GOLD BIOLEACHING OF ELECTRONIC SCRAP MATERIAL BY CYANOGENIC BACTERIA AND ITS ENHANCEMENT WITH BIOOXIDATION

PHAM VAN ANH (B. Eng. (Hons.), HUT)

A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE

2009

ACKNOWLEDGEMENTS

This thesis would not have been possible without help and support from many people.

First of all, I would like to express my gratitude to my supervisor, Associate Professor Ting Yen Peng, for his guidance, encouragement and support from the initial until the completion.

I wish to thank the National University of Singapore for financial support so that I can pursuit this research, to thank Cimelia Resource Recovery Pte Ltd for providing ESM as material for this study.

Many thanks to lab officers, Mr Sukianto, Ms Li Xiang, and particularly Ms Sylvia Wan for their assistance during the last two years.

I would like to thank all my lab mates, Vu Phuong Thanh, Ng Wenfa, Adriyan Harimawan, and Shailendra Mishra for suggestion, advice and help whenever I need during my course. Without your willingness and support, this thesis can not be submitted.

Last but not least, thank to my friends and my family for support and always being by my side.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS TABLE OF CONTENTS SUMMARY NOMENCLATURE LIST OF TABLES LIST OF FIGURES

i ii vi viii ix xii

CHAPTER 1: INTRODUCTION CHAPTER 2: LITERATURE REVIEW 2.1 Electronic Scrap Material (ESM) as a secondary gold ore 2.1.1 Economic value of gold 2.1.2 Gold in electronic devices 2.1.3 Electronic Scrap Material 2.2 Metallurgical recovery of metals from ESM 2.2.1 Definitions and classification 2.2.2 Process of gold recovery from ESM 2.2.3 Bioleaching in practice 2.3 Cyanogenic microorganisms and cyanide producing mechanism 2.3.1 General introduction 2.3.2 Chromobacterium violaceum 2.3.3 Pseudomonas fluorescens 2.4 Applying cyanogenic microorganisms in gold bioleaching 2.4.1 Potentials in bioleaching precious metals 2.4.2 Gold bioleaching by C. violaceum and P. fluorescens 2.5 Factors influence gold bioleaching efficiency 2.5.1 Growth conditions 2.5.1.1 Oxygen 2.5.1.2 pH 2.5.1.3 Temperature

1 4 4 4 6 7 9 9 11 12 13 13 14 15 16 16 17 18 18 18 19 19

ii

2.5.1.4 Glycine 2.5.2 ESM 2.5.2.1 Characteristics of ESM 2.5.2.2 Effects of ESM on microbial growth 2.6 Bio-oxidation of ESM before bioleaching gold 2.6.1 Bio-oxidation gold ores 2.6.2 Bio-oxidation ESM CHAPTER 3: MATERIALS AND METHODS 3.1 Materials 3.1.1 Electronic Scrap Material (ESM) 3.1.2 Bacteria 3.2 Experimental methods 3.2.1 Analysis methods 3.2.1.1 Particle size distribution 3.2.1.2 Specific Surface Area 3.2.1.3 Acid digestion 3.2.1.4 Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) 3.2.1.5 Scanning Electron Microscopy/Energy Dispersive using X-Ray (SEM/EDX) 3.2.1.6 Toxicity Characteristic Leaching Procedure (TCLP) 3.2.1.7 pH measurement 3.2.1.8 Optical Density 3.2.1.9 Free Cyanide Concentration Analysis 3.2.2 Bacterial culture 3.2.3 Bioleaching experiments 3.2.3.1 Shake flask bioleaching 3.2.3.2 Bioleaching in bioreactor 3.2.4 Bio-oxidation experiments 3.2.5 Chemical leaching

20 21 21 22 23 23 25 28 28 28 28 28 29 29 29 29 30

32

32 34 34 34 36 37 37 38 40 40

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CHAPTER 4: RESULTS AND DISCUSSION 4.1 Characterization of ESM 4.1.1 Particle size distribution 4.1.2 Specific surface area 4.1.3 ESM elemental composition 4.1.4 Toxicity Characteristic Leaching Procedure tests 4.2 Pretreatment ESM 4.2.1 Chemical leaching 4.2.1.1 Acid leaching 4.2.1.2 Ferric leaching 4.2.2 Bio-oxidation by Acidobacillus ferrooxidans 4.3 Culture Chromobacterium violaceum and Pseudomonas fluorescens in fresh medium in flasks 4.4 Bioleaching non-biooxidized ESM 4.4.1 One-step bioleaching 4.4.1.1 pH profiles 4.4.1.2 Free cyanide concentration 4.4.1.3 Gold leaching profiles 4.4.1.4 Copper leaching profiles 4.4.2 Two-step bioleaching 4.4.2.1 pH profiles 4.4.2.2 Free cyanide concentration 4.4.2.3 Gold leaching profiles 4.4.2.4 Copper leaching profiles 4.4.3 Comparison of Chromobacterium violaceum and Pseudomonas fluorescens, one-step and two-step in bioleaching non-biooxidized ESM 4.5 Bioleaching biooxidized ESM 4.5.1 pH profiles 4.5.2 Free cyanide concentration 4.5.3 Gold leaching profiles 4.5.4 Copper leaching profiles

42 42 42 42 43 45 46 46 47 47 51 54

59 59 59 63 68 73 78 78 82 86 91 94

97 98 102 105 109

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4.5.5 Comparison of bioleaching non-biooxidized ESM and biooxidized ESM 113 by Chromobacterium violaceum and Pseudomonas fluorescens 4.6 Bioleaching ESM by Pseudomonas fluorescens in bioreactor 4.6.1 Growth in fresh medium 4.6.2 Bioleaching ESM 4.6.3 Effects of aeration to gold leaching efficiency 4.6.4 Comparison of bioleaching in flasks and bioreactor CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions 5.2 Recommendations REFERENCES APPENDICES 115 115 116 119 121 122 122 124 126 133

SUMMARY

Bioleaching has been used for many years to recovery metals such as copper and zinc from low-grade ores or low-grade mineral resources. Electronic scrap materials, with its significant gold content, is recognized as a new emerging and fast-growing waste stream and could be considered as a secondary ore for gold due to its high concentration. The bioleaching mechanisms responsible for the metal recovery in mining operations may be also applied in the bio-mining of precious metals from such wastes. This project focused on the bioleaching of gold from ESM by cyanogenic bacteria and its enhancement by biooxidation. The ESM used in this project were fine particles, of size <75m. Its most valuable elements are gold (4.872 g/kg) and copper (28.32 g/kg). The ESM possessed a low specific surface area and a non-sporous structure.

When cultured in LB medium, C. violaceum and P. fluorescens biogenically produced cyanide. The highest free cyanide concentration produced by both bacteria was observed during the stationary phase. Both one-step and two-step bioleaching were investigated: in the former, bacteria were inoculated directly in the presence of the ESM, while in twostep bioleaching, the ESM was introduced into the culture one day after bacterial inoculation. C. violaceum and P. fluorescens were used in one-step bioleaching of Electronic Scrap Material (ESM) at pulp density range from 0.5-8%w/v. Results confirmed that both bacteria were capable of bioleaching gold when gold was solubilised from the solid ESM samples. In contrast, no gold was detected in the controls (without bacteria). Results suggested a higher metal resistance and a more favourable leaching kinetics of P. fluorescens than C. violaceum. One-step bioleaching showed the decreasing of leaching gold when increasing pulp density, and strongly inhibition of ESM on growth bacteria and cyanide production, particularly C. violaceum.

