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Effect of particle size on the kinetics of microbiological leaching of chalcopyrite Blancarte-Zurita, Martha Alicia 1983

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EFFECT OF PARTICLE SIZE ON THE KINETICS OF MICROBIOLOGICAL LEACHING OF CHALCOPYRITE  By  MARTHA ALICIA BLANCARTE-ZURITA IBQ I n s t i t u t o P o l i t e c n i c o Nacional, Mexico 1981  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Department o f Chemical Engineering)  We accept t h i s t h e s i s as conforming t o ^ t h ^ required standard  THE UNIVERSITY OF BRITISH COLUMBIA December  Martha  1983  Alicia  Blancarte-Zurita,  1983  In p r e s e n t i n g  this thesis in p a r t i a l  f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e for reference  and  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may  be  department o r by h i s or her  granted by  the head o f  representatives.  my  It i s  understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be  allowed w i t h o u t my  permission.  Department o f  Chemical Engineering  The U n i v e r s i t y of B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  'DecesnhisT  1 Qft ^  Columbia  written  - ii -  ABSTRACT  An experimental investigation was undertaken to study the microbiological leaching of chalcopyrite by Thiobacillus ferrooxidans.  Leach tests were conducted on a small scale using shake  flasks.  Tests were done which showed that the optimal time for transfer of inocula was between 6 and 8 days.  Attempts were made to c l a r i f y the complex media-mineral-bacteria interactions that occur i n the bioleaching process relating bacterial growth with the changes i n media composition as a result of the a c t i v i t i e s of the bacteria at the mineral surface.  In so doing i t was  observed that a significant amount of copper was leached during the stationary phase of bacterial growth.  A sedimentation technique was used to separate the chalcopyrite into various size fractions which were then used to determine bacterial leaching rates i n separate experiments.  The particle size ranges had  average diameters of 1.07, 1.78, 2.52, 3.56, 5.48 and 7.41 ym.  The  method used to measure particle size was based on the direct comparison of the particles with the scale of an eyepiece micrometer using a microscope.  - iii -  Measurements of particle size distribution made during the course of leaching showed that as the leach proceeded, particle size decreased and the particle size distribution moved i n the direction of more particles i n the smaller size ranges.  An attempt was made to apply Levenspiel's shrinking core model to the data obtained for leaching of the various sized particles.  Agreement  was reasonable but not perfect between the predicted and measured values of % copper extraction.  Better agreement was observed at lower  leaching times.  Electron micrographs are presented which i l l u s t r a t e the attack of the chalcopyrite particles by the organisms. of jarosite precipitation.  They also show the effects  - iv TABLE OF CONTENTS Page Abstract Table of Contents L i s t of Tables Li st of Figures Acknowledgements CHAPTER 1 1.1 1.1.1 CHAPTER II 2.1 CHAPTER III 3.1 3.2 3.2.1  Chapter IV  x l  2 Introduction  2  Objective  3  THE BACTERIA THIOBACILLUS FERROOXIDANS MORPHOLOGY AND CHEMICAL COMPOSITION SUBSTRATE DESCRIPTION MINERAL-MICROBE INTERACTIONS Mechanism of Oxidation of Ferrous Iron  3.2.2 Mechanism of Oxidation of Sulphides 3.3  ii iv vii vi i i  MINERAL-MEDIA INTERACTIONS FACTORS AFFECTING BIOLOGICAL LEACHING  4.1 PARTICLE SIZE AND SURFACE AREA 4.2 NUTRIENTS. 4.2.1 Carbon Source 4.2.2 Nitrogen Source 4.3 TEMPERATURE 4.4 pH 4.5 DISSOLVED OXYGEN 4.6 AGE OF CELLS  4 5 6 6 8 9 11 13 15 15 15 15 16 16 17 17 18  Chapter V  MODELLING BIOLOGICAL SYSTEMS  19  5.1 5.2 5.3  BACTERIAL KINETICS AND MODELLING MODELLING BIOLOGICAL LEACHING THE SHRINKING CORE MODEL  19 20 22  -  V  -  TABLE OF CONTENTS (Continued) Page CHAPTER VI  MATERIALS AND METHODS  6.1  BIOLOGICAL LEACHING TECHNIQUES  6.1.1 Sampling Techniques . . . 6.2  25 25 27  ANALYSIS  27  Metal Leach Rates Hydrogen Ion A c t i v i t y Oxidation-Reduction P o t e n t i a l B a c t e r i a l Growth  27 28 28 28  FRACTIONATION OF CONCENTRATE  30  6.3.1 D e f l o c c u l a n t S e l e c t i o n T e s t s  36  6.2.1 6.2.2 6.2.3 6.2.4 6.3  6.4  PARTICLE SIZE MEASUREMENT  6.4.1 P r e p a r a t i o n o f Concentrate f o r S i z e Measurement 6.4.2 Scanning E l e c t r o n Microscope Techniques CHAPTER VII EXPERIMENTAL RESULTS AND DISCUSSION 7.1 7.2 7.2.1 7.2.2 7.2.3 7.2.4 7.3  7.5  45 46  Copper E x t r a c t i o n Surface Area U t i l i z a t i o n S t e r i l e Run Leaching i n the Absence o f B a c t e r i a  46 50 55 57  EFFECTS OF PARTICLE SIZE ON THE LEACHING OF COPPER.  60  APPLICATION OF THE SHRINKING CORE MODEL OF LEVENSPIEL POST LEACHING OBSERVATIONS  CHAPTER V I I I 8.1  41 43 45  SELECTION OF THE INOCULUM AGE BACTERIAL GROWTH KINETICS  7.3.1 Changes i n the P a r t i c l e S i z e D i s t r i b u t i o n d u r i n g Leaching 7.4  40  SUMMARY AND CONCLUSIONS RECOMMENDATIONS FOR FUTURE STUDIES  65 66 73 79 81  - vi TABLE OF CONTENTS (Continued) Page CHAPTER IX  REFERENCES  82  Appendix 1  89  Appendix II  99  - vii LIST OF TABLES Page Table 1  Culture Media Composition  25  Table 2  Elemental A n a l y s i s o f the Copper Concentrate. . .  26  Table 3  F r a c t i o n s o f B a l I m i l i e d Concentrate: T h e o r e t i c a l S i z e Ranges, Free F a l l i n g V e l o c i t i e s and S e t t l i n g Times F r a c t i o n s o f B a l l m i l l e d Concentrate: Weight Percentages C o l l e c t e d  35  Average P a r t i c l e S i z e i n F r a c t i o n a t e d Concentrate  43  Table 4 Table 5  35  - viii LIST OF FIGURES Page Figure 1 2  7  Space L a t t i c e o f C h a l c o p y r i t e Ping Pong bi bi Mechanism o f Fe Jt Cytochrome +  C Reductase o f T h i o b a c i l l u s f e r r o o x i d a n s . . . . 3  Standard Curve f o r Nitrogen Determination  4  E f f e c t o f Aerosol i n the Growth o f  12  ....  31  J_. f e r r o o x i d a n s  37  5  Martin's Diameter  41  6  E f f e c t o f the Inoculum Age on Copper E x t r a c t i o n .  47  7  B a c t e r i a l Growth o f T h i o b a c i l l u s f e r r o o x i d a n s 49  i n Copper Concentrate 8  B i o l e a c h i n g Data Averages f o r Three Experiments  9  Average Values o f Copper/Iron Ratios f o r Three Leaches B a c t e r i a l Coverage o f the Mineral Surface . . . . Chemical Leaching o f C h a l c o p y r i t e E f f e c t of P a r t i c l e S i z e on the Copper E x t r a c t i o n . Copper E x t r a c t i o n Rates as a Function o f the P a r t i c l e Diameter E x t r a c t i o n Rate as a Function o f the Surface Area  10 11 12 13 14  .  .51 54 56 59 61 63 64  15  Changes i n the P a r t i c l e S i z e D i s t r i b u t i o n f o r the 1.78 nm Leach  67  16  Changes i n the P a r t i c l e S i z e D i s t r i b u t i o n f o r the 2.52 urn Leach  68  17  Changes i n the P a r t i c l e S i z e D i s t r i b u t i o n f o r the 5.48 pm Leach  69  18  Average P a r t i c l e S i z e f o r Three D i f f e r e n t Leaches vs. Time  70  - ix LIST OF FIGURES (Continued) Page 19  Experimental  and T h e o r e t i c a l Copper E x t r a c t i o n  Values f o r the Leaching o f C h a l c o p y r i t e . . . .  72  20  C h a l c o p y r i t e P a r t i c l e s before Handling  74  21 22  C h a l c o p y r i t e P a r t i c l e s a f t e r 200 h o f Leaching. . C h a l c o p y r i t e P a r t i c l e s a f t e r 300 h o f Leaching. .  75 77  23  Second Pass Leaching o f C h a l c o p y r i t e  78  -  X  -  APPENDIX I Page 1.1  B a c t e r i a l Growth Data  90  1.2 1.3  Average R e s u l t s f o r Three B i o l o g i c a l Leaching Experiments C h a l c o p y r i t e Concentrate S u r f a c e Area  91 92  1.4  B a c t e r i a l Coverage o f the Surface Area  92  1.5  S t e r i l e Run Data  93  1.6  Leach Using Monosized M a t e r i a l o f 1.07 urn  94  1.7  Leach Using Monosized M a t e r i a l o f 1.78 nm  95  1.8  Leach Using Monosized M a t e r i a l o f 2.52 pm  96  1.9  Leach Using Monosized M a t e r i a l o f 3.56 um  97  1.10  Leach Using Monosized M a t e r i a l o f 5.48 ym  98  1.11  Leach Using Monosized M a t e r i a l o f 7.41 ym  98  APPENDIX II 1.1  K i n e t i c Data f o r t h e S h r i n k i n g Core Model  102  1.2  C a l c u l a t e d Percentage E x t r a c t i o n A f t e r 50 h o f Leaching  103  1.3  Copper C o n c e n t r a t i o n and Percentage Copper E x t r a c t i o n f o r C h a l c o p y r i t e Leaches  104  - xi -  ACKNOWLEDGEMENTS  F i n a n c i a l support f o r t h i s research program was provided by t h e U n i v e r s i t y o f B r i t i s h Columbia, B.C. Research and E l Consejo Nacional de C i e n c i a y Tecnologia (Conacyt)  Mexico.  -  1 -  CHAPTER 1  - 2 -  CHAPTER I 1.1  INTRODUCTION  B i o l o g i c a l l e a c h i n g i s a process i n which metals a r e e x t r a c t e d from ores and m i n e r a l s by enzymic t r a n s f o r m a t i o n s .  The l e a c h i n g  o f metals u s i n g T h i o b a c i l l u s f e r r o o x i d a n s i s c u r r e n t l y a p p l i e d t o t h e l e a c h i n g o f low grade, and waste s u l p h i d e ores by dump and heap l e a c h i n g techniques f o r t h e recovery o f copper and uranium.  S t u d i e s conducted on a v a r i e t y o f s u l p h i d e m i n e r a l s have demonstrated  t h a t b a c t e r i a l o x i d a t i o n can a l s o be used f o r t h e  recovery o f l e a d , c o b a l t , n i c k e l , z i n c and other base metals.  The p o s s i b i l i t y o f using T h i o b a c i l l u s f e r r o o x i d a n s f o r t h e l e a c h i n g o f metals from o r e concentrate i s c u r r e n t l y being considered.  T h i s type o f l e a c h i n g would take place i n a batch  o r a continuous flow chemical r e a c t o r i n which a c o n t r o l l e d process c o u l d be e s t a b l i s h e d . The advantages o f b i o l o g i c a l l e a c h i n g over conventional p y r o m e t a l l u r g i c a l and hydrometal1urgical processes a r e claimed t o be lower c o s t s , t h e e l i m i n a t i o n o f t h e a i r p o l l u t i o n problems a s s o c i a t e d with smelting o p e r a t i o n s , and s u i t a b i l i t y f o r a s m a l l e r s c a l e o f operation.  - 3 Studies have been p r e v i o u s l y c a r r i e d out with t h e purpose o f o p t i m i z i n g t h e parameters t h a t a f f e c t t h e b i o l o g i c a l process such as temperature, pH, type and c o n c e n t r a t i o n o f n u t r i e n t s , a e r a t i o n , e t c . , and t o o b t a i n i n c r e a s e d metal y i e l d s .  It i s only r e c e n t l y t h a t attempts t o d e s c r i b e t h e b i o l o g i c a l l e a c h i n g process u s i n g mathematical models have been made.  Given t h a t t h e b i o l o g i c a l l e a c h i n g process i n v o l v e s complex, i n t e r a c t i o n s between t h e m i n e r a l , t h e b a c t e r i a and t h e aqueous phase, attempts a t modelling m u l t i p a r t i c l e l e a c h i n g systems have o n l y been p a r t i a l l y s u c c e s s f u l . Mathematical models a r e based on hypotheses about t h e mechanisms t h a t c o n t r o l t h e process and when t h e r e s u l t s o f such models a r e compared with r e s u l t s , a greater understanding  experimental  o f the process i s generated.  Mathematical models a r e a l s o used t o p r e d i c t t h e r e s u l t s o f changes i n the o p e r a t i n g c o n d i t i o n s thereby e l i m i n a t i n g lengthy and c o s t l y experiments.  1.1.1  Objective  The o b j e c t i v e s o f t h i s work were t o o b t a i n i n f o r m a t i o n about t h e mechanisms o f l e a c h i n g o f c h a l c o p y r i t e (copper-iron sulphide) by T h i o b a c i l l u s f e r r o o x i d a n s u s i n g monosize p a r t i c l e s i n batch r e a c t o r s , t o t r y t o q u a n t i f y t h e r e a c t i o n k i n e t i c s and v e r i f y the a p p l i c a b i l i t y o f t h e s h r i n k i n g core model t o t h e b i o l o g i c a l l e a c h i n g o f copper.  - 4 -  CHAPTER II THE BACTERIA THIOBACILLUS FERROOXIDANS  During the f i r s t h a l f o f the twentieth century l a r g e amounts o f copper were recovered from c h a l c o p y r i t e - p y r i t e heaps a t the Rio T i n t o o p e r a t i o n i n south-western Spain ( T r u s s e l l , 1964), but i t wasn't u n t i l 1947 t h a t b a c t e r i a were recognized as an i n t e g r a l p a r t o f t h i s process.  Colmer and Hinkle (1947) showed t h a t f e r r o u s i r o n o x i d a t i o n  o c c u r r i n g i n a c i d mine water was b i o l o g i c a l i n o r i g i n ; t h e i s o l a t i o n o f the organism r e s p o n s i b l e was made by Colmer, Temple and Hinkle i n 1949.  