 .04 2.5 2 .8 6.8 7 .5 7 .6 8.2 8.13 8.9 8.8 8.65
0 .25 d d 6.6 7 .5 7.5 8.3 8.2 8.3 8.4 8.6
0 .50 d d 5.4 7.4 7.4 7.8 8.4 8.5 8.3 8.5
0 .75 d d 4.8 7.0 7.2 7.7 8.0 8.5 8.6 7.8
1 .0 d d 3.6 6.2 6.8 7.1 7.2 7.8 7.8 7.4
1 .25 d d d 5.4 5.6 6.5 6.8 7.7 7.4 7.1
1 .50 d d 0.04 0.05 0.06 0.04 0.06 0.04 0.05 0.05
2 .0 d d d d d d d d d d
276 .
T a b l e A5.19 I n f l u e n c e o f EDTA on Zn t o x i c i t y t o w i l d - t y p e Anacystis.
Zn (mg 1 *) EDTA (mg 1 - 1 )
0.5 2 4 8 16 32 64
0 . 04 8.4 8 . 8 9.0 9.2 9 .6 9.9 10.2
0.5 8.2 8.6 8 .8 8.8 8.8 8.6 9.0
1 .0 6 . 4 7 .1 8.2 8.2 8.4 8.7 9.0
1 .25 4.5 4.8 5.6 8.2 8.3 8.7 9.0
1 .5 0.50 0.86 3 .8 8.3 8.2 8 . 2 8.8
2.0 d d d 8.0 8.2 8 . 0 8.4
3.0 d d d 3 . 6 6 .5 7.8 8.0
4.0 d d d d 4.5 7 .5 8.2
6.0 d d d d d 5.4 6.2
8.0 d d d d d 3 .6 5.6
Tabl e A5.20 I n f l u e n c e o f EDTA on Zn t o x i c i t y t o Z n - t 5 . 0
Zn (mg 1 ) EDTA (mg 1 - 1 )
0 .5 1 .0 2 4 8 16 32
0 .04 8 .0 8 .0 8 .2 8 .4 8 .8 8 .8 8 .8
2 7 .8 7 . 6 7 .8 7 .8 8 .8 8 .8 8 .8
4 5 .2 6 . 4 7 .4 7 .4 8.0 8 . 1 8 . 1
5 4 . 6 5 . 4 6 .2 6 .3 8 .0 8 . 1 8 .2
6 d 0 .05 0 . 0 4 4 . 4 6 .5 7 .2 7 .4
8 d d d 3 .2 4 . 8 7 .3 7 .5
10 d d d 0 .08 4 .5 7 .2 7 .3
12 d d d d 3 .6 6 . 4 7 .4
14 d d d d 2 .5 3 . 8 6 .4
16 d d d d d 1 .6 4 . 0
277.
T a b l e A5.21 I n f l u e n c e of EDTA on Zn t o x i c i t y to Zn - t l 2 . 0 Anacystis°
Zn (mg 1 _ 1 ) EDTA (mg 1 h 0.5 2.0 4 8 16 32 64
0.04 9.8 10.4 9.6 9.6 9.6 10.4 11.2
6.0 9.2 9.2 9.3 9.5 10.0 9.8 9.6
12.0 5.6 6.6 7.1 7.8 8.2 8.5 8.8
15.0 d d d 3.5 6.5 8.8 8.5
18.0 d d d d 5.8 8.8 8.4
21.0 d d d d 2.5 4.5 6.2
24.0 d d d d d 1.3 4.8
Table A5.22 Inf l u e n c e of EDTA on Cd t o x i c i t y t o w i l d - t y p e Anacystis.
Cd (mg 1 1 ) EDTA (mg I - 1 )
0 .5 2 .0 4 . 0 8 .0 16 .0 32 .0
C 9 8.8 9 9 . 2 9 . 0 9 .0
0 .15 6 . 4 8 . 0 8 .6 8 .2 8 .8 9 . 4
0 . 3 0 4 . 5 6 .2 8 .6 8 .8 8 .6 8 . 6
0 . 4 5 4 . 2 4 .5 8 . 0 8 .2 8 .8 8 .8
0 . 6 0 3 . 0 3 . 4 4 . 4 8 . 0 8 .6 8 .2
0 . 9 0 d d 3 .6 8 . 0 8 . 0 8 . 2
1.2 d d 2 . 8 6 . 4 7 . 4 8 . 0
1 .50 d d 1.2 6.5 7 .5 8 . 0
Table A5.23 I n f l u e n c e o f Zn on Cd t o x i c i t y t o w i l d - t y p e Anacysti
u n i t s ml ^ was used as a g r o w t h c r i t e r i o n .