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Two-step bioleaching ESM by the two bacteria was conducted at the same pulp densities. Gold recovery and copper recovery in both bacteria was improved; the highest gold concentration of 3.7mg/l at 2%w/v ESM was obtained with P. fluorescens.

In both one-step and two-step bioleaching, the copper/gold ratio in the leachates was very high was likely to be the reason for the poor gold recovery by the biogenically produced cyanide. Thus, bio-oxidation by At. ferrooxidans was applied to remove copper and other base metals from ESM, and to release gold from the mineral matrix. This work is the first reported attempt at using bio-oxidation as a pretreatment for bioleaching. It was found that biooxidation led to a reduction of more than 80% of copper and nearly 60% of aluminum from ESM. The copper/gold ratio was reduced from 5.8 to 3.1. Bioleaching of the bio-oxidized ESM resulted in an enhancement in the leaching of gold, in particularly by C. violaceum, and with a much lower copper/gold ratio in the leachate.

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NOMENCLATURE ATCC DO EDX ESM ICP-OES LB OD PCB ppb ppm rpm SEM TCLP US EPA vvm American Type Culture Collection Dissolved oxygen Energy Dispersive X-Ray Electronic Scrap Material Inductively Coupled Plasma Optical Emission Spectrometer Lysogeny broth Optical density Printed circuit board Parts-per-billion Parts-per-million Revolutions per minute Scanning Electron Microscopy Toxicity Characteristic Leaching Procedure United States Environmental Protection Agency Volume air per volume medium per minute

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LIST OF TABLES Table 2.1 Table 3.1 Table 3.2 Table 4.1 Table 4.2 Table 4.3 Table 4.4 Table 4.5 Table 4.6 Table B.1 Table B.2 Table B.3 Table B.4 Table B.5 Table B. 6 Table B.7 Table B.8 Table B.9 Table B.10 Table B.11 Table B.12 Improvement of leaching gold from gold ore by biooxidation Operating conditions of ICP-OES The wavelengths (nm) used for metal analysis by ICP-OES Metal composition of the liquor after acid digestion Metal concentration of the TCLP extract compared with Regulatory levels by US EPA Metal removal of ESM by ferric solution Comparison of metal composition in ESM before and after biooxidation by A. ferrooxidans Comparison of pH in bioleaching non-biooxidized and biooxidized ESM Increase in gold/copper ratio in leachate by bio-oxidation Size range of as-received ESM Particle size distribution (%volume) of original <75m ESM Particle size distribution (%volume) of acid digested <75m ESM Particle size distribution (%volume) of bio-oxiddized <75m ESM Particle size distribution (%volume) of original, acid digestion and bio-oxidized <75m ESM Elemental content of <75m (SEM/EDX) Elemental content of acid digested <75m (SEM/EDX) Elemental content of carbon tape Metal composition of ESM in literature Metal composition of the liquor after acid digestion pH profile of one-step bioleaching non-biooxidized ESM in shake flasks pH profile of two-step bioleaching non-biooxidized ESM in shake 24 30 31 44 46 51 54 101 113 137 139 141 143 145 155 157 158 160 161 162 163

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flasks Table B.13 Table B.14 Table B.15 Table B.16 Table B.17 Table B.18 Table B.19 Table B.20 Table B.21 Table B.22 Table B.23 Table B.24 Table B.25 Table B.26 Table B.27 Table B.28 Table B.29 Table B.30 pH profile of bioleaching bio-oxidized ESM in shake flasks Free cyanide concentration (mg/l) in one-step bioleaching nonbiooxidized ESM in shake flasks Free cyanide concentration (mg/l) in two-step bioleaching nonbiooxidized ESM in shake flasks Free cyanide concentration (mg/l) in bioleaching bio-oxidized ESM in shake flasks Gold concentration in cultures (mg/l) in one-step bioleaching nonbiooxidized ESM in shake flasks Gold concentration in cultures (mg/l) in two-step bioleaching nonbiooxidized ESM in shake flasks Gold concentration in cultures (mg/l) in bioleaching bio-oxidized ESM in shake flasks Gold recovery (%) in bioleaching ESM in shake flasks Copper concentration in cultures (mg/l) in one-step bioleaching nonbiooxidized ESM in shake flasks Copper concentration in cultures (mg/l) in two-step bioleaching nonbiooxidized ESM in shake flasks Copper concentration in cultures (mg/l) in bioleaching bio-oxidized ESM in shake flasks Copper recovery (%) in bioleaching ESM in shake flasks Growth of C. violaceum in fresh medium in shake flasks Growth of P. fluorescen in fresh medium in shake flasks Growth of P. fluorescens in fresh medium in bioreactor Metal removal (%) of bio-oxidation ESM by At. ferrooxidation Metal removal (%) of bio-oxidation ESM in control Growth and bioleaching ESM of P. fluorescens in bioreactor (with aeration) 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 179 180

Table B.31 Table B.32 Table B.33

Growth and bioleaching ESM of P. fluorescens in bioreactor (without aeration) Metal removal of acid leaching Metal removal of ferric leaching

180 181 182

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LIST OF FIGURES Figure 2.1 Figure 3.1 Figure 4.1 Figure 4.2 Figure 4.3 (a) Figure 4.3 (b) Figure 4.3 (c) Figure 4.3 (d) Figure 4.3 (e) Figure 4.4 (a) Figure 4.4(b) Figure 4.5 (a) Figure 4.5 (b) Figure 4.6 (a) Figure 4.6 (b) Figure 4.7 (a) Figure 4.7 (b) Figure 4.7 (c) Figure 4.7 (d) Figure 4.7 (e) Figure 4.8 Market price of gold over the last decade Experimental set up of bioreactor system Particle size distribution of < 75m ESM Images of < 75m ESM under Scanning Electron Microscope (SEM) Removal of metals from < 75m ESM by nitric acid Removal of metals from < 75m ESM by sulfuric acid Removal of metals from < 75m ESM by citric acid Removal of metals from < 75m ESM by oxalic acid Removal of metals from < 75m ESM by gluconic acid Bio-oxidation of ESM by A. ferrooxidans Control of bio-oxidation of ESM Growth of C. violaceum in shake flask in the absence of ESM pH profile and cyanide production of C. violaceum in shake flask in absence of ESM Growth of P. fluorescens in shake flask in absence of ESM pH profile and cyanide production of P. fluorescens in shake flask in absence of ESM pH profile in one-step bioleaching with 0.5% pulp density pH profile in one-step bioleaching with 1% pulp density pH profile in one-step bioleaching with 2% pulp density pH profile in one-step bioleaching with 4% pulp density pH profile in one-step bioleaching with 8% pulp density Influence of pulp density on pH value in one-step bioleaching nontreated ESM 6 39 42 43 48 48 49 49 50 52 52 57 57 58 58 60 61 61 62 62 63