Further s t u d i e s were conducted by Temple and Colmer (1951) t o  e s t a b l i s h t h a t the bacterium was an a u t o t r o p h i c i r o n o x i d i z e r ; they named i t T h i o b a c i l l u s f e r r o o x i d a n s . A u t o t r o p h i c b a c t e r i a can be d i v i d e d i n t o two groups on the b a s i s o f t h e i r source o f energy:  the f i r s t group i s named photosynthetic and  d e r i v e s i t s energy from l i g h t ; the second group i s c a l l e d chemosynthetic and d e r i v e s i t s energy from the o x i d a t i o n o f i n o r g a n i c compounds. T h i o b a c i l l u s f e r r o x i d a n s belongs t o t h i s second group o f b a c t e r i a which a l s o uses carbon d i o x i d e as a carbon source f o r t h e s y n t h e s i s o f i t s organic compounds.  I t has been f u r t h e r considered as  an o b l i g a t e chemoautotroph given i t s i n a b i l i t y t o use a l t e r n a t e carbon sources (more complex than carbon d i o x i d e ) .  - 5 -  2.1  MORPHOLOGY AND CHEMICAL COMPOSITION  T h i o b a c i l l u s f e r r o o x i d a n s i s a m o t i l e , non-spore forming, gram negative, rod-shaped organism which occurs s i n g l y o r o c c a s i o n a l l y i n p a i r s . I t s s i z e i s 0.6-1.0 u.m width by 1.0-1.6 ym i n l e n g t h and l i k e any other microorganism i t s b a s i c elemental composition i s C, H, 0, N, S, P. A l l o f these elements a r e then r e q u i r e d f o r i t s growth and have t o be s u p p l i e d i n forms u t i l i z a b l e by t h e b a c t e r i a .  In a d d i t i o n an energy source i s r e q u i r e d f o r t r a n s p o r t o f n u t r i e n t s , s y n t h e s i s , locomotion, e t c . T h i o b a c i l l u s f e r r o o x i d a n s can o b t a i n energy from t h e o x i d a t i o n o f sulphide m i n e r a l s such a s : c o p p e r - i r o n s u l p h i d e s , l e a d sulphide, nickel sulphide, zinc sulphide, e t c .  - 6 CHAPTER I I I SUBSTRATE  3.1  DESCRIPTION  C h a l c o p y r i t e i s p a r t o f a s e r i e s o f complex c o p p e r - i r o n s u l p h i d e s t h a t occur as m i n e r a l s .  I t i s a widely disseminated  mineral t h a t o c c u r r s i n m e t a l l i c veins and pockets f r e q u e n t l y a s s o c i a t e d with i r o n p y r i t e s , p y r r h o t i t e , s i d e r i t e , b o r n i t e and o t h e r m i n e r a l s ( M e l l o r , 1947). The mineral sometimes c o n t a i n s gold and s i l ver.  1  Burdick and E l l i s (1917) found t h a t the s p a c e - l a t t i c e o f c h a l c o p y r i t e i s t e t r a g o n a l with the a x i a l r a t i o s a:b:c = 1 :1 :0.985.  The i r o n and copper atoms a r e l o c a t e d so  t h a t they t o g e t h e r form a f a c e - c e n t r e d t e t r a g o n a l l a t t i c e , the planes p e r p e n d i c u l a r t o the t e t r a g o n a l a x i s being made up a l t e r n a t e l y o f copper atoms alone and i r o n atoms alone.  The  s u l p h u r atoms a r e l o c a t e d on a s i m i l a r f a c e c e n t r e d l a t t i c e with the planes o f the sulphur atoms l y i n g midway i n a l l three o f the a x i a l d i r e c t i o n s between the planes o f the i r o n and copper atoms (see F i g u r e 1 ) . The composition o f the mineral can be represented by the formula CuFeS , although i t has been suggested t h a t the atoms have no 2  +  f i x e d valences, but f l u c t u a t e between Cu Fe Cu  + +  Fe  + +  Sg ( P a u l i n g , 1932).  +++  S  2  and  -  Fig. 1  7 -  Space - Lattice of Chalcopyrite  - 8 3.2  MINERAL AND MICROBE INTERACTIONS  Chemical l e a c h i n g o f m e t a l l i c sulphides i s c a r r i e d o u t using f e r r i c - s u l p h a t e s o l u t i o n s i n s u l p h u r i c a c i d media.  The main  r e a c t i o n can be expressed a s :  MS + 2 F e  M"*" + S° + 2 F e  3 +  2  (3.1)  + +  where MS represents a m e t a l l i c s u l p h i d e .  The f e r r i c i r o n i s  reduced t o f e r r o u s i r o n and has t o be s u p p l i e d c o n s t a n t l y . F e r r i c sulphate a l s o hydrolyses a t low pH t o f e r r i c hydroxide according t o :  Fe  3 +  + 3H 0  Fe(0H) + 3H  2  (3.2)  +  3  It was f i r s t suggested (Silverman, 1961, 1963, Duncan, 1973, Lau, 1970) t h a t the r o l e o f b a c t e r i a i n the l e a c h i n g o f sulphide minerals was t o o x i d i z e the f e r r o u s i r o n produced i n r e a c t i o n 3.1 as f o l l o w s : 2 F e + l/20 + 2H + +  2  +  2Fe + H 0 3 +  2  (3.3)  This r e a c t i o n would r e p l e n i s h the f e r r i c i r o n (consumed i n r e a c t i o n 3.1 and 3.2).  The b a c t e r i a would a l s o produce  s u l p h u r i c a c i d from the o x i d a t i o n o f sulphur i n a d d i t i o n t o t h e  - 9 a c i d generated by the h y d r o l y s i s of f e r r i c i r o n according t o the following reaction: S° + 3/2 0  2  + H0 2  bacteria?-  H S0 2  4  (3.4)  Subsequently Duncan (1973) provided evidence o f the d i r e c t a t t a c k o f T h i o b a c i l l u s f e r r o o x i d a n s on the mineral s u r f a c e s of c h a l c o p y r i t e and p y r i t e .  The o x i d a t i o n o f i n s o l u b l e f e r r o u s i r o n and sulphide occurs simultaneously (Landesman, 1966) and independently  (Duncan,  1967) but the r e l a t i v e r a t e s depend on how the c e l l s are grown. I t has been demonstrated t h a t the a b i l i t y o f T h i o b a c i l l u s f e r r o o x i d a n s to grow on i r o n i s c o n s t i t u t i v e , w h i l e the use o f sulphide minerals seems to be s u b j e c t to s p e c i f i c adaptation mechanisms (Touvinen, 1972). A n a l y s i s o f the base r a t i o of DNA obtained from c e l l s growing on d i f f e r e n t s u b s t r a t e s conducted  by  Guay (1976) l e d him to suggest t h a t s u b c u l t u r i n g would cause the production of high metal c o n c e n t r a t i o n r e s i s t a n t s t r a i n s or new s p e c i e s by s e l e c t i o n and mutation mechanisms.  3.2.1  Mechanism of o x i d a t i o n o f f e r r o u s i r o n  I f T h i o b a c i l l u s f e r r o x i d a n s uses the o x i d a t i o n o f ions from the mineral surface o f sulphides to o b t a i n energy then the two p o s s i b l e energy t r a n s f e r mechanisms are t h a t e i t h e r the b a c t e r i a  - 10 a t t a c h t o the surface where the ions can be contacted w i t h enzymes a t the membrane l e v e l o r , the b a c t e r i a use e x t r a c e l l u l a r enzymes t o make these ions a v a i l a b l e and subsequently t r a n s p o r t them i n t o the c e l l .  Attachment o f T h i o b a c i l l u s f e r r o o x i d a n s t o  mineral surfaces has been demonstrated ( R a z z e l l , 1963, McGoran, 1969) and s t u d i e s o f the c e l l envelope o f T h i o b a c i l l u s . f e r r o o x i d a n s (Berry, 1980) have provided evidence f o r both mechanisms. According t o Lundgren (1978) the c e l l envelope o f T h i o b a c i l l u s f e r r o o x i d a n s c o n s i s t s o f three zones:  1) A cytoplasmic membrane which c o n s t i t u t e s the i n n e r l a y e r o f the envelope bordering on the cytoplasm. 2) A c e n t r a l zone comprising a r i g i d l a y e r o f peptidoglycan and a p e r i p l a s m i c space. 3) An outer l a y e r which c o n t a i n s l y p o p o l y s a c c h a r i d e and lipoprotein.  T h i s outer l a y e r might a c t as an i n i t i a l  b i n d i n g s i t e f o r f e r r o u s i r o n (Touvinen, 1972).  The t r a n s f e r  o f e l e c t r o n s i s c a r r i e d out a t the outer membrane o r a t the ++ ++ peroplasmic space l e v e l (2Fe ^ 2Fe + 2e ) w h i l e t h e energy a s s o c i a t e d r e a c t i o n (2e~ + 1/2 0^ + 2H  + v  r  H.,0) i s probably l o c a t e d w i t h i n the i n n e r C  membrane (Lundgren,  1978).  - 11 An e x t r a c e l l u l a r complex from the c u l t u r e f i l t r a t e o f T h i o b a c i l l u s f e r r o o x i d a n s has a l s o been i s o l a t e d (Agathe,  1968)  and suggested to a c t as a substrate f o r f e r r o u s i r o n o x i d a t i o n (donating e l e c t r o n s f o r e l e c t r o n t r a n s p o r t v i a a cytochrome system w i t h i n the c e l l ) or as a s o l v e n t (Touvinen, 1972).  The  cytochrome system r o l e i n i r o n o x i d a t i o n was f i r s t presented by Duncan (1967) who showed t h a t cytochrome i n h i b i t o r s i n h i b i t e d i r o n o x i d a t i o n ; furthermore cytochromes a and b were i s o l a t e d from T h i o b a c i l l u s f e r r o o x i d a n s by Din (1967a) who a l s o  suggested  a ping pong bi bi mechanism f o r i r o n o x i d a t i o n shown i n Figure 2 (Din, 1967b).  3.2.2  Mechanism o f Oxidation o f Sulphides Concerning the mechanism of sulphide o x i d a t i o n , Duncan (1967) found t h a t N-ethyl maleimide (NEM), a t h i o l - b i n d i n g i n h i b i t o r acted as an i n h i b i t o r o f sulphur o x i d a t i o n , showing t h a t t h i o l groups p a r t i c i p a t e d i n sulphur o x i d a t i o n .  A suggested mechanism  i s the o x i d a t i o n of sulphur to s u l p h i t e and to sulphate e i t h e r by a sulphite:cytochrome  c oxidoreductase or with the  intermediate formation of adenylyl sulphate.  The mechanism o f  c o n t a c t o f T h i o b a c i l l u s f e r r o o x i d a n s with sulphur i s not c l e a r but Agathe (1968) suggested t h a t the e x t r a c e l l u l a r complex might a c t as a wetting agent f o r sulphur  granules.  Fe +++  Fe  i  +++. ++ E (Fe ) Fe r  lr  E (Fe  r  1  cytochrome c  reduced cytochrome c  I E (Fe ) + +  • E ( F e ) cytochrome c + +  E (Fe  ) Fe  + + +  E = enzyme  Fig. 2  Ping Pong bi bi mechanism of Fe  ++  -cytochrome c  reductase of Thiobacillus ferrooxidans (Din, 1967b)  )  reduced cytochrome c  E (Fe  + + +  )  - 13 T r i b u t s c h (1981) suggested t h a t b a c t e r i a l a c t i v i t y on s u l p h i d e s was based on the chemical r e a c t i o n o f protons with the metal s u l p h i d e s u r f a c e which then caused a s h i f t i n the e l e c t r o n i c -S-  s t a t e s and produced surface s t a t e s d e s c r i b e d as -SH groups which were removed by b a c t e r i a l a c t i v i t y . The use of sulphide as a source o f energy by T h i o b a c i l l u s f e r r o o x i d a n s has been considered the r a t e l i m i t i n g step i n the l e a c h i n g process.  I t has been f u r t h e r suggested t h a t the high  i n i t i a l l e a c h i n g r a t e s are due t o o x i d a t i o n o f f e r r o u s i r o n atoms a v a i l a b l e a t the s u r f a c e o f the mineral (Duncan, 1967). When these atoms are exhausted, the use o f s u l p h i d e c o n t r o l s the r a t e o f d i s s o l u t i o n o f the mineral ( T r i b u t s c h , 1981). 3.3  MINERAL-MEDIA INTERACTIONS  During the b i o l o g i c a l l e a c h i n g o f sulphide m i n e r a l s a v a r i e t y o f i n o r g a n i c species i s present i n the media a t any time and secondary r e a c t i o n s take place between these s p e c i e s .  McGoran  (1969) found t h a t most o f the f e r r i c sulphate hydrolyzed would p r e c i p i t a t e as a b a s i c f e r r i c sulphate.  At pH <3 j a r o s i t e  w i l l normally form (Duncan, 1972; Sakaguchi, 1976) a c c o r d i n g t o the f o l l o w i n g r e a c t i o n :  3Fe (S0 ) 2  4  3  + 14H 0 2  >  2(H 0)Fe (S0 ) (0H) 3  3  4  2  6  + 5H S0 2  4  (3.5)  - 14 The j a r o s i t e s a l t s o f potassium, sodium, ammonium and hydronium a r e formed when these ions a r e a v a i l a b l e ( P i c k e r i n g , 1968).  The l e a c h i n g o f c h a l c o p y r i t e u s u a l l y y i e l d s 50-60% copper e x t r a c t i o n (Bruynesteyn,  1970).  T h i s incomplete s o l u b i l i z a t i o n  o f copper has been a t t r i b u t e d t o t h e formation o f an impermeable l a y e r o f sulphur around t h e p a r t i a l l y leached c h a l c o p y r i t e ( M i l l e r , 1979; C h a k r a b o r t i , 1979) and t o t h e accumulation o f gangue and p r e c i p i t a t i o n o f b a s i c f e r r i c sulphates and H [Fe ( S 0 ) 4  2  ((Fe(0H)S0  4  '2Fe(0H) ]) (Torma, 1973). T h i s l a t t e r 3  e x p l a n a t i o n i s supported by t h e f a c t t h a t increased y i e l d s can be obtained when t h e r e s i d u e s o f t h e l e a c h i n g a r e subject t o r e - g r i n d i n g and r e - l e a c h i n g (Torma, 1973, 1 977).  The o v e r a l l r e a c t i o n s o f b i o l o g i c a l l e a c h i n g o f c h a l c o p y r i t e can be expressed a s :  2 CuFeS. + 8 1/2 0- + H_S0. b a c t e r i a 3 Fe (S0 ) 2  4  3  + 14 H 0 2  CuFeS + 2 F e ( S 0 ) 2  2  4  2S° + 3 0 + 2H 0 2  2  3  2 CuSO. + Fe.fSO.),, + H 0 o  > 2(H 0)Fe (S0 ) 3  3  4  2  (0H) + 5H S0 g  2  4  > CuS0 + 5 F e S 0 + 2S°  bacteria  4  }  4  2H S0 2  4  The b i o l o g i c a l l e a c h i n g o f c h a l c o p y r i t e can then be considered as a s e r i e s o f events caused by i n t e r a c t i o n s o f b a c t e r i a , t h e mineral and t h e aqueous phase.  (3.6) (3.7) (3.8) (3.9)  - 15 CHAPTER IV FACTORS AFFECTING BIOLOGICAL LEACHING 4.1  PARTICLE SIZE AND SURFACE AREA  The e x t r a c t i o n r a t e i s a f u n c t i o n o f the surface area and w i l l have i t s maximum value a t the s t a r t o f the l e a c h and g r a d u a l l y decrease when accumulation transfer resistance.  