f. h. a v . shehata - coreu.v. ultra-violet x mean value filtrable capable of passing through filter...

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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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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 ....





Accumulation of Zn du r i n g batch c u l t u r e s growth o f . . .





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


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


7.6 Influence of pH on Zn t o x i c i t y t o Phormidium autumnale.... 201


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


7.8 Influence of Zn on Cd t o x i c i t y t o Phormidium autumnale ... 203


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 .


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 .


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



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


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 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 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 .


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



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

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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

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

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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.


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

http://A4.ll

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;


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 ;



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;


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



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


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



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




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



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



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



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


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



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



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



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




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




! 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


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


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


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


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


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



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


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


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




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




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


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

n

o

to I B

3 T3

< 0

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rC P o u cn

C o u c

V M

o •rH c 0

r H 0

u

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ft in P • H c D

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r H

ft

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a in p • H

c

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0 P c o u

0*>

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W r H P O • H O C D

en a

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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

I T ) in in m o O o O o o O O

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


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


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


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



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


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


-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


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


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


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


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 '

^ - - " ^

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4 1

V,

w

i\ '

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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 ;


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 ;


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


-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


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.

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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.

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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

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X Cn u E

0) E ~ •H X

u n i n CN CN CN CN CN

r - r - r -

m m 0 0 un Q o CO o CTi 2 • • • •

o o o '-'

O o o o u n r H

-1 CN m

r - 1 ^ r- r -

^ 0 \o t n c n oo c n Q o m c o CN r ~ 2 • •

c o r H r H CN

r H CN u n r ~ CO c n

r - r - r ~ r - r -

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


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



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


- 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


- 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


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



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


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


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

O CN O in o o X) o LO X) CD X) o CM CM en CO i n cn CM •sr

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

4J r C tn •

•H 0)

>1 p T3

CP E

Cn

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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)

o CO CM 00 CM 00 C m X) O X) CO c *-H CM CN CN CN CN CO

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


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.

u c rO U COJ

rH o 4-1 ,c

in 0) 4J •H to

o c •H N

x : CP

•H x :

g

0) 4-1 (0 rH O (ft •H

w

§ •H G rO CP 0

e o

4H

>1

rO -H 4-1 •H C •H

T) 01

O in •H

>l i—I

c •H n3 4- 1 5- J 0) u 4-1 in O 6

C rfl

C O •H 4-> O O)

X I

0) 4-> <0 u

•H •a c •H

w •H s to

•H c rO CP U O c (0

o 4-1

u c

•H N

'4-t O >i 4-1 •H O •H X! O EH

C O

•H 4J U 0) in

0 rH in c 0 rH 0

u o 01 •H c > 4-) •H 0) 0) <0 M u rH S4

3 4-1 0 4-> O c 4J rH c 0)

•H u 0) u N c u 0 G s u rO 0 0

u

VI rM rH X 0) 0)

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 i II

• P •H C U •H

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4-> 1 fl r-l XI CD £ Cn <£> • P B

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u •—i U

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CN O m m

• • • 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

CN m CN o CI IX < » • • • •

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

^ ^

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198.

03

Q

(D M 0)

3 + J 3

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i H M-l

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

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to > i rd T3

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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

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03 o o o o t-H

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i n

CN

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m in

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• "0* CN ro in m v£ >x>

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m v£> <£> r-

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r- oo 00 CO

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199.

0) I rtf rH

>x> r- s • m m C O CM

o ro ro L D • n <v • • • •

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(0 w m [3 •c ro ro 3 o I X •a T3 • • • •

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• 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

• - I X 0 o CM CM ro m in • P 0

•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->

.—. . • 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-

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200.

u

w (0

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0) <a 3

o r-H +J .G . H 0 3

U 0 >1 0) 01 x: u 4-1 c 0) u 3 3 o

i-l Ul IW (0 0) C o> CP M E (0

in co CM O ro ro

» • * • . (/) o o O O -H +1 •H + 1

O ro ro

o IX T> • • • • o CM ro

CM co in CM ro

• T) • • • » cn o o o o

o -H +1 -H -H ro

co CM in ix m T3 • » •

CM CO in in

1—1 < CN ,—( in 1 CM ro in o >£>

m C O o o o a O +1 +1 +1 +i +i +i

"— CM LO CO CM U3 CM

U I X CM CM ro 1" m in

, in CM m CM o CM r - >X> CO

W o o O o o o O +1 +1 +1 +i +i -H

CM CM in co o I X • • • • •

m in in in

t co in m m T3 CO ro W o o o -< O o

o +i +i -H +1 -H

in CM O CO in I X

r- uo

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7 3 7 3 73 • • O m

t i <X> m 7 3 O CN i£>

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+i + 1 +1

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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

+1 + 1 - H - H

i n I X 7 3 • • •

CN r - r -

LD O m o

>X> c o

X3 ro EH

202.