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Figure 4.9 (a) Figure 4.9 (b) Figure 4.9 (c) Figure 4.9 (d) Figure 4.9 (e) Figure 4.10 (a) Figure 4.10 (b) Figure 4.10 (c) Figure 4.10 (d) Figure 4.10 (e) Figure 4.11 Figure 4.12 (a) Figure 4.12 (b) Figure 4.12 (c) Figure 4.12 (d) Figure 4.12 (e) Figure 4.13 Figure 4.14 (a) Figure 4.14 (b) Figure 4.14 (c) Figure 4.14 (d) Figure 4.14 (e) Figure 4.15 (a)

Cyanide profile in one-step bioleaching with 0.5% pulp density Cyanide profile in one-step bioleaching with 1% pulp density Cyanide profile in one-step bioleaching with 2% pulp density Cyanide profile in one-step bioleaching with 4% pulp density Cyanide profile in one-step bioleaching with 8% pulp density Gold leaching in one-step bioleaching with 0.5% pulp density Gold leaching in one-step bioleaching with 1% pulp density Gold leaching in one-step bioleaching with 2% pulp density Gold leaching in one-step bioleaching with 4% pulp density Gold leaching in one-step bioleaching with 8% pulp density Influence of pulp density on one-step bioleaching gold from nontreated ESM Copper leaching in one-step bioleaching with 0.5% pulp density Copper leaching in one-step bioleaching with 1% pulp density Copper leaching in one-step bioleaching with 2% pulp density Copper leaching in one-step bioleaching with 4% pulp density Copper leaching in one-step bioleaching with 8% pulp density Influence of pulp density on leaching copper in one-step bioleaching pH profile in two-step bioleaching with 0.5% pulp density pH profile in two-step bioleaching with 1% pulp density pH profile in two-step bioleaching with 2% pulp density pH profile in two-step bioleaching with 4% pulp density pH profile in two-step bioleaching with 8% pulp density Cyanide production in two-step bioleaching with 0.5% pulp density

66 66 67 67 68 70 71 71 72 72 73 75 76 76 77 77 78 79 80 80 81 81 84

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Figure 4.15 (b) Figure 4.15 (c) Figure 4.15 (d) Figure 4.15 (e) Figure 4.16 (a) Figure 4.16 (b) Figure 4.16 (c) Figure 4.16 (d) Figure 4.16 (e) Figure 4.17 Figure 4.18 (a) Figure 4.18 (b) Figure 4.18 (c) Figure 4.18 (d) Figure 4.18 (e) Figure 4.19 Figure 4.20 Figure 4.21 (a) Figure 4.21 (b) Figure 4.21 (c) Figure 4.21 (d) Figure 4.21 (e) Figure 4.22 (a)

Cyanide production in two-step bioleaching with 1% pulp density Cyanide production in two-step bioleaching with 2% pulp density Cyanide production in two-step bioleaching with 4% pulp density Cyanide production in two-step bioleaching with 8% pulp density Gold leaching in two-step bioleaching with 0.5% pulp density Gold leaching in two-step bioleaching with 1% pulp density Gold leaching in two-step bioleaching with 2% pulp density Gold leaching in two-step bioleaching with 4% pulp density Gold leaching ESM in two-step bioleaching with 8% pulp density Influence of pulp density on gold leaching in two-step bioleaching Copper leaching in two-step bioleaching with 0.5% pulp density Copper leaching in two-step bioleaching with 1% pulp density Copper leaching in two-step bioleaching with 2% pulp density Copper leaching in two-step bioleaching with 4% pulp density Copper leaching in two-step bioleaching with 8% pulp density Comparison of gold recovery in one-step and two-step bioleaching Comparison of copper recovery in one-step and two-step bioleaching pH profile in bioleaching bio-oxidized ESM with 0.5% pulp density pH profile in bioleaching bio-oxidized ESM with 1% pulp density pH profile in bioleaching bio-oxidized ESM with 2% pulp density pH profile in bioleaching bio-oxidized ESM with 4% pulp density pH profile in bioleaching bio-oxidized ESM with 8% pulp density Cyanide production in bioleaching bio-oxidized ESM with 0.5% pulp density

84 85 85 86 88 88 89 89 90 90 92 92 93 93 94 95 96 99 99 100 100 101 103

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Figure 4.22 (b) Figure 4.22 (c) Figure 4.22 (d) Figure 4.22 (e) Figure 4.23 (a) Figure 4.23 (b) Figure 4.23 (c) Figure 4.23 (d) Figure 4.23 (e) Figure 4.24 (a) Figure 4.24 (b) Figure 4.24 (c) Figure 4.24 (d) Figure 4.24 (e) Figure 4.25 Figure 4.26 Figure 4.27 Figure 4.28 Figure 4.29 Figure 4.30

Cyanide production in bioleaching bio-oxidized ESM with 1% pulp density Cyanide production in bioleaching bio-oxidized ESM with 2% pulp density Cyanide production in bioleaching bio-oxidized ESM with 4% pulp density Cyanide production in bioleaching bio-oxidized ESM with 8% pulp density Bioleaching gold from 0.5%w/v bio-oxidized ESM Bioleaching gold from 1%w/v bio-oxidized ESM Bioleaching gold from 2%w/v bio-oxidized ESM Bioleaching gold from 4%w/v bio-oxidized ESM Bioleaching gold from 8%w/v bio-oxidized ESM Bioleaching copper from 0.5%w/v bio-oxidized ESM Bioleaching copper from 1%w/v bio-oxidized ESM Bioleaching copper from 2%w/v bio-oxidized ESM Bioleaching copper from 4%w/v bio-oxidized ESM Bioleaching copper from 8%w/v bio-oxidized ESM Comparison of bioleaching gold from non-biooxidized and biooxidized ESM Comparison of bioleaching copper from non-biooxidized and biooxidized ESM Monitoring growth of P. fluorescens in bioreactor, at 300C, 200 rpm, and 1 vvm Growth of P. fluorescens in bioleaching 2% ESM in bioreactor, at 300C, 200 rpm, and 1 vvm Bioleaching gold and copper from 2% ESM by P. fluorescens in bioreactor, at 300C, 200 rpm, and 1vvm Growth of P. fluorescens in bioleaching 2% ESM in bioreactor, at

103 104 104 105 107 107 108 108 109 110 111 111 112 112 114 114 115 117 118 120

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300C, 200 rpm, with no aeration Figure 4.31 Figure 4.32 Figure B.1 Figure B.2 (a) Figure B.2 (b) Figure B.3 Figure B.4 Figure B.5 Figure B.6 Figure B.7 Figure B.8 Figure B.9 Figure B.10 Bioleaching gold and copper from 2% ESM by P. fluorescens in bioreactor, at 300C, 200 rpm, with no aeration Comparison of bioleaching in shake flasks and bioreactor SEM image of as-received ESM at magnification of 50x SEM image (a) of as-received ESM at magnification of 250x SEM image (b) of as-received ESM at magnification of 250x SEM image of <75m ESM at magnification of 50x SEM image of <75m ESM at magnification of 250x SEM image of <75m ESM at magnification of 500x SEM image of <75m ESM at magnification of 2000x SEM image of acid digested <75m ESM at magnification of 50x SEM image of acid digested <75m ESM at magnification of 500x SEM image of acid digested <75m ESM at magnification of 1000x SEM image of acid digested <75m ESM at magnification of 2000x 120 121 148 148 149 149 150 150 151 151 152 152 153 154 154 156 156 157 158 159