o f by-products  i n c r e a s e s the mass  The s u r f a c e area a v a i l a b l e depends on the  p a r t i c l e s i z e and f o r a f i x e d amount o f material increases with the f i n e n e s s o f the m a t e r i a l .  Optimum p a r t i c l e s i z e s f o r  b i o l o g i c a l l e a c h i n g have been proposed a s : <325 mesh (Duncan, 1964), 42 nm (Torma, 1 977), <44 nm ( R a z z e l l , 1963). In theory, the optimum (minimum) p a r t i c l e s i z e w i l l be reached when the p a r t i c l e i s formed by a s i n g l e c r y s t a l (Touvinen, 1972).  4.2  NUTRIENTS  4.2.1 Carbon Source  T h i o b a c i l l u s f e r r o o x i d a n s being an autotroph uses carbon d i o x i d e as a carbon source.  The mechanisms o f carbon f i x a t i o n are the  C a l v i n r e d u c t i v e pentose phosphate c y c l e and the secondary c a r b o x y l a t i o n o f phosphoenolpyruvate (PEP) d e r i v e d from the carbon c y c l e (Touvinen, 1972).  - 16 The carbon d i o x i d e can be s u p p l i e d to the l i q u i d media through gas exchange with the atmosphere or sparged.  For the f i r s t case  Touvinen (1972) showed t h a t the carbon d i o x i d e consumption exceeds the maximum s o l u b l e amount present i n the media a t any time.  For t h i s reason the use o f a carbon d i o x i d e e n r i c h e d  atmosphere i s recommended.  4.2.2 Nitrogen Source  The primary source o f n i t r o g e n f o r T h i o b a c i l l u s f e r r o o x i d a n s i s ammonium i o n .  Mackintosh and Herbet (Duncan, 1972) b e l i e v e d t h a t the bacterium can f i x n i t r o g e n , but attempts to d e t e c t nitrogenase i n T h i o b a c i l l u s sp. f a i l e d (Tsuchiya, 1974).  Silverman (1959) found t h a t the media composition o f h i s 9K medium s u p p l i e d ammonia and other n u t r i e n t s (potassium, magnesium, calcium) i n adequate amounts to support the growth o f up to 5 x 10 4.3  o  cells/mL.  TEMPERATURE  The optimum temperature f o r T h i o b a c i l l u s f e r r o o x i d a n s growing on c h a l c o p y r i t e was determined to be 35°C (Duncan, 1964; Landesman, 1966, Sakaguchi, 1976).  - 17 4.4  pH The optimum pH has been determined t o be 2.0 (Landesman, 1966b) although i t grows well between 2.0-4.5 (Morrison, 1969).  4.5  DISSOLVED OXYGEN  The e f f e c t s o f the d i s s o l v e d oxygen c o n c e n t r a t i o n on the growth o f T h i o b a c i l l u s f e r r o x i d a n s were s t u d i e d by L i u (1973) who c a l c u l a t e d the s o l u b i l i t y o f oxygen i n 9K medium a t 35°C t o be 6.42 mg O2/L and found the c r i t i c a l oxygen c o n c e n t r a t i o n to be 0.29 mg 0 /L. 2  The maximum r e s p i r a t i o n r a t e f o r T h i o b a c i l l u s f e r r o o x i d a n s growing on c h a l c o p y r i t e was reported t o be Q0 (N) = 3200 uL 2  (STP)/mgN-h (Landesman, 1966). During the l e a c h i n g o f m e t a l l i c s u l p h i d e s , oxygen c o n c e n t r a t i o n s have been found to be as low as 0.2-0.55 ppm (Torma, 1973), which suggest a p o s s i b l e oxygen l i m i t a t i o n when oxygen i s provided only by surface exchange.  - 18 4.6  AGE OF CELLS There i s no agreement on the l i t e r a t u r e as t o the best inoculum age.  Some o f the reported values a r e : 3 days (Landesman,  1966), 4 days (Landesman, 1966b), l a t e l a g phase (Torma, 1977) and s t a t i o n a r y phase (Sakaguchi, 1976).  - 19 CHAPTER V MODELLING BIOLOGICAL SYSTEMS  5.1  BACTERIAL KINETICS AND MODELLING  In o r d e r t o use mathematical models t o d e s c r i b e m i c r o b i o l o g i c a l processes, i t i s necessary t o e s t a b l i s h r e l a t i o n s h i p s between the p h y s i c a l l y important v a r i a b l e s which w i l l best d e s c r i b e the phenomena.  M i c r o b i o l o g i c a l processes f o r the most p a r t are d e s c r i b e d by making use o f the r e l a t i o n s h i p s between biomass p r o d u c t i o n , s u b s t r a t e u t i l i z a t i o n and product y i e l d k i n e t i c s .  The knowledge o f these r e l a t i o n s h i p s i s then used t o s e t the process o p e r a t i n g c o n d i t i o n s i n which maximum y i e l d o f the product o f i n t e r e s t would be o b t a i n e d .  Most o f the models used f o r b i o l o g i c a l systems have been d e r i v e d f o r homogeneous systems i n which a l l the r e a c t i n g m a t e r i a l s are found w i t h i n a l i q u i d phase.  In the case o f heterogeneous systems, where the r e a c t i n g m a t e r i a l s are found i n more than one phase, k i n e t i c a n a l y s e s have been made to q u a n t i f y r e a c t i o n r a t e s i n m i c r o b i a l f i l m s ( A t k i n s o n , B. and Fowler, H.W.  1974), l i q u i d hydrocarbon  fermentations (Moo-Young, M. 1975) and f o r immobilized enzymes  - 20 ( B a i l e y , J.E. and O l l i s , O.F., 1977).  These a n a l y s e s are based  on mathematical models p r e v i o u s l y developed f o r n o n - b i o l o g i c a l reactions.  MODELLING BIOLOGICAL LEACHING  E a r l i e r k i n e t i c s t u d i e s o f b i o l o g i c a l l e a c h i n g processes were conducted t o i n v e s t i g a t e the r e l a t i o n s h i p s between e i t h e r product formation and c e l l growth (Landesman, 1966a, 1966b; McGoran, 1969) o r s u b s t r a t e u t i l i z a t i o n and product y i e l d (Torma, 1973; Bruynesteyn, 1974).  The r e s u l t s o f these  i n v e s t i g a t i o n s provided a l i m i t e d understanding o f the i n t e r a c t i o n s between the b a c t e r i a , the mineral and the l i q u i d phase i n a b i o l o g i c a l l e a c h i n g p r o c e s s . The importance o f those aspects r e l a t i n g t o the r o l e o f b a c t e r i a on the l e a c h i n g process has been underestimated f o r the most p a r t .  It has been proposed ( B a i l e y and O l l i s , 1977) t h a t f o r b a c t e r i a growing a t i n t e r f a c e s , two d i f f e r e n t growth r a t e s would be present depending on the r a t i o between c e l l s i z e and s u b s t r a t e size. One growth r a t e would be e v i d e n t f o r the case when the c e l l diameter was l e s s than the s u b s t r a t e diameter, and another growth r a t e when the s u b s t r a t e s i z e was l e s s than the c e l l s i z e and the s u b s t r a t e would be absorbed onto the c e l l s u r f a c e .  - 21 Based on surface area the k i n e t i c a n a l y s i s would show two d i f f e r e n t growth stages.  The f i r s t a f t e r i n t r o d u c t i o n o f the  inoculum when the b a c t e r i a would grow at i t s maximum growth r a t e ; and the second stage i n which growth would occur at expense o f s u b s t r a t e present a t the i n t e r f a c e between phases, when the s u r f a c e s were t o t a l l y covered by b a c t e r i a . These two stages combined produce a growth curve which i s l i n e a r a f t e r the surface i s saturated.  Gormely (1973) found t h a t when the  s u b s t r a t e surface was completely covered by b a c t e r i a , the l e a c h i n g r a t e o f z i n c from a z i n c sulphide concentrate was a f u n c t i o n of the s u b s t r a t e area.  He t r i e d to model t h i s u s i n g  the S h r i n k i n g Core Model o f Levenspiel (1972) f o r the case when chemical r e a c t i o n c o n t r o l s . Based on a constant l e a c h i n g r a t e per u n i t s u r f a c e area, he c a l c u l a t e d the l e a c h i n g r a t e s o f p a r t i c l e s o f d i f f e r e n t s i z e s . Estimates o f product formation using these values gave percentage e x t r a c t i o n s of z i n c which were low by a f a c t o r of t e n compared with the experimental v a l u e s .  He a l s o found t h a t the  r a t e o f l e a c h i n g reached a maximum soon a f t e r i n i t i a t i o n of the metal r e l e a s e , then the l e a c h curve became l i n e a r u n t i l c l o s e t o the completion o f the l e a c h , but he d i d not provide an e x p l a n a t i o n f o r t h i s phenomena.  Sanmugasunderam (1981) a l s o conducted s t u d i e s on z i n c sulphide leaching.  He determined e x p e r i m e n t a l l y the l e a c h i n g r a t e s f o r  d i f f e r e n t p a r t i c l e s i z e s and used them t o p r e d i c t e x t r a c t i o n o f  - 22 z i n c u s i n g the S h r i n k i n g Core Model.  His p r e d i c t i o n s o f  percentage e x t r a c t i o n o f z i n c were equal o r l e s s than the experimental values with a maximum d i f f e r e n c e o f 25 percent. The d i f f e r e n c e was a t t r i b u t e d t o the methods used to estimate the s u r f a c e area o f the p a r t i c l e s and i t s s i z e s .  THE SHRINKING CORE MODEL  For n o n - c a t a l y t i c r e a c t i o n s o f p a r t i c l e s with surrounding f l u i d there are two simple i d e a l i z e d models a c c o r d i n g to Levenspiel (1972):  1) The p r o g r e s s i v e c o n v e r s i o n model i n which the s o l i d r e a c t a n t i s converted c o n t i n u o u s l y and p r o g r e s s i v e l y through the particle.  2) The unreacted-core model i n which the r e a c t i o n zone moves i n t o the p a r t i c l e and the unreacted core s h r i n k s i n s i z e with time.  Given t h a t the b a c t e r i a l a c t i v i t y on s o l i d s u b s t r a t e s i s l i m i t e d to the s u r f a c e s i n c e the b a c t e r i a cannot penetrate the i n t e r i o r of the p a r t i c l e u n t i l the o u t e r l a y e r i s d i s s o l v e d , the unreacted core model suggests i t s e l f f o r a p p l i c a t i o n t o the m o d e l l i n g o f b i o l o g i c a l l e a c h i n g systems.  - 23 There a r e s e v e r a l conversion-time expressions f o r p a r t i c l e s o f d i f f e r e n t s i z e and shapes, and f o r r e a c t i o n s i n which the r a t e c o n t r o l l i n g step i s e i t h e r d i f f u s i o n c o n t r o l l e d o r chemical reaction controlled.  F o r the case o f s p h e r i c a l p a r t i c l e s when chemical r e a c t i o n c o n t r o l s , the i n t r i n s i c r e a c t i o n k i n e t i c data ( o r t h e time needed f o r complete r e a c t i o n o f a p a r t i c l e ) can be obtained f o r monosized p a r t i c l e s .  A t any time the extent o f c o n v e r s i o n o f  the s u b s t r a t e can be c a l c u l a t e d from the p a r t i c l e s i z e d i s t r i b u t i o n data and the o v e r a l l f r a c t i o n reacted f o r any g i v e n size.  The s h r i n k i n g core model was used s u c c e s s f u l l y f o r  m o d e l l i n g the chemical l e a c h i n g o f c h a l c o p y r i t e (Sepulveda, 1978) where the mechanism was suggested t o be an e l e c t r o c h e m i c a l r e a c t i o n i n which t h e conduction o f e l e c t r o n s through the s u l p h u r l a y e r was the r a t e l i m i t i n g step.  L e v e n s p i e l ' s s h r i n k i n g core model p r e d i c t s t h a t with mixed flow o f s i n g l e s i z e s o l i d s , the f r a c t i o n o f the s o l i d t h a t i s converted t o product i n a c e r t a i n time i s given by: Z = l - \f  T  ( 1 - X a ) -e "  t / £  dt  Xa <1  (1)  where: Z i s the average f r a c t i o n a l c o n v e r s i o n o f the s o l i d , T the time r e q u i r e d f o r complete c o n v e r s i o n o f a s i n g l e p a r t i c l e , Xa the f r a c t i o n a l c o n v e r s i o n f o r p a r t i c l e s i n time t + d t , t the mean residence time o f p a r t i c l e s i n the r e a c t o r and t time.  i  - 24 I f chemical r e a c t i o n i s t h e r a t e c o n t r o l l i n g step, then:  j  = (1-Xa)  (2)  1 / 3  S u b s t i t u t i n g and i n t e g r a t i n g we g e t : • Z = 3 ( j ) - 6 ( ^ ) '+ 6 ( ^ ) [l - exp ( - T / t ) ] 2  3  (3)  where T f o r m i c r o b i o l o g i c a l l e a c h i n g i s given by: T  (4)  *Vs  where: p i s t h e d e n s i t y o f t h e c o n c e n t r a t e , do i s t h e diameter o f t h e feed s o l i d p a r t i c l e s , and  r  f  z  s  i s t h e metal e x t r a c t i o n  per u n i t s o l i d surface area.  Thus, i f the metal e x t r a c t i o n r a t e f o r p a r t i c l e s i n each step i s known and t h e p a r t i c l e s i z e d i s t r i b u t i o n o f the concentrate i s a l s o known, Equation 3 can be used t o c a l c u l a t e t h e o v e r a l l e x t r a c t i o n a t any given time.  - 25 CHAPTER VI MATERIALS AND METHODS  BIOLOGICAL LEACHING TECHNIQUES  The b a c t e r i a used f o r the experiments was a s t r a i n o f T h i o b a c i l l u s f e r r o o x i d a n s o r i g i n a l l y i s o l a t e d from the B r i t a n n i a Mine near Vancouver (Razzell and T r u s s e l l , 1963), and r o u t i n e l y maintained on copper c o n c e n t r a t e a t B.C. Research.  The l i q u i d medium used was the medium 9K d e s c r i b e d by Silverman (1959) i n which the copper c o n c e n t r a t e r e p l a c e d F e S 0 as the 4  energy source. The medium had the f o l l o w i n g composition (Table 1 ) .  