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 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 -<*

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d) M <0

3

3

n E 3

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tn

(1) N •H Ui

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r-4 3 U o C •H

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u

>1

a>

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0) n 3

3 U

o Q) CP (0

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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

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rd

in rd

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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

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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

metal. When c u l t u r i n g i n the presence of a metal i s repeated, the

resistance of the c u l t u r e becomes more s t a b l e . R e p e t i t i o n o f t r a i n i n g

CH +3 0 0 • CD rH

W a C P rH •H

rd 4-) (0

•P CD CD CD U S XI S P •O U CD 3 P U 0 rd UH Xi „ — CJ

u T 1 •A CD 1 JC .C % •P o CP rH

rd CD 4J

CD

P rH e rd e 0 P 4H P CD 0 S rH rH

(d 4-4 CD +J 0 > CD CD

S rH <H CD

M > CD rd CD si

i H rH 4J

U > 1 >f •H >H XI P 0 >H 4-> T3 <d • H CD ft X! 4-1 ft •H (d CD CJ

C C -rH O •H T3 C O > i •H

•P rH CP CO

4 J C •H

S 0 rd > i U -P 4-> » CD (0 •rH s i-l o p 0 0 •H •H +1 4J X -D 0 CD

w CP P E c c •H •H CH rH

rd •a O rd H u CO

•P 0 C rd to o 0 £1 o w m rd •H C 0 H •H

T3 rd CD CD ft 4->

u S S id c rd o x; <d C u 4-> 4-> CO CD <*>

•H U • o CO rd c LT) CD 0 M CO •H o 1 G P P CO •H rd LO rd rH CD O H O •U h •P CO rd U W •H U

CO (D

r—I

•9

& T3 0 CD 4-1 4-> •H ft X) id •H T3 r C rd c •rH ch

> 1 •H rH X! CP

c 0 0 n 4-> 4-) CO r-l , — . rd T-H a •P 1 0 CD

rH )H a MH a CP 144

,B s 0 ,B H r-l rH

rH 3 CD id CJ > P 0 CD CD rH

B •H

HH o CD > CD

U

•r-l X O P

a 3 •H

T ) CD

a rd co rd -Q e o M

I P

r H 3 D O C •H

U o <*H

"° <D >i CO rd 3 CO

co c (d •H rd SH •P CO

3 O

•rH

2

o o

73

3 U

- H

z

0 o

CN ro

«—1 CN r H o CN

ro in in o o o o O o

in o to in in m CO co CN

r H o O o r H «—1

CN

o r H

r> o

to o

m r H

00 o CN

o o o o o o O

en o CN

co t£) r H

CO o

r-o in CN

o o o o o o o

to «-H CN

r-o r-CN

CN m to o r H o o o o o

CO o CN r H o CN CN r H CN CN CN

O O O O o O r H

in o in in o o r~- r> CN m m CP. r H o O r H CO o

O o CO m CN o r H r H O r H r H r H

r H O O o O O o

CP. r H 00 CN CN CN

O r H r- r H r H o r H

O o o o O o O

CN m CO CN O m tO r H co ro CN CN r H

O o O O o O o

CD ft

K V I

T J rH •H 3

O CO o in o • O • * • 0 CN • r H o in r H CN

•P •P P P P •P 1

0 1

•H 1

3 1

c C 1 T ) u 2 0 N N U

208.

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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214

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 .

http://A4.ll

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 )


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


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)


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


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

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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*., Ôtxz ^ o i x e - E ôixrs ^0ix« g 0 1 » V l e o i x z

{ O W S 9 o i « z ÔTXl ^ 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 ÔIXS ^01x9 9 0 1 » £ 1 { 0 1 X 9 £ , 0 1 - E - l ÔIXE'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

Ôixs 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 ÔIXE'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 ôixe-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>


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 )


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


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


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


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)


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

http://atr.my.ly

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)


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 )


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 )


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 )


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



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

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