Figure B.11 (a) Selected area in elemental analysis by EDX/SEM of <75m ESM Figure B.11 (b) SEM/EDX spectra of <75m ESM Figure B.12 (a) Selected area in elemental analysis by EDX/SEM of acid digested <75m ESM Figure B.12 (b) SEM/EDX spectra of acid digested <75m ESM Figure B.13 (a) Selected area in elemental analysis by EDX/SEM of carbon tape Figure B.13 (b) SEM/EDX spectra of carbon tape Figure B.18 Analysis of specific surface area

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Introduction CHAPTER 1: INTRODUCTION Over the last few decades, there has been concern that gold, an irreplaceable element in many industries, may be depleted in the first half of the 21st century (Tateda et al., 1997). It is recognized that there is a need to find replacement for the traditional gold ores in order to delay gold depletion and to avoid the lack of gold in the future. Electronic Scrap Materials (ESM) has attracted interest as a good alternative source of gold, with its significant gold content and its fast increasing amount. Electronic products have high content of gold and other metals. When they are discarded, the high value metals present in the electronic materials is wasted if they are not recovered. At the same time, the toxic metals present in the discarded waste may leach into the groundwater and threaten life in the biota. Mining of gold as well as other metals has been carried out using hydrometallurgical and pyrometallurgical processes for a long time. However, this traditional method has been replaced gradually by environmentally friendly processes. Bioleaching using

microorganisms to leach metals from solid materials is considered a green way to recover metals from waste nowadays. Bioleaching of base metals such as Cu, Al, Mo, Ni, Mn, Zn, Pb, Fe has been studied for decades (Rohwerder et al., 2003). Meanwhile, gold is an inert element which is unable to be dissolved, and chemical leaching of gold (the most well known being cyanidation) is still being used widely to recover gold. Recovery of gold by cyanidation causes a lot of environmental problem as cyanide, a highly poisonous substance, is used at high concentration.

Introduction There are so-called cyanogenic microorganisms which can produce cyanide at the concentration in the ppm range. There are studies on application of these microorganisms on bioleaching gold. However, only few papers are found with ESM up to date. Thus, the first objective of this project is to investigate the potential of leaching gold from ESM by two cyanogenic bacteria: Pseudomonas fluorescens and Chromobacterium violaceum. It should be mentioned that there had been preliminary work from our group (Tan, 2006) which explored bioleaching ESM by the two bacteria. Tan used two different media for P. fluorescens and C. violaceum and compared bioleaching by these organisms. Apart from this, ESM used in this study and the previous one were from different sources. The second objective of this study is to improve gold recovery. Although biooxidation is used widely to improve gold recovery by (chemical) cyanidation in the mining of gold from mineral ores, there is no report on applying biooxidation before bioleaching. This approach is new and novel in gold bioleaching. Thus, effects of biooxidation by

Acidithiobacillus ferrooxidans prior to the bioleaching gold by C. violaceum and P. fluorescens were determined. Biooxidation should help to remove the mineral matrix, release gold and reduce unnecessary cyanide consumed in the complexation with metals other than gold. Moreover, the possibility of using bioreactor in bioleaching was initially studied to test potentials of scale up of the bioleaching processes. The research work reported here includes:

Introduction Determination some characteristics of ESM (elemental composition, toxicity, particle size distribution, specific surface area); Characterisation of growth of C. violaceum and P. fluorescens in experimental system; Investigation of bioleaching gold from ESM by C. violaceum and P. fluorescens in a range of pulp density 0.5-8%w/v; Biooxidation ESM by At. ferrooxidans in comparison with chemical leaching; Determination of the effects of biooxidation on bioleaching gold from ESM by C. violaceum and P. fluorescens in a range of pulp density 0.5-8%w/v; and Evaluation of the potential in the use of a bioreactor in bioleaching ESM.

Literature Review CHAPTER 2: LITERATURE REVIEW 2.1 2.1.1 Electronic Scrap Material (ESM) as a secondary gold ore Economic value of gold

Sources of gold Gold is found in nature in two major types of deposits: gold-quartz lodes and fossil placers. Contribution of other deposits is very small. Gold usually exists in the form of native gold and electrum (alloy of gold and silver). It also occurs in some others minerals (about 40 types), but rarely (Gasparrini, 1993). In the Earths crust, the average economic abundance of gold in the ores is in the range of 3.42-6.84 g gold/ton ore (Gasparrini, 1993). In many mining operations world wide, gold is being recovered from ores at yields of 0.5 to 13.5 g gold/ton ore (Korte et al., 2000). Gold is obtained as a by-product during the refining of copper or lead with significant amount, approximately one-fifth of the total resources of gold in the world. It is also present in sea water, but in too low concentration (0.0011-0.05 ppb) to be recovered economically (Gasparrini, 1993). Demand for gold Gold is used widely in monetary exchange, jewelry, industrial uses (electrical and electronics, dental bridges), award medals and coins. The rarity of gold makes it an expensive element. However, its use is still significant since there are few lower cost alternatives (Goodman, 2002).

Literature Review The steady annual demand for gold currently stands high at around 3000 tons, 350-400 tons (about 12%) of which is for industrial uses. In addition, increasing applications are being found for gold in other sectors such as biomedicine and catalysis (Corti and Holliday, 2004) which results in an increase in the demand for gold. Gold depletion In recent years, the depleting concentrations of gold in remaining gold ores is becoming a problem since more easily mined gold ores at existing higher concentration are being depleted (Olson, 1994). It is estimated that the total amount of gold yet to be retrieved from the Earth is 100,000 tons. If the gold is only obtained from mining gold ore, with the gold demand staying at 3000 tons annually, gold will be depleted in 30-40 years. This agrees with Tateda et al. (1997) who postulated that gold will be depleted in the year 2000-2049. Market value of gold As can be seen in Figure 2.1, the price of gold over the period of November 1998 to November 2008 had been increasing dramatically from around 600 $US per ounce up to 1000 $US per ounce, which is three times higher than the gold price in the last decade. Market value of gold as well as other metals varies with availability and demand (Gasparrini, 1993). Thus, increasing demand and decreasing availability (as discussed above) will lead to higher and higher price of gold in the near future.

Literature Review

Figure 2.1 Market price of gold in the last decade (http://www.goldprice.org/gold-price-history.html#10_year_gold_price) 2.1.2 Gold in electronic devices Gold in electronic devices contributes the largest part in the industrial demand for gold which is about 350-400 tons annually (Corti and Holliday, 2004). It is widely used in electronics because of its unique physical and chemical properties, such as excellent resistance to corrosion and oxidation, very low electrical resistivity (just higher than silver and copper), low current, low voltage conditions and low contact force. The largest use of gold in electronics is as an electroplated coating on connectors and contacts. This is followed by gold bonding within semiconductor packages. Smaller quantities are used in hybrid circuits, solderable coatings for printed circuit boards and components, as gold based solders and for metal layers in semiconductors as conductor tracks and contact pads (Goodman, 2002).