Table 1 C u l t u r e Media Composition  Component  Concentration (g/L)  (NH ) S0  3.0 0.1 0.5 0.5 0.01  4  2  4  KC1  K2HPO4  MgSOA • 7H?0 Ca ( N 0 ) 3  2  The c h a l c o p y r i t e c o n c e n t r a t e used i n t h i s study was a commercial f l o t a t i o n c o n c e n t r a t e s u p p l i e d by Newmont Mines L i m i t e d , Similkameen D i v i s i o n , P r i n c e t o n , B.C.  - 26 A l l l e a c h i n g experiments were c a r r i e d out on concentrate from a 2 kg grab sample drawn from a b a r r e l c o n t a i n i n g approximately 120 kg of thoroughly mixed concentrate which had p r e v i o u s l y been b a l l m i l l e d a t 55 percent s o l i d s f o r 1 h to 91.8 percent -400 T y l e r mesh, f i l t e r e d and d r i e d a t 60°C.  The a n a l y s i s of the  c o n c e n t r a t e i s t a b u l a t e d i n Table 2.  Table 2 Elemental A n a l y s i s o f the Copper Concentrate (B.C. Research Data) Element  Percentage by Weight  Copper Iron Sulphur Insol.  27.8 28.0 31.1 5.5  T h i s study was c a r r i e d out u s i n g the shake f l a s k l e a c h technique where 7.5 g of concentrate were placed i n bottom-baffled, 250 mL, Erlenmeyer f l a s k s ; 70 mL of i r o n - f r e e 9K medium s o l u t i o n were added and the pH of the suspension was adjusted to 2.0. The f l a s k s were l o o s e l y stoppered with a c o t t o n p l u g , and incubated on a g y r a t o r y shaker (Model 591-70, New  Brunswick  S c i e n t i f i c , N.J.).  The shaker was l o c a t e d i n a dark room with a carbon d i o x i d e e n r i c h e d atmosphere provided by bubbling carbon d i o x i d e through water i n a c o n t a i n e r open to the atmosphere (dry carbon d i o x i d e , Canadian L i q u i d A i r L t d . ) .  - 27 The temperature o f the room was c o n t r o l l e d a t 35°C by means o f a temperature c o n t r o l l e r (Honeywell type RP 908). Before i n o c u l a t i o n t h e f l a s k s were incubated f o r 24 h t o a l l o w f o r a c i d consumption caused by a l k a l i n e gangue present i n the c o n c e n t r a t e ; a f t e r t h i s p e r i o d the pH was a d j u s t e d back t o 2.0 u s i n g s u l p h u r i c a c i d and t h e f l a s k s were s t o r e d i n a r e f r i g e r a t o r a t 4°C u n t i l they were used.  6.1.1 Sampling Techniques  D i f f e r e n t sampling techniques were used depending on t h e type o f a n a l y s i s t o be c a r r i e d out. The s o l u b l e metal content was determined i n l mL o f supernatant drawn from the f l a s k a f t e r 10-15 min s e t t l i n g time.  For the a n a l y s i s o f c e l l s and p a r t i c l e  s i z e , a sample o f s l u r r y was used; t h e sample was o b t a i n e d u s i n g a p i p e t t e t o draw the r e q u i r e d amount o f s l u r r y from the f l a s k , a f t e r i t s c o n t e n t s were thoroughly mixed by shaking.  6.2  Analyses  6.2.1 Metal Leach Rates The copper and i r o n c o n t e n t s o f t h e medium were determined by atomic a b s o r p t i o n spectrophotometry (Atomic A b s o r p t i o n Spectrophotometer, P e r k i n Elmer 306).  - 28 1 mL samples, a f t e r the necessary d i l u t i o n s , were analysed f o l l o w i n g the standard p r a c t i c e s recommended i n Perkin-Elmer's manual  (1973).  The copper e x t r a c t i o n rate was c a l c u l a t e d as the slope o f the l i n e a r p o r t i o n o f the copper c o n c e n t r a t i o n vs. time curve u s i n g a l e a s t squares curve f i t t i n g method.  6.2.2 Hydrogen Ion A c t i v i t y  Measurements o f hydrogen i o n a c t i v i t y (pH) were made by i n t r o d u c i n g a pH e l e c t r o d e i n t o the s l u r r y . T h i s was connected to a pH meter ( F i s h e r Accumet model 61 OA).  6.2.3  Oxidation-Reduction P o t e n t i a l  Measurements o f the Eh p o t e n t i a l were made d i r e c t l y i n the f l a s k u s i n g a platinum e l e c t r o d e connected t o a pH meter (model 28 Radiometer, Copenhagen). 6.2.4 B a c t e r i a l Growth  In o r d e r t o o b t a i n i n f o r m a t i o n on the r o l e o f T h i o b a c i l l u s f e r r o o x i d a n s i n the l e a c h i n g o f m e t a l l i c sulphides and o f t h e r e l a t i o n s h i p between b a c t e r i a l growth and s u b s t r a t e u t i l i z a t i o n , a method o f e s t i m a t i o n o f c e l l numbers o r biomass i s necessary. A l i t e r a t u r e search showed t h a t measurements o f l e a c h i n g  - 29 k i n e t i c s r e l a t e d t o b a c t e r i a l growth have been n e i t h e r c o n s i s t e n t nor systematic.  T h i s a r i s e s from the f a c t t h a t no  s i n g l e proven method of e s t i m a t i n g b a c t e r i a l numbers has been established.  When T h i o b a c i l l u s f e r r o o x i d a n s i s c u l t u r e d on s o l i d s u b s t r a t e s , i t attaches t o the s o l i d surface (Gormely and Duncan, 1974). Because o f t h i s attachment the conventional methods o f e s t i m a t i n g c e l l numbers or c e l l mass ( t u r b i d i t y , dry weight, d i r e c t count) cannot be used. 14 Measurements o f  C0  2  f i x a t i o n , oxygen u t i l i z a t i o n , ATP  and  DNA l e v e l s have been used as e s t i m a t o r s o f biomass, but r e q u i r e the use o f complex instruments and  techniques.  A simple and widely used method f o r e s t i m a t i n g biomass i s the measurement o f organic n i t r o g e n or p r o t e i n content; t h i s l a t t e r method u s u a l l y i n v o l v e s a c o l o r i m e t r i c determination and cannot be used when c o l o r e d substances are present.  Organic  nitrogen  i s normally measured u s i n g the t o t a l K j e l d a h l n i t r o g e n a n a l y s i s (AOAC, 1965), but Gormely and Duncan (1974) found t h a t the ammoniojarosite p r e c i p i t a t e , produced during the l e a c h i n g o f m e t a l l i c s u l p h i d e s , would be i n c l u d e d as b a c t e r i a l n i t r o g e n . They suggested t h a t a method based on the d i f f e r e n c e between the organic and i n o r g a n i c n i t r o g e n contents o f the media would e l i m i n a t e t h i s problem.  - 30 B a c t e r i a l n i t r o g e n i s then c a l c u l a t e d as the d i f f e r e n c e between Kjeldahl n i t r o g e n (organic and i n o r g a n i c nitrogen) and t h e d i s t i l l able ammonium i o n c o n c e n t r a t i o n ( i n o r g a n i c n i t r o g e n ) . Samples o f 1 mL o f s l u r r y were used t o measure the K j e l d a h l n i t r o g e n (Micro-Kjeldahl technique (AOAC, 1965)) and the d i s t i l l able ammonium i o n c o n c e n t r a t i o n was measured by t h e a c i d i m e t r i c method using a p r e l i m i n a r y d i s t i l l a t i o n step (APHA, 1973).  Using L-Alanine (15.69%N) a standard curve f o r the t e s t  was obtained.  Samples c o n t a i n i n g 0.6, 0.8, 1.0, 1.2, 1.4, 1.6  and 1.8 mL o f a s o l u t i o n o f L-Alanine (1 mgN/mL) were analysed f o r n i t r o g e n content.  The standard curve i s shown i n Figure 3.  Twenty i d e n t i c a l samples o f s l u r r y were used t o measure the accuracy o f the t e s t . The probable e r r o r s were c a l c u l a t e d t o be 2.0U f o r the K j e l d a h l t e s t and 3.98% f o r the d i s t i l l a b l e ammonia t e s t .  FRACTIONATION OF CONCENTRATE  Methods f o r s e p a r a t i o n by s i z e o f p a r t i c l e s i n the subsieve range are based on d i f f e r e n c e s i n the terminal s e t t l i n g v e l o c i t i e s o f the p a r t i c l e s .  C e n t r i f u g a t i o n , e l u t r i a t i o n and  sedimentation methods a r e a l l based on the p r i n c i p l e o f g r a v i t y sedimentation.  Fig. 3  Standard curve for nitrogen determination.  - 32 E l u t r i a t i o n grades p a r t i c l e s by means of an upward current f l u i d , u s u a l l y water or a i r .  of  The process i s the reverse o f  g r a v i t y sedimentation ( A l l e n , 1968).  C e n t r i f u g a l methods speed  up the g r a v i t a t i o n a l s e t t l i n g and are useful f o r s i z e s <5pm where the s e t t l i n g times are long. p a r t i c l e s i z e separation  A number o f apparatus f o r  has been designed based on these  p r i n c i p l e s such as the C y c l o s i z e r (hydraulic c y c l o n e e l u t r i a t o r ) , and the Bahco m i c r o p a r t i c l e c l a s s i f i e r ( a i r e l u t r i a t o r combined w i t h a c e n t r i f u g e ) .  These apparatus are  normally used f o r determination" of p a r t i c l e s i z e d i s t r i b u t i o n i n f i n e l y d i v i d e d m a t e r i a l s and are not s u i t a b l e f o r the recovery o f l a r g e amounts of  material.  Beaker decantation (Pryor, 1965)  i s a simple sedimentation  method which does not r e q u i r e any s p e c i a l equipment.  The parameter by which p a r t i c l e s are c l a s s i f i e d i s t h e i r f a l l i n g speed which i s not uniquely r e l a t e d to t h e i r s i z e ( A l l e n , 1968), but beaker d e c a n t a t i o n can be used t o obtain the f r a c t i o n s  and  subsequently t h e i r s i z e could be measured by some other means. The beaker d e c a n t a t i o n method i s based on the d i f f e r e n c e i n the f r e e terminal  v e l o c i t i e s of s p h e r i c a l p a r t i c l e s o f d i f f e r e n t  s i z e s f a l l i n g through a f l u i d a t such r a t e s that the Reynolds' number i s l e s s than  0.2.  - 33 T h e o r e t i c a l s i z e ranges were i n i t i a l l y a r b i t r a r i l y d e f i n e d and the f r e e f a l l i n g v e l o c i t i e s (V) o f t h e s m a l l e s t d e f i n e d p a r t i c l e s i n each f r a c t i o n were c a l c u l a t e d on the b a s i s o f the o v e r a l l d e n s i t y o f the concentrate a c c o r d i n g t o Stokes' Law:  _ d g 2  v  (g-P)  rEr"—  where: V = f r e e f a l l i n g v e l o c i t y cm/sec 3  a = d e n s i t y o f t h e p a r t i c l e g/cm P  = d e n s i t y o f the f l u i d  g/cnr 2  g  = g r a v i t a t i o n a l a c c e l e r a t i o n cm/sec  n = absolute v i s c o s i t y d  g/cm'sec  = Stokes diameter o f t h e p a r t i c l e cm  T h e o r e t i c a l s e t t l i n g times f o r 6 f r a c t i o n s o f diameters  <40um  were then c a l c u l a t e d f o r a l i q u i d height o f 13.5 cm employed i n the beaker d e c a n t a t i o n method. Using a mechanical s t i r r e r 100 g o f the concentrate were d i s p e r s e d i n a l i t r e o f water with the a i d o f a d e f l o c c u l a n t . When t h e suspension was uniform t h e o u t s i d e o f the beaker was sharply tapped with a g l a s s rod covered with rubber t u b i n g , and the suspended p a r t i c l e s allowed t o s e t t l e f o r t h e c a l c u l a t e d time, a f t e r which the supernatant pulp was poured q u i c k l y i n t o a second beaker.  The s e t t l e d p a r t i c l e s remained as a compact cake  - 34 which was a g a i n d i s p e r s e d i n water and the procedure repeated f i v e more times t o e l i m i n a t e the m a t e r i a l o f s m a l l e r s i z e s t h a t was entrapped during s e t t l i n g .  The recovered m a t e r i a l was then  d r i e d and weighed.  The c a l c u l a t e d v e l o c i t i e s and t h e o r e t i c a l s e t t l i n g times f o r t h e d i f f e r e n t f r a c t i o n s obtained a r e shown i n Table 3.  The p a r t i c l e s i z e d i s t r i b u t i o n f o r the b a l l m i l l e d concentrate was obtained from the weight o f the d i f f e r e n t f r a c t i o n s and i s shown i n Table 4. The weight percentage of f r a c t i o n seven was c a l c u l a t e d by d i f f e r e n c e s i n c e t h i s , the f i n e s t f r a c t i o n , forms a very s t a b l e suspension.  The beaker d e c a n t a t i o n method r e q u i r e s the use o f a d e f l o c c u l a n t i n order t o o b t a i n a uniform suspension.  A v a r i e t y of  d i s p e r s i n g agents a r e suggested i n the l i t e r a t u r e : O.T., sodium l i n o l e a t e , sodium a r s e n i t e , sodium  Aerosol  pyrophosphate,  sodium s i l i c a t e , potassium c i t r a t e , sodium o x a l a t e (Skinner e t a l , 1965), aerosol N.Y. (Pryor, 1965) and sodium hexametaphosphate (Pinches, 1972). The c r i t e r i a used t o s e l e c t the d e f l o c c u l a n t were as f o l l o w s :  1) S o l u b i l i t y o f the d i s p e r s a n t i n water. 2) Amount o f f i n e s obtained during the f r a c t i o n a t i o n (the amount o f f i n e s i n d i c a t e s the e f f e c t i v e n e s s o f the d i s p e r s a n t ) .  - 35 Table 3 F r a c t i o n s o f B a l l m i l l e d Concentrate: T h e o r e t i c a l S i z e Ranges, Free F a l l i n g V e l o c i t i e s and S e t t l i n g Times Fraction No.  1 2 3 4 5 6 7  Stokes Diameter f o r C a l c u l a t e d Assumed P a r t i c l e Velocity Density (4.3 g/cm ) (nm) (cm/sec)  .  Theoretical S e t t l i n g Time (sec) For L i q u i d Height=13.5 cm  3  >40 >32 <40 >24 <32 >16 <24 >8 <16 >4 <8 .<4  0.992 0.635 0.357 0.159 0.0396 0.009  13.60 21.26 37.81 84.90 340.90 1500  Table 4 F r a c t i o n s o f B a l l m i l l e d Concentrate: Weight Percentages C o l l e c t e d Fraction No. 1 2 3 4 5 6 7  Weight Percentage  -  11.55 2.33 3.86 11.95 32.62 18.33 19.36  - 36 3) P o s s i b l e e f f e c t s of r e s i d u a l d i s p e r s a n t on the growth of the 1eaching b a c t e r i a . Based on these c r i t e r i a , sodium a r s e n i t e was r u l e d out f o r p o s s i b l e t o x i c i t y . T r i v a l e n t a r s e n i c ( a r s e n i t e s ) are reported t o be t o x i c f o r b a c t e r i a ( P o r t e r , 1946).  