Cd (mg 1 *) Zn (mg r 1 ) 0 .04 0 . 2 0 .4 0 . 6 0 .8 1 .0
0 8 .6 8.7 8 . 9 9 . 0 8.3 8 .7
0 .15 3 .7 8 .2 8 .8 8 . 6 8.3 4 . 8
0 .24 4 . 1 8 .9 8 .5 8.7 8.7 a
0 . 3 0 3 .8 8 .2 8 .5 8 . 1 8 .5 a
0 .36 3 .3 8 . 1 8 . 0 8 . 0 6 . 2 a
0 .42 a 8 . 6 8.3 8 .2 a a
Table A5.24 I n f l u e n c e o f Zn on Cd t o x i c i t y t o w i l d - t y p e Anacysti
c h l a was used as a g r o w t h c r i t e r i o n .
Cd (mg 1 Zn (mg r 1 ) 0 .04 0 .2 0 . 4 0 . 6 0 .8 1 .0
0 0 .68 0 .74 0 .57 0 . 6 8 0 . 7 8 0 .68
0 .15 0 . 3 4 0 .68 0 . 7 8 0 . 7 8 0 . 7 8 0 . 6 8
0 .24 0 . 2 4 0 .85 0 .57 0 . 7 8 0 .57 0 .40
0 . 3 0 0 . 1 8 0 .78 0 .68 0 .92 0 .57 ND*
0 . 3 6 0 .23 0 .78 0 . 7 8 0 .57 0 . 4 6 ND*
0 .42 ND* 0 .68 0 .57 0 . 4 6 0 . 5 7 ND*
Table A5.25 I n f l u e n c e o f low Zn l e v e l s on Cd t o x i c i t y t o Z n - t 5 . 0 2 7 9
Anacystis; c h l a was used as a g r o w t h c r i t e r i o n .
Cd (mg 1 1 ) Zn (mg r 1 ) 0. 04 0 .25 0 . 5 1 .0
0 2 . 1 4 2 .21 1 .92 2 .21
0 .15 2 . 1 4 1 .91 1 .71 1 .71
0 . 3 0 2 .14 1 .88 1.71 1 .85
0 .45 2 .14 1 .71 1 .71 1 .71
0 . 6 0 1 .85 1 .71 1 .71 1 .71
0 .75 1.14 1 .57 1 .77 1 .71
0 .90 1 .14 1 .43 1 .78 1 .87
1 .05 1 .07 1 .14 1 .71 1 .07
1 .20 ND 1 .14 1 .86 1 .43
1 .35 ND ND ND 1 .43
T a b l e A5.26 I n f l u e n c e o f h i g h Zn l e v e l s on Cd t o x i c i t y t o Zn-t5.0
Anacystis; c h l a was used as a g r o w t h c r i t e r i o n .
•1 Cd (mg 1 )
0
0 .10
0 . 2 0
0 . 3 0
0 .40
0 . 5 0
0 . 6 0
0 . 7 0
0 . 8 0
1.0
1.2
1.4
1 .6
Zn (mg 1 - 1 .
0 . 04
1 .21
1 .20
1 .21
1. 14
1.20
1 .21
1 .12
1.14
1.14
0.57
ND
ND
ND
2 .5 5 .0 7 .5 10 12 .5
1.14 0 .92 0 .14 d d
0 . 7 1 0 . 7 1 0 . 1 4 d d
0 .85 0 . 7 1 0 .14 d d
0 .85 0 . 7 6 d o d d
0 .85 0 . 7 1 d d d
0 . 9 4 0 .87 d d d
0 . 7 1 0 . 7 1 d d d
0 .93 0 . 7 1 d d d
0 .93 0 . 7 8 d d d
0 .93 0 . 8 8 d d d
0 .87 0 . 8 6 d d d
0 . 5 7 0 .57 d d d
0 . 2 8 0 .57 d d d
280.