Literature Review 2.1.3 Electronic Scrap Material Definition When electronic products are discarded, they become electronic waste (e-waste). These wastes are also called electronic scrap, or electronic scrap material (ESM). ESM can be defined as a mixture of various metals, particularly copper, aluminum, and steel, attached to, covered with, or mixed with various types of plastics and ceramics (Hoffman, 1992). Its metal composition varies considerably with its age, origin and manufacturer and there is no average scrap composition (Cui and Zhang, 2008). Metal and non-metal composition of different sources of ESM from literature can be found in Table B.9. Amongst the metals in ESM, gold has the highest market value. In October 2008, the gold price was around 800 $US per ounce, and silver price was around 9.7 $US per ounce (The London Bullion Market Association (LBMA), http://www.thebulliondesk.com/). This gold price is about 10000 times higher than the price of base metals which is in the range of thousands of $US per tonne (The London Metal Exchange (LME), http://www.lme.co.uk/). Considering the market value, gold content in ESM and the environment, it has been pointed out that gold has the highest value distribution among the metals in ESM and that the attention on recycling should focus firstly on gold (Groot and Pistorius, 2008; Cui and Zhang, 2008).

Literature Review Benefits of gold recovery from ESM When electronic products are discarded, the high value of gold present in the electronic materials is devalued into a waste stream. As technology advances and the life cycle of electronic products become shorter and shorter (Solomon et al, 2000), this waste stream is expected to become increasingly significant with time. The amount of e-waste generated annually has been reported for many countries (Terazono et al., 2006). In Asia, China generates the largest amount of PC waste at 4,480,000 tons (Terazono et al., 2006). In UK, 50,000 tons of PCB scraps are disposed annually (Goosey and Kellner, 2002). Gold recovered from this source can delay gold depletion in the future. Compared with the gold content in gold ore of 0.5 to 13.5 g gold/ton (Korte et al., 2000), ESM has significantly higher gold content (10-1000 g gold/ton, see Table B.9). Thus recovery gold from ESM is much more attractive, and easier, and thus cheaper than from gold ores. Most, if not all of the gold used in electronic materials come from gold mining nowadays. Like many other mining operations, there are numerous environmental problems associated with gold mining. For instance, after a mining operation, the land is usually abandoned, leading to the formation of mining lakes. There are changes in the chemistry of millions of tons of natural ores during grinding for gold recovery, including the ruining of natural landscape and wildlife habitats (Korte et al., 2002). Besides the highly toxic chemicals used in mining gold, the residual unwanted metal tailings left behind contains metals such as zinc and lead which contribute in causing serious health problems to the local people. Hence, recovery gold from ESM has not only economic benefits; gold

Literature Review recovered from ESM can serve as another source of gold, hence reducing the demand on gold mining operations. Recovery gold and other metals from ESM also help to reduce the volume of waste for landfill or thermal destruction. With the above benefits, identifying and comparison of existing as well as potential processes that can efficiently recover the gold in electronic scrap will be discussed in the next sections. 2.2 Metallurgical recovery of metals from ESM 2.2.1 Definitions and classification Metal recovery from ESM can be carried out by pyrometallurgical processes, hydrometallurgical processes, and/ or biohydrometallurgical processes (Goosey and Kellner, 2002; Cui and Zhang, 2008). Pyrometallurgical processes are mostly conducted at high temperatures and often involve the melting of materials. Pyrometallurgical processes are subdivided into two large groups: roasting and metallurgical smelting, based on the temperature involved and the nature of the reactants (Volsky and Sergievskaya, 1971). Roasting is a metallurgical process conducted at high temperatures, but mostly without even a partial melting of the reacting phases; it involves reactions between solid and gaseous phases at temperatures of the order of 500-12000C. There are varieties of

Literature Review roasting processes such as calcining, oxidizing roasting of sulphide ores and concentrates, reducing roasting, chlorination and fluorination. Metallurgical smelting is a process in which liquid phases play a major part. It involves not just melting, but other complex chemical reactions. The solid materials interact and react with the gaseous phase, giving rise to a number of liquid phases and altered gaseous phases. The liquid mixtures possess a poor mutual solubility and, therefore, separate. Metallurgical smelting is subdivided into ore smelting (including reducing smelting, oxidizing concentration smelting, electrolytic smelting, metallothermic smelting, and reaction smelting) and refining smelting (liquation refining, distillation refining, oxidizing refining, chlorine refining, sulphidizing refining, carbonyl refining). Hydrometallurgical processes take place at temperatures between 10-3000C on the interface between solid and liquid phases. Hydrometallurgical processes are subdivided into leaching, refining of solutions from impurities, and precipitation of elemental metal from solution (Volsky and Sergievskaya, 1971). Leaching is the process of extracting a soluble constituent from a solid by leaching agents. The most common leaching agents used in recovery gold include cyanide, halide, thiourea, and thiosulphate (Cui and Zhang, 2008). Refining of solutions from impurities is carried out by precipitation with additives, extraction with organic solvents, adsorption or ion-exchange processes, or crystallization. Precipitation of elemental metal from solutions is applied with electrolytic precipitation (electrowinning), cementation, or solid reducing agent under pressure.

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Literature Review Some common definitions for bioleaching found in literature are listed below:

Bioleaching is a term used for extraction of metals (direct) or removal of constituents that interfere with the extraction of metal elements (indirect) through the mediation of microorganisms (Agate, 1996).

Bioleaching processes are based on ability of microorganisms (bacteria, fungi) to transform solid compounds resulting in soluble and extractable elements which can be recovered (Krebs et al., 1997).

Bioleaching is the biomediated recovery of precious metals from their ores (Nill, 2006).

2.2.2 Process of gold recovery from ESM Recycling metals from ESM can be broadly divided into three major steps: disassembly, upgrading and refining (Cui and Zhang, 2008). Disassembly is an indispensable process in recycling e-waste. ESM is dismantled and manually sorted into reusable, valuable for recovery or hazardous components requiring treatment before disposal (Brandl et al., 2001; Cui and Zhang, 2008). Upgrading is important to reduce the material volume, liberate metals or to obtain one or more concentrates (Groot and Pistorius, 2008). It is noticed that the maximum yield of precious metals was attained via shredding of boards without any additional comminution to reduce bulk volume (Goosey and Kellner, 2002). In refining, metals are melted (by pyrometalurgical processing) or dissolved (by hydrometallurgical processing) for removal of impurities. Lastly, recovery of the gold

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Literature Review back into a solid state is done through processes such as electrowining and precipitation (Cui and Zhang, 2008). Although the conventional method of pyrometallurgy is still used to recover metals, it has some limits, particularly in case of ESM. These include high cost due to high demand of energy, and the formation of some toxic substances (such as dioxins) due to very high temperature applied (Sum, 1991; Cui and Zhang, 2008). Hydrometallurgical processing is usually cheaper and more common nowadays than pyrometallurgical methods (e.g. lower power consumption and possible recycling of chemical reagents) (Sum, 1991). In the past two decades, the most active research area in the recovery of metals from electronic scraps is precious metal recovery using hydrometallurgical techniques. Unfortunately, these techniques suffer from several disadvantages such as the release of highly toxic substances (e.g. cyanide, chlorine), and the high cost due to high consumption of leaching agents (thiourea, thiosulphate) (Korte et al., 2000; Cui and Zhang, 2008). 2.2.3 Bioleaching in practice Bioleaching has been successfully applied in recovery of metals from metallic sulfides, which are the major bearing minerals for many base and precious metals, using sulfuroxidizing and iron-oxidizing bacteria. However, for the recovery of gold, leaching bacteria is industrially applied only to remove interfering metal sulfides from ores bearing the precious metals prior to cyanidation (see the discussion in Section 2.6.1).