R a z z e l l ' s (1963) and Landesman's (1966) s t u d i e s have shown t h a t c a r b o x y l i c a c i d s and f a t t y a c i d s are i n h i b i t o r s of i r o n o x i d a t i o n by T h i o b a c i l l u s f e r r o o x i d a n s ; based on these s t u d i e s sodium l i n o l e a t e , potassium c i t r a t e and sodium o x a l a t e were a l s o r u l e d out.  The use of sodium s i l i c a t e was not considered due t o  the d i f f i c u l t y o f i t s removal by simple washing a f t e r the fractionation. 6.3.1  D e f l o c c u l a n t S e l e c t i o n Tests  The f i r s t d i s p e r s i n g agent used was aerosol G.P.G. (sodium d i o c t y l s u l f o s u c c i n a t e i n ethanol and water.  Cyanamid Canada  Inc.).  A 0.1% s o l u t i o n o f t h i s d i s p e r s a n t was prepared and added to a l e a c h t e s t to detect any e f f e c t s on the a b i l i t y of the b a c t e r i a t o e x t r a c t copper from c h a l c o p y r i t e .  The r e s u l t of t h i s t e s t i s  shown i n Figure 4 compared with a s i m i l a r t e s t i n which no d i s p e r s a n t was added.  -  37  -  ispersant  i  2  r-  r  4  6 TIME (DAYS)  ispersant  ispersant  2  4  6 TIME (DAYS)  . 4  Effect of aerosol in the growth of T._ ferrooxidans  - 38 The r e s u l t s i n d i c a t e t h a t t h e copper e x t r a c t i o n over a s i x day p e r i o d was only 20% o f t h e e x t r a c t i o n i n t h e blank; t h i s d i f f e r e n c e was a t t r i b u t e d t o t h e presence o f t h e d i s p e r s a n t aerosol G.P.G. C o n s i d e r i n g t h a t t h e a e r o s o l ' s chemical s t r u c t u r e has two l a r g e hydrocarbon chains with hydrophobic p r o p e r t i e s , t h e molecule then has an a f f i n i t y f o r organic s o l v e n t s .  Acetone, chloroform  and isopropanol were used t o remove t h e r e s i d u e s o f t h e aerosol from t h e f r a c t i o n a t e d concentrate.  10 g o f concentrate were  placed i n a separatory funnel and 50 mL o f t h e s o l v e n t added. The funnel was v i g o r o u s l y shaken f o r 10 minutes, and t h e phases allowed t o separate; then a small p o r t i o n o f concentrate was withdrawn and t e s t e d f o r MBAS (Methylene Blue A c t i v e Substances) (APHA, 1973).  The MBAS t e s t i n d i c a t e d t h a t t h e f o l l o w i n g  amounts o f d i s p e r s a n t had been e x t r a c t e d by t h e v a r i o u s s o l v e n t s used:  acetone-96%, chloroform-95% and isopropanol-98%.  Isopropanol was the most e f f e c t i v e .  Concentrate t r e a t e d with isopropanol was then washed with d i s t i l l e d water and d r i e d a t 50°C f o r 24 h, then t h e b i o l o g i c a l l e a c h t e s t was repeated.  T h i s time t h e copper e x t r a c t i o n was  33% o f t h a t o f t h e blank, showing t h a t t h e aerosol  maintained  i t s i n h i b i t o r y e f f e c t even a t very low c o n c e n t r a t i o n s .  - 39 At t h i s p o i n t two other d i s p e r s i n g agents were c o n s i d e r e d ; sodium hexametaphosphate and Tween 40 (polyoxyethylene (20) s o r b i t a n monopalmitate) both from J.T. Baker Chemical Co., N.J. B i o l o g i c a l l e a c h i n g i n h i b i t i o n t e s t s using 0.1% v/v media o f d i s p e r s a n t showed no e f f e c t s on the copper e x t r a c t i o n r a t e by Tween 40; hence t h i s d i s p e r s i n g agent was used i n the f r a c t i o n a t i o n procedures.  Subsequent t e s t s with f r a c t i o n s o f  concentrate obtained by f r a c t i o n a t i o n showed an unexpected r e t a r d a t i o n i n t h e copper e x t r a c t i o n r a t e , suggesting t h a t b i n d i n g o f the d i s p e r s i n g agent t o the s u r f a c e o f t h e mineral during f r a c t i o n a t i o n had taken p l a c e and a f f e c t e d i t s l e a c h i n g properties.  T h i s apparently d i d not occur d u r i n g the e a r l i e r  inhibition test.  A more d r a s t i c treatment t o remove r e s i d u a l Tween 40 was then implemented based on the 0ECD method (1976).  This method i s  used f o r t h e e x t r a c t i o n o f s u r f a c t a n t s from detergents, and i n v o l v e s a binary e x t r a c t i o n with isopropanol i n t h e presence o f K C0 . 2  3  Using t h i s method, 99% e x t r a c t i o n s o f the d i s p e r s a n t  were obtained.  To remove I^CO^, a s e r i e s o f washing steps  followed by c e n t r i f u g a t i o n were used u n t i l the l i q u i d medium showed no s i g n s o f carbonates; no i n h i b i t i o n occurred when t h e m a t e r i a l obtained by t h i s procedure was subjected t o l e a c h i n g .  - 40 6.4  PARTICLE SIZE MEASUREMENT The measurement o f p a r t i c l e s i z e i n t h e subsieve range has been analyzed e x t e n s i v e l y  i n the l i t e r a t u r e (Schweyer and Work, 1941;  Loveland, 1958; Chamot and Mason, 1938; A l l e n , 1968).  The most  widely used methods a r e based on measurements o f p h y s i c a l p r o p e r t i e s o f the material  p r e v i o u s l y c o r r e l a t e d with the s i z e ,  such a s : l i g h t s c a t t e r i n g , a b s o r p t i o n o f l i g h t , f i l t r a t i o n by media o f known pore s i z e , sedimentation, c e n t r i f u g i n g , e t c .  The  s i z e that i s measured w i l l then depend on t h e method employed.  The use o f the microscope i s the only method i n which the i n d i v i d u a l p a r t i c l e s a r e measured and i t i s often used as a standard f o r comparison with other methods.  Direct observation  and measurement can be made down t o l e s s than one micron  (Pryor,  1965).  The measured p a r t i c l e s i z e depends on the p a r t i c l e shape. The diameter o f an equidimensional p a r t i c l e (sphere o r cube) has a s i n g l e value but f o r i r r e g u l a r p a r t i c l e s the s i z e may be expressed by any one o f several dimensions (Pryor, 1965) such as the length o f the c i r c u m s c r i b i n g  rectangle, when the p a r t i c l e i s  i n i t s most s t a b l e p o s i t i o n ; by the diameter o f a sphere having the same terminal  v e l o c i t y ; by the diameter o f a sphere having  the same volume; and so f o r t h .  - 41 M a r t i n ' s diameter i s the simplest expression of the diameter o f i r r e g u l a r p a r t i c l e s and i s s u f f i c i e n t l y accurate when averaged f o r a large number of measurements.  I t i s the horizontal  dimension b i s e c t i n g the projected area of the p a r t i c l e as shown i n Figure 5 (Welcher, 1963).  Experimental comparison of various  proposed diameters are reported (Chamot and Mason, (1938) t o have shown s a t i s f a c t o r y agreement between M a r t i n ' s diameter and the three actual dimensions of the i n d i v i d u a l p a r t i c l e s ( l e n g t h , breadth and t h i c k n e s s ) .  Fig.  5  MARTIN'S DIAMETER  d  d  d  d  In t h i s study, a microscope eyepiece micrometer c a l i b r a t e d f o r the objective i n use against a stage micrometer (Ann Arbor, U . S . A . ) , was used t o measure M a r t i n ' s diameter i n a Leitz-Wetzlar microscope (HM-Lux, Germany).  . 1 Preparation of Concentrate f o r Size Measurement  A small sample of concentrate was placed on a microscope s l i d e and a few drops of water were added.  The powder was worked i n t o  the f l u i d using a small glass rod and a cover s l i p was put  - 42 c a r e f u l l y i n p l a c e so as to exclude a i r bubbles.  Two s l i d e s  were prepared f o r each sample and observed under the microscope.  The number o f p a r t i c l e s counted was 100 c o n s i d e r i n g the standard p r a c t i c e o f a model c l a s s c o n t a i n i n g a t l e a s t 25 p a r t i c l e s .  For  the narrow range o f s i z e s examined the count w i l l g i v e a standard e r r o r  10  i n the mean s i z e ( A l l e n , 1968; ASTM, 1974).  The average p a r t i c l e diameter was then c a l c u l a t e d by the Sauter mean (mean diameter based on s u r f a c e ) u s i n g the f o l l o w i n g expression (Coulson and Richardson, 1976). ds =  z z  z S  3  z  where: n = number o f p a r t i c l e s d = measured Martin's diameter S = t o t a l s u r f a c e o f u n i t mass m a t e r i a l The computed p a r t i c l e s s i z e s f o r the d i f f e r e n t f r a c t i o n s o f c o n c e n t r a t e are shown i n Table 5.  - 43 -  Table 5 Average P a r t i c l e S i z e i n F r a c t i o n a t e d Concentrate Particle Size  Size Class Limits Based on Stokes diameter >32 >24 >16 >8 >4  <4  vim  <40 <32 <24 <16 <8  7.41 5.48 3.56 2.52 1.78 1.07  The values o f p a r t i c l e s i z e s were a l s o used t o c a l c u l a t e t h e s p e c i f i c s u r f a c e area o f t h e p a r t i c l e s with t h e f o l l o w i n g expression assuming s p h e r i c a l p a r t i c l e s : 6  6.4.2 Scanning E l e c t r o n Microscope Techniques For p r e - l e a c h i n g observations samples o f c h a l c o p y r i t e were obtained from several f r a c t i o n s o f the dry-concentrate. f o r p o s t - l e a c h i n g observations were obtained as f o l l o w s :  Samples A t the  end o f t h e l e a c h t h e contents o f the f l a s k s were f i l t e r e d through Whatman f i l t e r paper #1 and d r i e d i n a i r i n a covered c o n t a i n e r f o r 48 h a t 35°C.  The specimens t o be examined i n t h e  scanning e l e c t r o n microscope were drawn from t h e s u r f a c e o f t h e f i l t e r using a m e t a l l i c s p a t u l a .  - 44 A l l specimens were then mounted on aluminium  stubs  (14 mm x 14 mm) over a t h i n l a y e r o f g r a p h i t e i n ethanol (20% g r a p h i t e (Dag 154) Acheson C o l l o i d s Canada Limited) and coated with g o l d u s i n g a Hummer g o l d s p o t t e r c o a t e r f o r 4 minutes a t 170 m i l l i t o r r vacuum and 9 m i l l i a m p e r s D.C. The mounted specimens were examined i n a scanning e l e c t r o n microscope (Autoscan number 26, Etec Corporation) operated a t 20 kV i n the secondary e l e c t r o n emission mode.  - 45 CHAPTER VII EXPERIMENTAL RESULTS AND DISCUSSION  SELECTION OF THE INOCULUM AGE When f r e s h medium i n a c l o s e d system i s i n o c u l a t e d with c e l l s a number o f changes take p l a c e .  A f t e r a l a g phase where no  i n c r e a s e i n c e l l numbers occurs, t h e c u l t u r e enters a phase o f exponential growth u n t i l some n u t r i e n t i s exhausted o r some by-products reach t o x i c l e v e l s .  At t h i s p o i n t t h e r a t e s o f  death and growth a r e i n e q u i l i b r i u m and t h e c u l t u r e i s i n t h e s t a t i o n a r y phase. What f o l l o w s i s a d e c l i n e phase when t h e death r a t e exceeds t h e growth r a t e .  The length o f t h e l a g phase i s r e l a t e d t o t h e age and s i z e o f t h e inoculum. What i s meant by age i s t h e time between t h e s t a r t o f growth o f the parent c u l t u r e and t h e t r a n s f e r o f t h e inoculum t o t h e subculture (Dean and Hinshelwood, 1966).  Bailey  and O l l i s (1977) found t h a t when young c e l l s a r e t r a n s f e r r e d , s h o r t l a g phases a r e obtained and when o l d e r populations a r e t r a n s f e r r e d long l a g phases r e s u l t , because o l d e r  populations  have a slower growth r a t e due t o n u t r i e n t d e p l e t i o n i n t h e media and/or t h e accumulation o f t o x i c products.  It i s c l e a r then, t h a t t h e i n t r o d u c t i o n o f an a c t i v e c u l t u r e growing i n t h e exponential phase w i l l reduce t h e l a g phase. F o r t h i s study t h e s e l e c t i o n o f t h e inoculum age was based on t h e  - 46 r e s u l t s o f a s e r i e s o f batch leaches c a r r i e d o u t u s i n g i n o c u l a o f v a r i o u s ages between 1-10 days.  The copper c o n c e n t r a t i o n was  monitored during t h e leaches and t h e r e s u l t s used as a measure o f inoculum performance.  The r e s u l t s a r e shown i n F i g u r e 6a, b.  F i g u r e 6a shows t h e time elapsed between i n o c u l a t i o n and t h e maximum copper c o n c e n t r a t i o n found i n s o l u t i o n f o r t h e d i f f e r e n t inoculum ages.  The gradual decrease i n t h e number o f days  needed t o reach t h e maximum copper c o n c e n t r a t i o n i n d i c a t e d a s h o r t e n i n g o f t h e l a g phase (assuming a d i r e c t r e l a t i o n s h i p between copper e x t r a c t i o n and c e l l growth), and hence a minimum o f 6 days between t r a n s f e r s would ensure s h o r t l a g times. Figure 6b shows t h e copper e x t r a c t i o n r a t e as a f u n c t i o n o f t h e inoculum age.  