Table A5.2 7 I n f l u e n c e of pH on Zn t o x i c i t y to wi l d - t y p e Anacystis.
Zn (mg 1 1 ) pH
6.0 6.5 7.0 7.5 8.0
0.04 3.2 5.6 8.6 8.8 9.0
0.25 2.4 4.8 7.8 8.5 8.8
0.50 1.5 4.6 7.5 8.5 8.6
0.75 0.6 4.5 7.4 8.5 8.5
1.0 d 3.8 7.2 8.2 8.1
1.25 d 3.5 6.8 7.2 7.5
1.5 d d 0.60 6.8 7 .2
2.0 d d d 6.5 6.8
2.5 d d d 2.7 4.3
Table A5.28 I n f l u e n c e of pH on Zn t o x i c i t y to Zn-t5.0 Anacystis.
Zn (mg 1 1 ) pH
6.0 6.5 7 .0 7.5 8.0
0.04 6.4 7.6 8.0 8.0 8.0
2 6.2 7 .4 7.7 7.6 7.4
4 4.8 5.2 5.3 4.8 4.8
5 4.5 4.6 3.4 2 .2 d
6 4.2 4.4 d d d
8 1.2 3.6 d d d
10 d d d d d
2 8 1 .
Table A5.29 I n f l u e n c e of pH on Zn t o x i c i t y to Zn-tl2.0 Anacystis
Zn Cmg 1~ ) PH
6.0 6.5 7.0 7.5 8.0
0.04 5.5 7.0 7.4 7.2 7.2
4 4.2 6.3 6.7 4.7 4.7
6 2.6 5.8 6.4 3.2 3.2
8 1.0 5.4 6.0 1.8 1.8
12 d 5.0 2.0 0.05 0.05
16 d 4.3 d d d
20 d 2.0 d d d
24 d d d d d
Table A 5 . 3 0 I n f l u e n c e of pH on Cd t o x i c i t y t o w i l d - t y p e
A n a c y s t i s .
Cd (mg 1 1 ) pH
6 .0 6 .5 7 . 0 7 .5 8 .0
0 .04 3 .5 5 .8 8 .8 9 .5 10 .2
0 . 1 5 d 0 .8 6 . 4 8 .8 9 .4
0 . 3 0 d 0 .03 4 . 2 8 . 4 8 .5
0 . 4 5 d d 3 . 8 8 . 6 8 .6
0 . 6 d d 3 .2 6 . 2 7 .2
0 . 9 d d d 5 .8 6 .2
1.2 d d d 4 . 5 5 .8
T a b l e A5.31 I n f l u e n c e of inoculum s i z e on Zn t o x i c i t y to wi l d - t y p e
Anacystis
Zn (mg 1 ) Inoculum s i z e ( u n i t s ml )
4 2x10 2 x l 0 5 2 x l 0 6 2 x l 0 ?
0.04 6.8x10 7 8 . 4 x l 0 7 1 2 . 2 x l 0 7 1 2 . 5 x l 0 8
0.5 5 . 4 x l 0 7 8 . 5 x l 0 7 9 . 2 x l 0 7 I 0 . 4 x l 0 8
1.0 3 . 6 x l 0 ? r , , 1
6.4x10 8.8xlO ? 9 . 8 x l 0 8
1.25 1 . 8 x l 0 ? 1 . 8 x l 0 7 6.5xlO ? 7 . 8 x l 0 8
1.5 d d d Q
4.0x10
2,0 d d d d
Table A5.32 I n f l u e n c e of inoculum s i z e on Zn t o x i c i t y to w i l d - t y p e
Anacystis
Zn (mg 1 ) Inoculum s i z e ( c h l a mg 1 )
0.001 0.01 0.10 1.0
0.04 1.3+0.0 1.38+0.07 1.67+0.07 2.30+0.18
0.5 1.14+0.0 1.21+0.12 1.44+0.17 1.83+0.14
1.0 0.75+0.15 0.75+0.12 1.36+0.07 1.40+0.06
1.25 0.25+0.03 0.28+0.04 0.88+0.08 1.35+0.08
1.5 d d 0.35+0.005 0.85+0.12
2.0 d d d d
3.0 d d d d
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