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Literature Review Bioleaching has been used for the recovery of precious metals and copper from ores for many years. However, limited research has been carried out on the bioleaching of base metals (see Section 2.6.2) and precious metals from electronic waste (see Section 2.4). 2.3 Cyanogenic microorganisms and cyanide producing mechanism 2.3.1 General introduction

Cyanide is formed by a variety of bacteria (e.g. Chromobacterium violaceum, Pseudomonas fluorescens, P. aureofaciens, P. aeruginosa, P. plecoglossicida, P. putida, P. syringae, Bacillus megaterium), and fungi (e.g. Marasmius oreades, Clitocybe sp., Polysporus sp.), (Patty, 1929; Castric, 1975; Megathan and Castric, 1977; Askeland and Morrison, 1983; Knowles and Bunch, 1986; Kremmer and Souissi, 2001; Faramarzi et al., 2004; Faramarzi and Brandl, 2006; Brandl et al., 2008). They are called cyanogenic microorganisms. It has been generally believed that cyanide formation has an advantage for the organism by inhibiting competing microorganisms (Blumer and Haas, 2000). In cyanogenic bacteria, cyanide is considered a secondary metabolite because its production is independent of the growth phase (Castric, 1975). Cyanide is formed during growth only during a short time period (early stationary phase) (Knowles, 1976) in the presence of glycine (Castric, 1981; Michaels and Corpe, 1965). The maximum cyanide production of C. violaceum occurred in the onset of the stationary phase (Lawson et al., 1999). Glycine is a precursor of cyanide which is formed by an oxidative decarboxylation catalyzed by HCN synthase which is membrane-associated in both C. violaceum and P.

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Literature Review fluorescens (Knowles and Bunch, 1986). This enzyme is encoded by hcnABC cluster which was cloned and sequenced (Laville et al., 1998) and its role was demonstrated (Flaishman et al., 1996; Voisard et al., 1989). Induction of this gene of P. fluorescens by oxygen limitation requires the FNR-like transcriptional regulator ANR, an ANR recognition sequence in the -40 region of the hcn promoter, and non limiting amounts of iron (Blumer and Haas, 2000). Glycine is first oxidized to iminoacetic acid. Then, the C-C bond is split, with a concomitant second dehydrogenase reaction, which produces HCN and CO2 (Wissing, 1974; Knowles and Bunch, 1986) via Equation 2.1:

(2.1) 2.3.2 Chromobacterium violaceum Chromobacterium violaceum belongs to the genus Chromobacterium; it is rod-shaped with round ends, motile, and Gram-negative. This genus has four interesting charateristics: indole metabolism and biosynthesis of violacein, production of cyanide, occurrence of unusual sugar compounds, and the production of extracellular polysaccharide (Buchana and Gibbons et al., 1975). C. violaceum is rod-shaped, 0.6-0.9 by 1.5-3 um, often coccobacillary. It is facultatively anaerobic, although the oxidative strains grow slowly anaerobically. It is mesophilic and

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Literature Review can grow in the temperature range from 10-40 0C, and optimum temperature is at 30350C (Buchana and Gibbons et al., 1975). The optimum pH for its growth is 7-8 (Sneath, 1956). C. violaceum can produce violacein in the presence of tryptophan, it has antibiotic properties and is violet in colour. The bacterium is a soil and water organism, common in tropical and subtropical countries, occasionally causing serious pyogenic or septicemic infection in mammals, including man (Buchana and Gibbons et al., 1975). C. violaceum can produce extracellular cyanide during mid- to late- logarithmic and briefly in early stationary phase, and converts cyanide into -cyanoalanine during the stationary phase and death phase (Rodgers, 1978; Kita et al., 2006). Highest cyanide production occurs at the start of the stationary phase (Sneath, 1956; Michaels and Corpe, 1965; Castric, 1975; Knowles, 1976; Smith and Hunt, 1985), and can reach 45 ppm (without glycine supplement) (Lawson, 1999). 2.3.3 Pseudomonas fluorescens P. fluorescens belongs to the genus Pseudomonas, family Pseudomonadaceae. The genus Pseudomomas is traits of curved rods, motile, and obligate aerobe (Buchana and Gibbons et al., 1975). P. fluorescens is rod-shaped, 0.7-0.8 by 2.3-2.8 um during exponential phase (it becomes shorter and thinner in old cultures), motile with polar multitrichous flagellation, and occasionally non-motile. The bacterium produces diffusible fluorescent pigment, particularly in iron-deficient media. It is able to use from 60 to more 80 different carbon

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Literature Review sources for growth. Optimum temperature for growth is 25-300C (Buchana and Gibbons et al., 1975). Optimum pH for cyanogenesis by an unknown strain of P.fluorescens was established to be 8.3 in Tris/HCl buffer and between 7.3 and 7.8 in other buffers (Wissing, 1968). P. fluorescens can be found in soil and water. It in commonly associated with spoiled food and can be isolated from clinical specimens, and diseased plants (Buchana and Gibbons et al., 1975). 2.4 Applying cyanogenic microorganisms in gold bioleaching 2.4.1 Potential in bioleaching precious metals Cyanide can react with many metals forming highly water soluble complexes with very high chemical stability. Cyanide is one of few chemical that can dissolve gold (Chadwick and Sharpe, 1966); the reaction is shown in the following equation (Haque, 1992): 2Au + 4CN- + H2O + 1/2O2 2Au(CN)-2 + 2OH(2.2)

Thus, cyanogenic microorganisms can be used in bioleaching precious metals (gold, silver). Cyanide generated by cyanogenic bacteria is very tightly regulated as it cannot exceed the threshold of these microorganisms, and the local concentrations are usually below 1mM (Blummer and Haas, 2000), which is lower when compared with amount of cyanide used in chemical cyanide leaching. However, cyanogenic bacteria may form biofilm on the surface of materials (Campbell et al., 2001) that helps cyanide to