The curve shows t h a t t h e h i g h e s t r a t e s were  obtained f o r between 6 and 8 day o l d i n o c u l a . Based on these r e s u l t s a s e r i a l s u b c u l t u r e was s t a r t e d , and r o u t i n e l y performed throughout t h e experimental study, making t r a n s f e r s every 8 days t o provide i n o c u l a f o r a l l t h e experiments.  7.2  BACTERIAL GROWTH KINETICS  7.2.1  B a c t e r i a l Growth  In t h i s s e c t i o n t h e r e l a t i o n s h i p between b a c t e r i a l growth and copper e x t r a c t i o n i s explored.  I t i s shown t h a t b a c t e r i a l  growth was l i m i t e d t o t h e e a r l y stages o f l e a c h i n g .  - 47 -  T  1  1  1  1  1  1  1  1 2 3 4 5 6 7 8 9  1  r  10  INOCULUM AGE (DAYS) Fig. 6  Effect of the inoculum age on copper extraction  - 48 An estimate o f the mineral s u r f a c e area c a l c u l a t e d from the mean diameter of the p a r t i c l e s and t h e i r s i z e d i s t r i b u t i o n i n the concentrate was used t o c a l c u l a t e the f r a c t i o n o f the t o t a l surface area a v a i l a b l e f o r b a c t e r i a l coverage.  In order t o c a l c u l a t e the number o f b a c t e r i a present a t any given time during the l e a c h , the n o n - d i s t i l l a b l e n i t r o g e n (n.d.n.) c o n c e n t r a t i o n was measured during fermentation i n duplicate  experiments.  The n.d.n. measurements were c o r r e l a t e d to b a c t e r i a l numbers using the values d e r i v e d by Gormely and Duncan (1974). 10 workers c a l c u l a t e d t h a t 10 c e l l nitrogen.  These  c e l l s are e q u i v a l e n t to 0.191  mg  T h i s g i v e s a lower estimate o f b a c t e r i a l numbers  compared to those c a l c u l a t e d using Beck's y i e l d (Touvinen,  1972)  who reported a n i t r o g e n content o f only 0.033 mgN/10^ c e l l s . A t y p i c a l growth curve f o r T h i o b a c i l l u s f e r r o o x i d a n s growing on c h a l c o p y r i t e i s shown i n Figure 7 with the data t a b u l a t e d i n Table I (Appendix I ) . The curve shows a l a g phase o f 20 h followed by an exponential i n c r e a s e i n c e l l numbers f o r 30 h and a s t a t i o n a r y phase up to 200 h.  o  1400  t-H  o x 1200  A  1000  A  00  IX)  800 c_>  2  600 400  A  200  A T  20  40  1  1  60  80  1  1—  1  100  120  140  r  160  180  100  TIME (HOURS)  Fig. 7  B a c t e r i a l Growth o f J_ L f e r r o j ^ i d _ a n s i n c o p p e r c o n c e n t r a t e . • B a c t e r i a l numbers vs time.  - 50 7.2.2  Copper E x t r a c t i o n A s e r i e s o f experiments were conducted t o i n v e s t i g a t e the r e l a t i o n s h i p between b a c t e r i a l growth and copper e x t r a c t i o n . C e l l numbers, copper c o n c e n t r a t i o n , i r o n c o n c e n t r a t i o n , Eh, and pH were c a r e f u l l y monitored i n b i o l o g i c a l leaches.  Figure 8  shows the average values determined i n t h r e e o f such experiments.  The data are presented i n Table II (Appendix I ) .  A n a l y s i s o f F i g u r e 8 shows t h a t the exponential growth was  again  l i m i t e d t o the e a r l y stages o f the l e a c h .  During t h i s p e r i o d a c l o s e c o r r e l a t i o n between growth and copper and i r o n e x t r a c t i o n s was found.  McGoran (1969) conducted  s i m i l a r experiments and found t h a t the l o g a r i t h m i c r a t e s o f copper r e l e a s e and c e l l m u l t i p l i c a t i o n were i d e n t i c a l , but he f u r t h e r s t a t e d t h a t copper e x t r a c t i o n c o u l d be used as an i n d i c a t o r o f growth r a t e .  Figure 8 i n d i c a t e s t h a t the bulk o f  the copper e x t r a c t i o n occurs d u r i n g the l a t e l a g phase and s t a t i o n a r y phase.  This d i f f e r e n c e i n the growth p a t t e r n s o f  T h i o b a c i l l u s f e r r o o x i d a n s a r i s e s from the f a c t t h a t McGoran estimated h i s b a c t e r i a l c o n c e n t r a t i o n s using a K j e l d a h l a n a l y s i s which probably i n c l u d e d the n i t r o g e n content o f j a r o s i t e p r e c i p i t a t e formed during the l e a c h .  -  T  u  1  1  2  4  6  8  51  -  1 10  1 12  l 14  i 16  " 18  • 20  TIME (DAYS)  Fig. 8  Bioleaching data average f o r three experiments Soluble copper and iron concentrations Cell  numbers,  pH, Eh  - 52 During the leach the pH g r a d u a l l y decreased, i n d i c a t i n g bacterial activity.  A c i d production stops (minimum pH i s  reached) a t the same time t h a t the maximum copper c o n c e n t r a t i o n i s reached. The Eh i n c r e a s e i s a l s o an i n d i c a t o r of b a c t e r i a l a c t i v i t y .  It  i s caused by the o x i d a t i o n o f f e r r o u s i r o n . T h i s r i s e i n p o t e n t i a l has been p r e v i o u s l y r e l a t e d to l o g a r i t h m i c growth (Touvinen, 1972).  The t r a n s i t i o n from the l o g a r i t h m i c growth phase to the s t a t i o n a r y phase shown i n Figures 7 and 8 could be caused by a number o f environmental accumulation  f a c t o r s . N u t r i e n t exhaustion  and  of by-products are known t o cause the change from  l o g a r i t h m i c growth to s t a t i o n a r y phase i n m i c r o b i a l c u l t u r e s .  For the b i o l e a c h i n g o f c h a l c o p y r i t e , the by-products  are  s u l p h u r i c a c i d and j a r o s i t e s . The pH values i n d i c a t e t h a t the production of s u l p h u r i c a c i d had lowered the pH but i t was  still  w i t h i n the normal range o f growth of T h i o b a c i l l u s f e r r o o x i d a n s .  J a r o s i t e p r e c i p i t a t i o n becomes s i g n i f i c a n t above Eh values o f 500 mv (Torma, 1977), where most of the i r o n present i s i n the f e r r i c form from which hydroxides and j a r o s i t e s are formed. T h i s change o f growth phase from l o g a r i t h m i c to s t a t i o n a r y occurred when the Eh values were below 500 mV and so cannot be a t t r i b u t e d to t h i s phenomena.  - 53 It i s assumed then, t h a t n u t r i e n t l i m i t a t i o n s caused t h e change o f growth phase, i n t h i s case t h e a v a i l a b i l i t y o f an energy source which f o r t h e b i o l e a c h i n g o f c h a l c o p y r i t e i s provided by t h e concentrate  surface.  Above 500 mV o f Eh t h e p r e c i p i t a t i o n o f j a r o s i t e s becomes s i g n i f i c a n t and may e x p l a i n t h e decrease i n t h e copper e x t r a c t i o n r a t e between days 4-6 and u l t i m a t e l y t h e t e r m i n a t i o n of copper e x t r a c t i o n .  This may be because t h e ammonium and  potassium ions a r e s t r i p p e d out o f s o l u t i o n when t h e j a r o s i t e s are formed.  The j a r o s i t e t h a t p r e c i p i t a t e s over t h e mineral  s u r f a c e (see photographs o f leached p a r t i c l e s , s e c t i o n 7.5), w i l l cause mass t r a n s f e r l i m i t a t i o n s o f carbon d i o x i d e , oxygen and n u t r i e n t s from t h e bulk o f t h e s o l u t i o n t o t h e b a c t e r i a attached t o t h e s u r f a c e .  An estimate o f t h e magnitude o f p r e c i p i t a t e s o f b a s i c f e r r i c sulphates and j a r o s i t e can be obtained when the i r o n and copper c o n c e n t r a t i o n s i n s o l u t i o n a r e compared. The chemical composition  o f the c h a l c o p y r i t e CuFeSg i n d i c a t e s  t h a t equal amounts o f copper and i r o n a r e produced when t h e mineral d i s s o l v e s .  F i g u r e 9 shows t h e copper/iron r a t i o  (average values f o r 3 leaches) and i n d i c a t e s t h a t h y d r o l y s i s and p r e c i p i t a t i o n r e a c t i o n s a r e t a k i n g p l a c e because t h e r a t i o i s always >1.  -  2.4  54  -  J  — i  6  1  8  1  10  1—  12  TIME (DAYS)  Fig. 9  Average Values of Copper/Iron Ratios for 3 Leaches  - 55 7.2.3 Surface Area U t i l i z a t i o n  The t o t a l s u r f a c e area f o r t h e 7.5 g o f concentrate used i n each of the b i o l o g i c a l leaches was c a l c u l a t e d from the mean diameters of t h e p a r t i c l e s and t h e s i z e d i s t r i b u t i o n o f t h e c o n c e n t r a t e , 2 and has a value o f 4.96 m  (Table I I I , Appendix I ) . The  average dimensions o f one bacterium ( l e n g t h 1.0 urn and breadth 0.6 urn) can be used t o c a l c u l a t e t h e s u r f a c e area covered by a s i n g l e bacterium, assuming an e l l i p t i c a l form f o r i t s p r o j e c t e d area.  Thus, t h e s u r f a c e area covered by a s i n g l e bacterium 2  would be approximately 0.5 ym . The number o f b a c t e r i a times t h e s u r f a c e area used by one b a c t e r i a would then g i v e an estimate o f the s u r f a c e covered by b a c t e r i a a t any given time. It i s assumed t h a t a l l b a c t e r i a are found a s s o c i a t e d w i t h t h e mineral and none can be found i n the l i q u i d phase.  Pinches  (1972) has shown t h i s t o be t r u e up t o t h e end o f t h e exponential phase. F i g u r e 10 shows t h e percentage o f t h e t o t a l s u r f a c e covered by b a c t e r i a a g a i n s t time f o r the average v a l u e s o f c e l l numbers o b t a i n e d i n t h r e e l e a c h e s . The data a r e presented i n Table IV (Appendix I ) . The curve i n F i g u r e 10 shows a maximum coverage a f t e r 4 days when 88% o f the s u r f a c e i s covered by b a c t e r i a .  The chemical  - 56 -  Fig. 10  Bacterial coverage of the mineral surface  - 57 a n a l y s i s data o f t h i s concentrate showed t h a t 86.9% o f t h e m a t e r i a l i s s u l p h i d e , which suggests t h a t t h e b a c t e r i a a t t a c h s e l e c t i v e l y t o t h e sulphide phase over gangue m a t e r i a l s present i n t h e concentrate. Berry (1978) showed t h a t t h e attachment o f T h i o b a c i l l u s f e r r o o x i d a n s t o low-grade waste-rock s u r f a c e s was s p e c i f i c t o exposed F e S and CuFeS r e g i o n s . 2  2  Myerson and K l i n e (1983),  a r r i v e d a t t h e same c o n c l u s i o n a f t e r c a l c u l a t i n g a s u r f a c e 2 u t i l i z a t i o n value o f 122 ym / c e l l f o r coal with a s u l p h i d e content o f 1.66% when they compared t h i s value with t h e sulphide 2  u t i l i z a t i o n value per bacterium (0.5 ym ). Our experiments then i n d i c a t e t h a t most o f t h e a v a i l a b l e s u r f a c e i s covered by b a c t e r i a 4 days a f t e r i n o c u l a t i o n and f u r t h e r supports t h e hypothesis o f a change i n growth phase due t o s u r f a c e area l i m i t a t i o n s . 7.2.4 Leaching i n the Absence o f B a c t e r i a To e v a l u a t e t h e c o n t r i b u t i o n o f chemical l e a c h i n g t o t h e copper e x t r a c t i o n obtained i n the b i o l o g i c a l l e a c h i n g experiments, a s t e r i l e l e a c h was s e t up.  - 58 No b a c t e r i a were introduced and s t e r i l e c o n d i t i o n s were provided by adding phenol which i s a known germicide.  P o r t e r (1946)  reported t h a t a r a t i o o f 1:70 v/v phenol:media was e f f e c t i v e i n 10 minutes f o r Staphylococcus four s p e c i e s he examined.  aureus, the most r e s i s t a n t o f t h e  This same r a t i o was used i n t h e  s t e r i l e c o n t r o l ; microscopic examination  f a i l e d t o d e t e c t any  microorganism i n t h e media c o n t a i n i n g phenol.  The r e s u l t s f o r  the a n a l y s i s o f samples f o r t h e s t e r i l e run a r e t a b u l a t e d i n Table V i n Appendix I and shown i n Figure 11.  During t h e s t e r i l e run t h e Eh values remained constant, an i n d i c a t i o n o f the low l e v e l s o f o x i d a t i o n o f f e r r o u s i r o n . Duncan (1972) i n d i c a t e d t h a t there i s seldom any f e r r o u s i r o n present when t h e b a c t e r i a a r e a l i v e . The h y d r o l y s i s o f f e r r i c i r o n which generates a c i d was t h e r e f o r e almost n i l and no a c i d was formed.  The pH curve i n d i c a t e s  consumption o f a c i d , probably due t o a l k a l i n e gangue and t o chemical e x t r a c t i o n o f copper and i r o n . Given t h e low values o f copper e x t r a c t i o n (2.95%) obtained during t h e s t e r i l e experiment, no account f o r chemical l e a c h i n g was introduced i n the c a l c u l a t i o n s o f copper e x t r a c t i o n i n t h e b i o l o g i c a l l e a c h i n g experiments.  -  Fig. 11  59  -  Chemical Leaching of Chalcopyrite  - 60 EFFECTS OF PARTICLE SIZE ON THE LEACHING OF COPPER A s e r i e s o f experiments u s i n g monosized f r a c t i o n s o f copper concentrate was conducted.  Results of these experiments  t a b u l a t e d i n Tables VI-XI (Appendix  are  I ) , and the equations f o r  copper c o n c e n t r a t i o n as a f u n c t i o n of time, f i t t e d by the method o f l e a s t squares are g i v e n .  F i g u r e 12 presents the r e s u l t s f o r  the l e a c h i n g o f copper using c h a l c o p y r i t e concentrates the f o l l o w i n g mean p a r t i c l e s i z e s : 1.07, 1.78, and 7.41  having  2.52, 3.56,  5.48  nm.  