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Literature Review accumulate inside the biofilm and have close contact with the material, thus enhancing gold leaching efficiency even with low concentration of cyanidees. It was stated that cyanide production by C. violaceum was sufficient to dissolve gold, while maintaining a high cyanide concentration did not enhance gold dissolution (Kita et al., 2006). Although cyanide formation by bacteria is known for many years, there are very few reports on bioleaching precious metals by cyanogenic microorganisms: silver from silver containing jewelry waste, platinum from spent platinum containing automobile catalytic converter, gold from ESM, by P. plecoglossicida, (Faramarzi and Brandl, 2006; Brandl et al., 2008); and gold by C. violaceum and P. fluorescens which will be listed in next sections). 2.4.2 Gold bioleaching by C. violaceum and P. fluorescens To date, there are only few studies on bioleaching gold of Chromobacterium violaceum Pseudomonas fluorescens in the literature. C. violaceum has been studied more extensively than P. fluorescens. There are some studies on bioleaching gold by C. violaceum from gold-coated glass slides (Campbell et al., 2001), gold ores (Lawson et al., 1999; Campbell et al., 2001), electronic scrap (Faramarzi et al., 2004; Brandl et al., 2008), gold powder (Kita et al., 2006). There are few studies on bioleaching gold by P. fluorescens from electronic scrap (Brandl et al., 2008) In a study on bioleaching by C. violaceum and P. fluorescens, the former was found to be more efficient than the latter in leaching nickel from nickel powder (Faramarzi et al., 2004). Gold recovery by the two bacteria varies, depending on the type of materials (even different with same material from different source), experimental conditions (growth

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Literature Review medium, addition of glycine, etc.). Gold leaching by C. violaceum is highly variable depending on ore type and its gold content (Lawson et al., 1999). For example, after seven days bioleaching, C. violaceum ATCC 12472 could dissolve 83% of gold from gold coated glass slides, reaching concentration of nearly 40 mg/l, but only 28% gold from gold concentrate ore with concentration of 0.25 mg/l in the same paper of Campbell et al, 2001. 2.5 Factors influence gold bioleaching efficiency 2.5.1 Growth conditions There are many factors (e.g. pH, temperature, the concentration of oxygen and glycine) which can affect to the growth of bacteria as well as cyanide production, thus indirectly affecting its bioleaching efficiency. 2.5.1.1 Oxygen Oxygen is needed for the growth of both the aerobic bacteria C. violaceum and P. fluorescens, for cyanide production (Castric, 1975), as well as for the dissolution of gold (see Equation 2.2). Dissolved oxygen is the main factor affecting the efficiency of cyanide leaching of gold by bacteria. Increased oxygen concentration enhanced gold leaching from gold powder by C. violaceum four folds (Kita et al., 2006). However, the cyanogenic enzyme system, HCN synthase (HCS), is rapidly inactivated, both in vivo and in vitro, in the presence of oxygen (Castric et al., 1981). HCN production by Pseudomonas aeruginosa operated maximally at low oxygen levels, whereas moderate oxygen levels limited HCS activity (Castric, 1994).

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Literature Review 2.5.1.2 pH HCN has a pKa of 9.4 and the concentration of cyanide in solution is highly dependent on pH (Haque, 1992). Equilibrium of aqueous and gaseous cyanide can be describes as in Equation 2.3: H+ + CN- HCN (2.3)

Thus, low pH (ie high concentration of H+) shifts the equilibrium to the right, and thus promotes the production of HCN rather than CN-. Commercial leaching operations are conducted at a pH greater than 10.3 to prevent loss of cyanide via volatilization. However, gold bioleaching by the two cyanogenic bacteria takes place in physiosical pH, 7-8, suitable for growth of the bacteria. Although at the range of pH 7-8, cyanide may lost via volatilization, the maximum growth of C. violaceum as well as its highest cyanide production occurred at this initial pH range (Lawson et al., 1999). pH affects not only on growth of bacteria and its cyanide production, but also the chemical interaction of substances in solutions (e.g. stability of complexes), and hence affects gold dissolution. 2.5.1.3 Temperature Temperature significantly influences the reaction rate of enzyme-mediated chemical reactions which result in bacterial growth, on the structure activity of enzymes and proteins in bacterial cells, and on cell structures and functions (Caldwell, 1995). There is

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Literature Review a small range of optimal temperature for growth of bacteria. Growth of bacteria decrease rapidly when the temperature is out of this range, and increasing temperature from this range is more harmful than decreasing (Caldwell, 1995). In leaching gold by cyanogenic bacteria, temperature also affects cyanide production as well as volatilization of cyanide and the equilibrium of cyanide metal complexes in solution. All these factors affect the leaching efficiency of the bacteria. Optimum temperature for growth of C. violaceum and P.fluorescens is 30-350C and 25300C respectively (Buchana and Gibbons et al., 1975). All reported work on the bioleaching gold by the two bacteria that are listed in Section 2.4.2 were carried out at 300C. 2.5.1.4 Glycine Glycine is a precursor of cyanide. Thus, glycine concentration in the culture directly affects the bacterial cyanide production. Cyanide concentration as high as 200 ppm was observed when glycine (8-10 g/l) was added into the culture of C. violaceum, with nickel at 1 g/l in LB medium. However, increasing glycine addition (>10 g/l) at the time of inoculation can inhibit cell growth (Faramarzi et al., 2004). Maximum leaching of nickel from nickel powder (at1% pulp density) by P. plecoglossicida was attained with a glycine concentration of 5 g/l (Farramarzi and Brandl, 2006).

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Literature Review However, it should be noted that the amount of glycine added is different from the amount of glycine in the culture, since most complex medium contains glycine as an amino acid or in the protein structure. Moreover, the time of addition of glycine is as important as the glycine concentration. It was proved that delaying glycine supplementation until the start of the stationary phase resulted in higher cyanide production. Early glycine supplementation inhibited growth of bacteria (Campbell et al., 2001). Besides, there are many other substances that have been reported to affect gold leaching. For instance, ferrous sulphate and disodium hydrogen orthophosphate improved cyanide production by C. violaceum but reduced gold leaching efficiency (Lawson et al., 1999). Cyanogenesis is also stimulated by increasing iron (30 M) and phosphate content in the medium (Castric, 1975b; Rodgers and Knowles, 1978). 2.5.2 ESM 2.5.2.1 Characteristics of ESM Compositions As can be seen from the composition of ESM in the literature (Table B.9), heavy metal concentrations (e.g. iron, copper, etc.) are typically quite high. For instances, iron content varies from 3-62% and copper content varies from 3-27%. Although living organisms require trace amounts of some heavy metals, microorganisms in general may be resistant to these metals to different extent.

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Literature Review Heterogeneity Recycling of electronic scrap is still quite limited due to the heterogeneity of the materials present in the products and the complexity of the production of this material (Veit et al., 2005). 2.5.2.2 Effects of ESM on microbial growth Inhibition It has earlier been shown that although ESM in excess of 1%w/v inhibits the growth of microorganisms, growth still occurs until the concentration reaches 10%w/v (Brandl et al., 2001). The inhibition is likely to be caused by the toxic effect of the heavy metals in the ESM. In order to reduce the toxic effects of the heavy metals, a two-step process is usually applied. In the first step, microbial cells are grown in the absence of the ESM until a high enough biomass is obtained and/or until the production of chemical leaching agents in significant amounts have occurred (Brandl et al., 2001). The ESM is then introduced as a second step. Enhancement Campbell et al., 2001 suggested that the presence of gold may have a beneficial physiological effect on C. violaceum with the possibility that gold complexing with cyanide reduces the toxic effect of cyanide on the cell. Faramarzi and Brandl, 2006, observed addition of nickel up to 4 g/l stimulated growth of C. violaceum. However,

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Literature Review when pulp density was increased to over 4 g/l, the growth was reduced. The reason proposed was the toxic effects and/ or mechanical stress with increasing pulp density (Faramarzi and Brandl, 2006). 2.6 Bio-oxidation of ESM before bioleaching gold 2.6.1 Bio-oxidation gold ores In leaching gold from ores by cyanidation, bio-oxidation has been used to improve the leaching efficiency. In this process, bacteria are utilized to degrade the mineral matrix surrounding the gold, thus improving accessibility of gold in the subsequent cyanidation process (Norman and Raforth, 1995, Olson, 1994). Bio-oxidation has been industrially applied to pretreat refractory gold concentrates (in a commercialized process under the name BIOX, Aswegen et al., 2007) and sulfidic gold-bearing ores (BIOPRO process, Logan et al., 2007). Following bio-oxidation pretreatment, the gold is extracted and recovered by hydrometallurgical processes such as cyanide leaching and recovery on carbon or precipitation on zinc (Olson et al., 2003; Reith et al., 2007). It has been shown that gold extraction increased significantly after bio-oxidation (see Table 2.1).