Experiments u s i n g the 3.56 nm f r a c t i o n were conducted i n t r i p l i c a t e t o v e r i f y the accuracy of the measurements, and the r e s u l t s are p l o t t e d together i n Figure 12.  The s e r i e s o f curves  obtained i n d i c a t e s t h a t the amount of copper e x t r a c t e d i n c r e a s e d when the p a r t i c l e s i z e decreased from 5.48 nm t o 1.07  nm,  with the maximum being obtained f o r the 1.07 nm f r a c t i o n . extent o f the copper e x t r a c t i o n was maximum f o r the 1.07  The nm  f r a c t i o n and c o u l d not be reached with any other p a r t i c l e s i z e . This suggests t h a t f o r the case when the ore p a r t i c l e s have s i z e s s m a l l e r o r equal t o the bacterium s i z e the attachment of p a r t i c l e s t o the c e l l s u r f a c e i n c r e a s e s the l e a c h i n g r a t e . M i c r o s c o p i c examination o f the c u l t u r e showed a t a n g l e d mass o f b a c t e r i a and p a r t i c l e s i n the fermentation broth which was not found f o r any other p a r t i c l e s i z e .  - 61 -  - i  20  1  1  1  1  1  1  1  1  1  1  40.  60  80  100  120  140  160  180  200  220  1  240  TIME (h)  Fig. 12  Effect of particle size on the copper extraction  1  260  - 62 -  The e x t r a c t i o n rate was approximately for the larger sizes  (7.41, 5.48  and  constant during t h e l e a c h 3.56  urn),  rate curve was obtained f o r the s m a l l e r s i z e s 1.07  while a two  (2.52, 1.78  and  um).  Two rate copper e x t r a c t i o n curves would r e s u l t from t h e two phases o f growth o f the b a c t e r i a .  The f i r s t rate would occur  when t h e c e l l s a r e growing l o g a r i t h m i c a l l y and surface i s available.  The second lower rate would occur f o r the s t a t i o n a r y  phase when t h e surface becomes l i m i t i n g .  For the p a r t i c l e s i n the l a r g e r s i z e s  (>3.56 ym)  the surface  was r a p i d l y covered with b a c t e r i a during the f i r s t few hours o f the l e a c h and surface l i m i t a t i o n occurred e a r l i e r i n the l e a c h ; consequently  the curves showed a trend towards a s i n g l e copper  extraction rate. Copper e x t r a c t i o n r a t e s a s a f u n c t i o n o f t h e p a r t i c l e diameter are shown i n Figure 13. The copper e x t r a c t i o n rate i n c r e a s e s as the p a r t i c l e s i z e decreases.  The upper p a r t o f Figure 13 shows how the surface i n c r e a s e s with reduction o f t h e p a r t i c l e diameter.  I f t h e e x t r a c t i o n rate was  a unique f u n c t i o n o f t h e s u r f a c e area a v a i l a b l e , then t h e e x t r a c t i o n rate would increase i n a s i m i l a r f a s h i o n .  A plot of  copper e x t r a c t i o n versus s p e c i f i c surface would produce a straight line. data.  Figure 14 shows such a p l o t f o r the experimental  - 63 11  1  i  1 Fig. 13  1  1  2  3  1  1  1  1  4 5 6 7 PARTICLE DIAMETER ( ym)  Copper extraction rates as a function of the particle diameter  r  8  —i .2  1  1  1  .4  .6  .8  1  1.0  —  1  1.2  SPECIFIC SURFACE AREA ( m / g ) 2  F i g . 14  Extraction rate as a function of the surface area.  r~ 1.4  - 65 Using f r a c t i o n s o f d i f f e r e n t s i z e s o f z i n c sulphide  concentrate  i n s i m i l a r experiments Sanmugasunderam (1981) found a p r o p o r t i o n a l i n c r e a s e between z i n c e x t r a c t i o n r a t e and s p e c i f i c 2 s u r f a c e area up t o s p e c i f i c surface area values o f 1.1 m /g and reported t h a t beyond t h i s value i n c r e a s e s i n s p e c i f i c surface area had almost no e f f e c t on z i n c e x t r a c t i o n r a t e .  The  r e s u l t s o f t h i s study show t h a t the maximum i n c r e a s e i n the copper e x t r a c t i o n r a t e of c h a l c o p y r i t e occurred when the 2 s p e c i f i c surface area increased from 0.78 to 1.3 m /g. 7.3.1  Changes i n the P a r t i c l e S i z e D i s t r i b u t i o n During  Leaching  The p a r t i c l e s i z e used to c h a r a c t e r i z e each f r a c t i o n of the concentrate, was i n f a c t the average s i z e o f a group o f p a r t i c l e s w i t h i n a small range of s i z e s . The d i s t r i b u t i o n o f s i z e s i s l i k e l y to change as the leach progresses when i n c r e a s i n g amounts of material are d i s s o l v e d and oxidized.  In order to determine these changes a s e r i e s o f measurements o f p a r t i c l e s i z e d i s t r i b u t i o n s i n d i f f e r e n t leaches was made. Samples o f concentrate were withdrawn from the f l a s k s using a Pasteur p i p e t t e and t h e i r s i z e d i s t r i b u t i o n was determined using the technique described i n m a t e r i a l s and methods.  - 66 F i g u r e s 15, 16 and 17 show the p a r t i c l e s i z e d i s t r i b u t i o n f o r the 1.78,  2.52  and 5.48  nm l e a c h e s .  A gradual s h i f t towards  the s m a l l e r s i z e s i s e v i d e n t (see Figure 18).  It was  not  p o s s i b l e to quantify the amount o f p a r t i c l e s of s i z e below 0.5 pm which appear to increase r a p i d l y during the fermentation,  Ps a r e s u l t the average p a r t i c l e diameter  c a l c u l a t e d from the s i z e d i s t r i b u t i o n did not appear to change very much during the l e a c h (see Figure 18).  The changes i n s i z e  could only be e x a c t l y q u a n t i f i e d i f the t o t a l contents of the f l a s k were analyzed, or a mass-based d i s t r i b u t i o n determined and some c o r r e c t i o n f a c t o r introduced to account f o r the increase  in  mass due to the o x i d a t i o n and j a r o s i t e p r e c i p i t a t i o n processes.  APPLICATION OF THE SHRINKING CORE MODEL OF LEVENSPIEL The r e s u l t s o f the t e s t s to determine the copper e x t r a c t i o n  rate  obtained i n leaches of s i x monosize f r a c t i o n s presented i n s e c t i o n 7.3,  and the p a r t i c l e diameter and surface area data f o r  the same leaches were used to c a l c u l a t e the o v e r a l l  extraction  a g a i n s t time curve f o r the b i o l o g i c a l l e a c h i n g of a copper c o n c e n t r a t e of known p a r t i c l e s i z e d i s t r i b u t i o n by using the Levenspiel  model.  Data f o r t h i s c a l c u l a t i o n are presented i n  Table II.1 (Appendix I I ) .  •  DAY 1  DAY 3  3.225 urn  2.9506 y m  DAY 7 2.5427  30  5 Q.  20 15 10 5  DAY 12  DAY 10 2.051  F i g . 15  vim  1.9789 p m  DAY 14 1.7815  Changes i n the p a r t i c l e s i z e d i s t r i b u t i o n for the 1.78 ym Leach (Percentage vs p a r t i c l e s i z e )  DAY 1  DAY 3  3.2768 ym  3.024 ym  DAY 7 2.517 ym  35 . 30 £ 25 20 . 15 . 10 5  DAY 10  DAY 12  3.031 ym  2.04 ym  DAY 14 1.3494 ym  JZL ym  F i g . 16  Changes in the p a r t i c l e s i z e d i s t r i b u t i o n for the 2.52 ym leach. (Percentage v s : p a r t i c l e  size)  CO  35 .  DAY 1  DAY 0  30 .  6.1514 ym  5.3763 ym  6.1284 ym  25 .  DAY 3  20 . 15 • 10 • 5•  tL  ~1—I 40  DAY 7  DAY 5  35 • 30-  6.1515 ym  DAY 10  4.7941 ym  25 • 2015 • 10. 5•  ZL DAY 17  DAY 14  40 • 35 .  6.1136 ym  6.2368 ym  3025' 201510' 5lO  M  CO  >t  O  lO  CM  PO  lO  KO  00  O  f-t  T—<  1 I  CO t I  ym  F i g . 17  Changes i n the p a r t i c l e size d i s t r i b u t i o n f o r the 5.48 ym leach. (Percentage vs p a r t i c l e s i z e )  6.1641 ym  - 70 -  • 2.52 ym leach  11  o 5.48 ym leach A  1.78 ym leach  10 9 -  1=  3  8-  e> jcvj Q. Qc ! c  7 -  LU M  1  2  4  6  8  10  12  14  16  18  TIME (DAYS) F i g . 18  Average p a r t i c l e s i z e for 3 d i f f e r e n t leaches vs time.  - 71 A sample c a l c u l a t i o n o f the copper e x t r a c t i o n a f t e r 48 h o f b i o l o g i c a l l e a c h i n g p r e d i c t e d by the s h r i n k i n g core model i s shown i n Appendix I I .  The p r e d i c t e d values o f copper e x t r a c t i o n versus time are shown i n F i g u r e 19.  Experimental r e s u l t s from a s e r i e s o f leaches  u s i n g copper concentrate o f known p a r t i c l e s i z e d i s t r i b u t i o n are a l s o shown i n F i g u r e 19 f o r comparison purposes with data shown i n Table 11.3 (Appendix I I ) .  Previous attempts t o use the s h r i n k i n g core model can be summarized as f o l l o w s :  1) Gormely (1973) working with z i n c s u l p h i d e c o n c e n t r a t e s , p r e d i c t e d values o f z i n c e x t r a c t i o n u s i n g the s h r i n k i n g core model t h a t were below the experimental e x t r a c t i o n s by a factor of ten.  He estimated the l e a c h i n g r a t e s f o r d i f f e r e n t  p a r t i c l e s i z e s based on a constant l e a c h i n g r a t e per u n i t s u r f a c e area.  T h i s study has shown t h a t the l e a c h i n g r a t e s  need t o be determined e x p e r i m e n t a l l y .  2) Samugasunderam (1981) obtained z i n c e x t r a c t i o n s w i t h i n 10% o f the p r e d i c t e d values (with a maximum d i f f e r e n c e o f 25%).  He  a t t r i b u t e d t h i s d i f f e r e n c e t o the method he used t o estimate s u r f a c e area and p a r t i c l e diameter.  Although the method used  f o r s u r f a c e area and p a r t i c l e diameter determination i n t h i s  -  • •  72  -  Experimental Value Predicted values using the shrinking core model  50 -  o  40 -  cn  UA CL.  ex. CD  I 30 -  CJ3  o  cm  20 -  10 •  — i  100  1  200  1—  300  LEACHING TIME (h) Fig. 19  Experimental and theoretical copper extraction values for the leaching of chalcopyrite.  - 73 study was d i f f e r e n t , the model p r e d i c t i o n s f e l l i n the same range, which suggests the e x i s t e n c e o f a f a c t o r r e l a t e d t o m i c r o b i a l physiology t h a t has not been i n c o r p o r a t e d i n the model and causes these d e v i a t i o n s .  POST LEACHING OBSERVATIONS A s e r i e s o f scanning e l e c t r o n photographs were taken o f several f r a c t i o n s of the concentrate p r i o r t o and a f t e r l e a c h i n g .  The  o b j e c t i v e was t o o b t a i n i n f o r m a t i o n on the nature of the attack by T h i o b a c i l l u s f e r r o o x i d a n s on the s u r f a c e o f the m i n e r a l . F i g u r e 20 shows p a r t i c l e s o f c h a l c o p y r i t e before l e a c h i n g .  In  g e n e r a l , the faces o f the p a r t i c l e s are smooth s u r f a c e s , w h i l e the edges are h i g h l y i r r e g u l a r .  F i g u r e 21 shows c h a l c o p y r i t e p a r t i c l e s a f t e r 200 h o f l e a c h i n g . A c a v i t y with dimensions s i m i l a r t o the dimensions o f one bacterium i s present i n the c e n t r e o f the p a r t i c l e shown i n F i g u r e 21a.  A s e r i e s o f c a v i t i e s c o v e r i n g s u r f a c e areas  approximately equal t o the areas covered by one bacterium are present i n F i g u r e 21b.  The b a c t e r i a seem t o a t t a c h only t o the  c e n t r a l p o r t i o n o f the p a r t i c l e s f a r from the edges. T h i s same phenomena was p r e v i o u s l y a t t r i b u t e d t o s u r f a c e t e n s i o n e f f e c t s (Berry, 1978).  I t i s c l e a r t h a t the edges of the p a r t i c l e s w i l l  F i g . 20  C h a l c o p y r i t e p a r t i c l e s before leaching  - 75 -  j F i g . 21  10  ym  10  ym  C h a l c o p y r i t e p a r t i c l e s a f t e r 200  1  h o f leaching  - 76 be s u b j e c t t o f r i c t i o n a g a i n s t other p a r t i c l e s when t h e suspension o f p a r t i c l e s i s a g i t a t e d . The shear f o r c e s generated during a g i t a t i o n w i l l make i t d i f f i c u l t f o r the b a c t e r i a t o a t t a c h t o t h e edges o f t h e p a r t i c l e s .  Small d e p o s i t s o f hexagonal c r y s t a l s s i m i l a r t o j a r o s i t e c r y s t a l s appear as small granules a t t h e s u r f a c e s o f t h e particles.  The amount o f these d e p o s i t s i n c r e a s e s with time.  P a r t i c l e s o f c h a l c o p y r i t e a f t e r 300 h o f l e a c h i n g a r e almost covered by these d e p o s i t s (Figure 22).  The presence o f j a r o s i t e d e p o s i t s on t o p o f t h e c h a l c o p y r i t e p a r t i c l e s prevents t h e contact between the b a c t e r i a and t h e n u t r i e n t s found i n t h e l i q u i d phase,  when m a t e r i a l subjected t o  300 h o f l e a c h i n g was recovered and n u t r i e n t s o l u t i o n and inoculum added, no f u r t h e r e x t r a c t i o n c o u l d be obtained.  See  r e s u l t s i n F i g u r e 23.  If j a r o s i t e p r e c i p i t a t i o n was avoided o r t h e p r e c i p i t a t e removed by r e - g r i n d i n g t h e m a t e r i a l , then the copper y i e l d o f the process would i n c r e a s e .  F i g . 22  C h a l c o p y r i t e p a r t i c l e s a f t e r 300 h o f l e a c h i n g  -  Fig  23  78  -  Second - Pass Leaching of Chalcopyrite  - 79 CHAPTER VIII  1.  An experimental  SUMMARY AND CONCLUSIONS  i n v e s t i g a t i o n was undertaken t o study t h e  a p p l i c a t i o n o f t h e s h r i n k i n g core model o f Levenspiel t o t h e modelling o f copper e x t r a c t i o n from c h a l c o p y r i t e by T h i o b a c i l l u s ferrooxidans.  2.  M i c r o s c o p i c a l examination o f t h e p a r t i c l e s subject t o l e a c h i n g supported t h e idea o f a s h r i n k i n g core type o f r e a c t i o n .  3.  The p r e d i c t e d e x t r a c t i o n s using t h e s h r i n k i n g core model f i t t h e experimental  r e s u l t s and a r e useful t o p r e d i c t t h e copper  e x t r a c t i o n up t o 30-35% e x t r a c t i o n l e v e l s .  Since no account f o r t h e p h y s i o l o g i c a l s t a t e o f t h e bacterium i s i n c l u d e d i n t h e model, i t tends t o overestimate  t h e copper  e x t r a c t i o n which l e v e l s o f f a f t e r 120 h o f l e a c h i n g .  The d e p o s i t o f a s o l i d l a y e r o f o x i d a t i o n products which l i m i t e d the r a t e o f d i f f u s i o n o f n u t r i e n t s and metabolic products t o and from t h e c e l l s a t t h e r e a c t i n g s u r f a c e , was found t o be r e s p o n s i b l e f o r t h e incomplete e x t r a c t i o n .  4.  Using e l e c t r o n microphotographs t h e s o l i d r e a c t i o n product d e p o s i t s were i d e n t i f i e d as j a r o s i t e s and t h e i r appearance on the mineral surface was found t o be d i r e c t l y r e l a t e d t o t h e end of t h e copper e x t r a c t i o n .  - 80  5.  -  In order t o determine t h e e f f e c t s o f t h e p a r t i c l e s i z e on t h e l e a c h i n g o f copper, t h e <400 mesh concentrate was f r a c t i o n a t e d i n t o 7 p a r t i c l e s i z e s and each f r a c t i o n was leached s e p a r a t e l y . The mineral p a r t i c l e s o f v a r i o u s s i z e s were o x i d i z e d simultaneously and independently with v a r y i n g l e a c h i n g r a t e s dependent on t h e i r s u r f a c e area.  The highest r a t e was obtained  f o r p a r t i c l e s o f 1 urn s i z e and had a value o f 28.3 mg Cu/l.h.  These r e s u l t s i n d i c a t e t h a t t h e optimum p a r t i c l e s i z e was reached when t h e p a r t i c l e s o f c h a l c o p y r i t e had a s i z e comparable t o t h e b a c t e r i a l s i z e and t h a t t h e extent o f t h e e x t r a c t i o n obtained a t t h i s s i z e (97%) c o u l d not be reached with any other particle size. 6.  To understand t h e r o l e o f t h e b a c t e r i a i n t h e l e a c h i n g process, the growth p a t t e r n s o f T h i o b a c i l l u s f e r r o o x i d a n s were determined.  Using o r g a n i c and i n o r g a n i c n i t r o g e n determinations  to measure b a c t e r i a l growth, t h e l a g time was shortened t o <1 day when inoculum from a 6-8 days o l d c u l t u r e was used t o seed the new f l a s k s .  T h i s study provided evidence t h a t t h e bulk o f  the copper e x t r a c t i o n (62% o f the t o t a l e x t r a c t i o n ) occurred once t h e c u l t u r e had entered the s t a t i o n a r y phase and t h a t no d i r e c t r e l a t i o n s h i p e x i s t e d between metal e x t r a c t i o n r a t e s and b a c t e r i a l growth.  - 81 RECOMMENDATIONS FOR FUTURE STUDIES  Studying the k i n e t i c properties; o f i n d i v i d u a l mineral p a r t i c l e s provides an i n s i g h t t o the l e v e l o f m i n e r a l - b a c t e r i a i n t e r a c t i o n s , which i n t u r n serves t o c l a r i f y the mechanism o f leaching.  In a d d i t i o n t o what was done i n t h i s work, f u r t h e r  s t u d i e s a r e r e q u i r e d regarding t h e e f f e c t s o f the changes i n pH and redox p o t e n t i a l produced by t h e b a c t e r i a l metabolism and t h e leached products because the d i r e c t i o n and i n t e n s i t y o f b a c t e r i a l s y n t h e s i s i s known t o depend on these parameters.  The d i s s o l v e d oxygen c o n c e n t r a t i o n should a l s o be s t u d i e d s i n c e i t plays a very important r o l e given the o x i d a t i v e c h a r a c t e r i s t i c s o f t h i s process.  High oxygen demands would  l i k e l y r e s u l t i n oxygen d e p l e t i o n given t h e l i m i t e d oxygen s o l u b i l i t y o f the medium.  S t u d i e s a r e a l s o r e q u i r e d t o i n v e s t i g a t e the chemical  reactions  r e s u l t i n g i n the p r e c i p i t a t i o n o f j a r o s i t e type m a t e r i a l s and i t s e f f e c t s on b a c t e r i a l metabolism.  The use o f a continuous  c u l t u r e c o u l d prove v a l u a b l e i n studying the e f f e c t s o f these and other parameters and i n so doing would increase the c o n t r o l over t h e process and s e l e c t t h e optimum operating c o n d i t i o n s f o r the l e a c h i n g o f copper from c h a l c o p y r i t e .  - 82 CHAPTER IX  REFERENCES  Agathe, A.D., K o r c z y n s k i , M.S., and Lundgren, D.G. E x t r a c e l l u l a r Complex from the C u l t u r e F i l t r a t e o f F e r r o b a c i l l u s f e r r o o x i d a n s . Canadian Journal o f M i c r o b i o l o g y . , l b , ZbD ( i y b 8 ) . A i b a , S., Humphrey, A.E., and Mil l i s , N.F. 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Semi-conductor-Electrochemical Aspects o f B a c t e r i a l Leaching. I. Oxidation o f Metal Sulphides with Large Energy Gaps. Journal o f Chemical Technology and Biotechnology 3^, 565 (1981 ).  - 88 Touvinen, O.H., and K e l l y , D.P. Biology o f T h i o b a c i l l u s f e r r o o x i d a n s i n R e l a t i o n t o the M i c r o b i o l o g i c a l Leaching o f Sulphide Ores. Z e i t s c h r i f t f u r A l l g . Mikrobiologue. 12, 311 (1972). T r u d i n g e r , P.A. Microbes, Metals and M i n e r a l s . M i n e r a l s Science and Engineering 3, 13 (1971). T r u s s e l l , P.C., Duncan, D.W., and Walden, C C . B i o l o g i c a l Mining, Canadian Mining J o u r n a l , pp. 1, March (1964). T s u c h i y a , M.M., T r i v e d i , N.C, and S c h u l e r , M.L. M i c r o b i a l Mutualism i n Ore Leaching. Biotechnology and B i o e n g i n e e r i n g ]_6_, 991 (1974). Welcher, F . J . Standard Methods o f Chemical A n a l y s i s . D. Van Nostrand Company Inc. 6th E d i t i o n 351 (1963).  - 89 -  TABLE 1.1 BACTERIAL GROWTH DATA  Time (hours)  21 27 44.5 50.5 71.0 97.0 121.0 145.0 193.5  Kjeldahl Nitrogen (ppm)  Ammonia Nitrogen (ppm)  Cell Nitrogen (ppm)  Cell Numbers x lO"  1 0  (1)  (2)  (1)  (2)  (1)  (2)  (1)  (2)  1886.26 1672.89 1578/05 1554.34 1435.80 1269.84 1103.88 1151.3 1103.88  1862.56 1672.89 1554.34 1530.64 1412.09 1317.26 1222.42 1198.72 1103.88  1886.26 1815.14 1767.72 1791.43 1625.47 1471.36 1317.26  1886 .26 1767 .72 1744 .01 1767 .72 1649 .18 1542 .49 1459 .51 1412 .09 1317 .26  0 142.25 189.67 237.09 189.67 201.52 213.88  23.7 94.83 189.67 237.09 213.38 225.23 237.09 213.37 213.88  0 906 1208 1208 1208 1283  151 604 1208 1510 1359 1434 1510 1359 1359  -  1317.26  -  213.88  -  1359  - 91 -  TABLE 1.2 AVERAGE RESULTS FOR THREE BIOLOGICAL LEACHING EXPERIMENTS Time (days) 2 4 6 9 11 13 15 17  pH  Eh  Cu (ppm)  Fe (ppm)  1.96 1.91 1.69 1.52 1.50 1.52 1.48 1 .50  386 460 567 665 676 676 680 683  4033 6750 7875 8000 7933 7833 6750 5766  2533 4150 3537 4350 4375 4366 4158 3550  Time (days) 1 2 4 6 8 10 14 16  Cell x 10" 360 730 880 790 760 760 790 630  - 92 TABLE 1.3 CHALCOPYRITE CONCENTRATE SURFACE AREA  Fraction No.  Surface Area (m )  Fraction Percentage  1 2 3 4 5 6  9.78045 5.87925 4.15275 2.93925 1.90950 1.37475  0.1936 0.1833 0.3262 0.1195 0.0742 0.1032  2  Area 1.8934 1.07766 0.35124 0.35124 0.14168 0.14187  Total area i n 7.5 g o f concentrate = 4.96047 m  2  TABLE 1.4 BACTERIAL COVERAGE OF THE SURFACE AREA DATA Time (days) 1 2 4 6 8 10  Area Occupied By B a c t e r i a x 1 0 " ynr I U  180 365 440 395 380 380  Percentage o f Total Surface Covered By B a c t e r i a 36.29 73.58 88.70 79.63 76.61 76.61  Cell Numbei x 10" 360 730 880 790 760 760  - 93 -  TABLE 1.5 STERILE RUN DATA Time (h)  0 44.5 97.5 143.75 215.25  pH  Eh  Copper Cone. (ppm)  Iron Cone. (ppm)  2.0 2.17 2.17 2.25 2.26  355 355 355 355 355  0 795 820 880 880  0 532 525 550 520  J  - 94 -  TABLE 1.6 LEACH DATA USING MONOSIZED MATERIAL OF 1.07 m P  Time (h)  pH  Eh  Cu (ppm)  Fe (ppm)  28 69.5 115 158.5 208 256.5 305  2.03 1.81 1.63 1.49 1.49 1.58 1.46 1.57  385 415 575 610 650 660 660 660  2125 3825 7000 8300 8750 9700 10000  540 1650 3850 5550 6350 6500 6450  [Cu] = 0.02833 t + 2.4834  TABLE 1.7 LEACH DATA USING MONOSIZED MATERIAL OF 1.78 m u  Time (h) 24 72.25 168 240.5 286 337  pH  2.06 1.89 1.52 1.47 1.53 1.52  Eh  Cu (ppm)  Fe (ppm)  345 540 560 560 550 570  1357 3532 4157 4357 4507 4807  785 1750 3900 4225 4225 4325  [Cu] = 0.00894 + 2.1053  Cells x 10-10 178 446 446 535 446 535  i cn  i  TABLE 1.8 LEACH DATA USING MONOSIZED MATERIAL OF 2.52 Time (h)  pH  22 70.25 166 238.5 284 335  2.19 1.93 1.49 1.47 1.52 1.47  . Eh  355 530 560 545 550 575  w  Cu (ppm)  Fe (ppm)  Cells xlO  1057 2407 3107 3408 3508 3807  785 1650 3325 3500 3500 3550  446 713 446 535 535 535  [Cu] = .00766 t + 1.4576  - 1 0  i <£>  CTi  I  - 97 -  TABLE 1.9 LEACH DATA USING MONOSIZED MATERIAL OF 3.56 um  Time (h)  pH  Eh  Cu (ppm)  28 69.5 115 158.5 208 256.5 305  2.05 1.74 1.51 1.43 1.39 1.47 1.40  505 560 620 625 640 640 660  1450 1950 2250 2400 2700 2800 2900  [Cu] = 0.004971 t + 1 .5399  Fe (ppm) 830 1950 2450 2350 2900 2875 3050  - 98 TABLE 1.10 LEACH DATA USING MONOSIZED MATERIAL OF 5.48 um  Time (h) 18 73 124.4 171.65 241.9 337.65 409.15  pH  Eh  Cu (ppm)  Fe (ppm)  1.98 1.83 1.73 1.60 1.56 1.46 1.42  525 565 565 560 560 555 550  572 922 1172 1321 1421 1771 1872  532 1025 1900 1900 2000 2300 2350  [Cu] = .00315 t + 0.6730  -  TABLE 1.11 LEACH DATA USING MONOSIZED MATERIAL OF 7.41 m u  Time (h) 24 72.25 160 240.5  pH  Eh  Cu (ppm)  Fe (ppm)  1.95 1.88 1.57 1.47  395 535 550 545  782 1307 1807 2108  717 1050 1300 1400  [Cu] = .00315 t + 0.6730  - 99 -  APPENDIX II  - 100 -  APPENDIX II Sample C a l c u l a t i o n f o r The Copper E x t r a c t i o n Using The S h r i n k i n g Core Model o f Levenspiel  C a l c u l a t i o n s f o r the percentage copper e x t r a c t i o n are done f o r 50 h o f l e a c h i n g time.  The s i z e d i s t r i b u t i o n o f the concentrate i s given i n  Table 4. Percentage Copper E x t r a c t i o n  For the f i r s t f r a c t i o n with mean diameter 1.07  v  m 2  ssa  = s p e c i f i c surface area of the p a r t i c l e s = 1.30 m /g  r  = copper e x t r a c t i o n r a t e = 28.33 mg/l.h.  z  w  = weight f r a c t i o n = 0.1936.  then  S  surface area c o n c e n t r a t i o n of the p a r t i c l e s  S  feed pulp d e n s i t y x weight f r a c t i o n x ssa  S  Z i L a x 1™0J! ! .30406 m = 27.049 m /L 70 mL L ^ g copper e x t r a c t i o n rate per u n i t s o l i d surface area  r z/s r z/s  2  x  .001047  g/h.m  0 i 1 9 3 6  x  2  - 101 -  with the density o f the c h a l c o p y r i t e concentrate 4.3 x 1 0  being  g/m .  6  3  The time f o r complete r e a c t i o n o f one p a r t i c l e i s given by:  T  =  = 2196.47 h  The f r a c t i o n a l e x t r a c t i o n w i l l then be c a l c u l a t e d using equation 3, page 24: 2 3 3 (|)6 (^) + 6 (|) £l - exp (-T/t)J z = T  z = 3 ( )- 6 ( V 2196.47 21 96.47 5 0  z =  5 0  +  6 (  ) f l - exp ( 21 96.47 L  5 0  3  2 1 9 6  50  4 7  '0.065  The f r a c t i o n a l e x t r a c t i o n o f the r e s t o f the 6 f r a c t i o n s o f the copper concentrate a r e s i m i l a r l y c a l c u l a t e d and t a b u l a t e d i n Table I I . 2 .  The t o t a l copper e x t r a c t i o n a f t e r 50 h o f l e a c h i n g i s found t o be 16.15%.  - 102 -  TABLE II.1 KINETIC DATA FOR THE SHRINKING CORE MODEL  Particle Size (nm)  Specific Surface Area (m /g)  Surface Area Concentration (m /L)  1.07 1.78 2.52 3.56 5.48 7.41  1.30406 0.7839 0.5537 0.3919 0.2546 0.1883  27.049 15.395 19.352 5.0176 2.024 2.026  2  2  Release Rate o f Copper (mg/Lh) 28.33 8.94 7.66 4.31 3.15 5.89  TABLE II.2 CALCULATED PERCENTAGE EXTRACTION AFTER 50 h OF LEACHING  Fraction  1 2 3 4 5 6  Diameter ssa nm m /g 2  1.07 1.78 2.52 3.56 5.48 7.41  1.3040 0.7839 0.5537 0.3919 0.2546 0.1883  r / mg/h.m  Weight F r a c t i o n  T (h)  Contribution  1.047 0.580 0.645 0.859 1.550 2.907  0.1936 0.1833 0.3262 0.1195 0.0742 0.1032  2196.47 6590.23 8387.9 8900.28 7570.4 5480.0  0.06525 0.02241 0.01767 0.01666 0.01954 0.02686  z  s  2  C a l c u l a t e d Total E x t r a c t i o n = 16.15%  - 104 -  TABLE 11.3 COPPER CONCENTRATION DATA FOR BIOLOGICAL LEACHING OF CHALCOPYRITE  ppm o f copper  Time (h)  24 47.8 69.25 116.8 144 183 214.5 239.25 284.8  3199 5120 6921 9581 9781 9581 10181 9581 9781  3059 4460 5460 7179 9981 10781 11182 9380 11582  3259 5040 6661 8780 8780 9581 9380 9581 9781  3079 4860 5981 9781 9781 10181 11182 10181 10381  PERCENTAGES OF COPPER EXTRACTION FOR BIOLOGICAL LEACHING OF CHALCOPYRITE ppm o f copper  Time (h)  24 47.8 69.25 116.8 144 183 214.5 239.25 284.8  10.74 17.18 23.23 32.16 32.83 32.16 34.18 32.16 32.83  10.27 14.97 18.33 24.10 33.50 36.19 37.54 31.49 38.88  10.94 16.92 22.36 29.47 29.47 32.16 31.49 32.16 32.83  10.33 16.31 20.08 26.79 32.83 34.18 37.54 34.18 34.85  

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