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Literature Review

Table 2.1 Improvement of leaching gold from gold ore by bio-oxidation Gold extraction Material Organism(s) Without biooxidation Sulfidic gold ore Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans Sulfidic gold ore Sulfidic gold ore Refractory gold concentrates Sulfidic gold ore At. ferrooxidans, L. Sb. 71.3% Acidianus and ~ 45 ~ 70-95% Brierley, 2001 Sulfobacillus bacteria ~ 45 ~ 70-85% Brierley, 2001 ~ 45% ~ 70-90% Brierley, 2001 With biooxidation Reference

Metallospheara species At. ferrooxidans, L. 97% Aswegen 2007 (BIOX) Logan et al., 2007 (BIOPRO) et al.,

ferrooxidans

ferrooxidans, thermosulfidooxidans,

Acidianus, Metallosphaera Acid mine At. ferrooxidans, At. 56% 97% Nagy et al., 2007

Drainage generating tailings

thioxidans, L. ferrooxidans

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Literature Review 2.6.2 Bio-oxidation ESM Although bio-oxidation of gold ore has been widely commercialized in may parts of the world (Olson et al., 1994), there are few reports on leaching base metals from ESM (Brandl et al., 2001; Ten and Ting, 2003; Choi et al., 2005; Ilyas et al., 2007; Willscher et al., 2007) by At. thiooxidans, At. ferrooxidans, Sb. thermosulfidooxidans, L.ferrooxidans, Aspergillus niger, Penicillium simplicissimum). Some fungi such as A. niger, P. simplicissimum produce organic acids which can dissolve copper well (Brandl et al., 2001; Ten and Ting, 2006). However, chemolithoautotrophic bacteria such as At. ferrooxidans and At. thiooxidans have advantages in leaching metals as they require only inorganic carbon growth which is cheaper as compared with organic carbon. In addition, bioaccumulation of the bioleached metals by the fungal mycelia may lead to the loss of erstwhile bioleached metals. Besides, the mixture of At. ferrooxidans and At. thiooxidans using sulfur as the energy source, also showed better copper removal than A. niger or P. simplicissimum (Brandl et al., 2001). As can be seen from the components of ESM (Table B.9), although the content of metals can varies, nickel, zinc, and particularly copper, contribute the largest amount, and hence should be the focus in the bio-oxidation step. Acidithiobacillus ferrooxidans The physiology and classification of Acidithiobacillus ferrooxidans have been well summaried by Schipper, 2007: Species of Acidithiobacillus ferrooxidans (or At. Ferrooxidans, in short) formerly called Thiobacillus ferrooxidans, belongs to the genus

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Literature Review Acidithiobacillus which are obligately acidophilic (pH < 4.0), Gram- negative, and motile rods. At. ferrooxidans is an obligate autotroph, and derives energy from oxidization of ferrous iron (Fe2+), various sulfur compounds (e.g. elemental sulfur, thiosulfate, trithionate, tetrathinate and sulfide), and hydrogen. Since At. ferrooxidans grows in an environment with high metal ion concentration, it is resistant to many highly toxic metal. For example, it has been reported that the metabolic activity of this bacteria still occurs at metal concentration of 84 mM As(III), 800 mM Cu (II), 1071 mM Zn (II), 500 mM Cd (II) and 1000 mM Ni(II) (Schippers, 2007). At. ferrooxidans grows in a pH range of 1.3-4.5 (optimum pH at 2.5) and the temperature range of 10-370C (optimum temperature 30-350C). Bio-oxidation of ESM by At. ferrooxidans It was shown that even without the addition of ferrous ion, At. ferrooxidans could still leach copper from printed circuit board (PCB) waste at a concentration of 2550 mg/l (Choi et al., 2005). This is due to the presence of significant amount of iron in PCB as well as other ESM. The acidic condition of the medium (pH around 2-3) brought about the dissolution of ferrous ion for bacterial use as an energy source, following Equation 2.4 (Rossi, 1990): 2Fe2+ +2H+ +1/2O2 At. ferrooxidans 2Fe3+ + H2O (2.4)

Fe2(SO4)3 formed by At. ferrooxidans could leach elemental copper in the material via Equation 2.5: Fe2(SO4)3 + Cu0 Cu2+ + SO42- + 2FeSO4 (2.5)

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Literature Review When Fe2+ was added at 7g/l, the copper concentration of the leachate was more than 5000 mg/l. This is equivalent to the maximum amount of copper can be dissolved in 2.5%w/v with a copper content of 20% (Choi et al., 2004). Learning from two processes BIOX and BIOPRO, At. ferrooxidans is always used with high efficiency. Our primary study confirmed higher copper recovery by At. ferrooxidans than At. thiooxidans at 1% pulp density with the same source of ESM, and the highest copper recovery was attained with At. ferrooxidans at 9g/l ferrous (Teo, 2007). Thus, ESM was bio-oxidized using At. ferrooxidans with 9g/l ferrous before the bioleaching of gold with the cyanide generating bacteria, i.e. C. violaceum and P. fluorescens.

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Materials and Methods CHAPTER 3: MATERIALS AND METHODS 3.1 Materials 3.1.1 Electronic Scrap Material (ESM) 3 kg electronic scrap material was supplied by Cimelia Resource Recovery Pte Ltd. ESM was stored in plastic bottles, kept a dry cabinet (Weifo), with humidity at 50%, and at room temperature (250C). The ESM was ground in a blender and screened using a 75m-sieve (Retsch). ESM of particle size <75m fraction (i.e. particle size up to 75 m) was used for this study. 3.1.2 Bacteria Chromobacterium violaceum (ATCC 12472) and Pseudomonas fluorescens Migula (BAA 477) were purchased from the American Type Culture Collection (ATCC). The cultures were supplied freeze-dried. C. violaceum was activated in the Difco Nutrient Broth and P. fluorescens was activated in Lysogeny broth (LB). These bacterial cultures were stored in a deep freezer (at about 750C) with their medium, supplemented with 15% glycerol. Acidithiobacillus ferrooxidans (tfH6) was kindly provided by Professor Natarajan K.A., IISc, Bangalore, India. This culture was maintained by subculturing in fresh 9K medium every two weeks. 3.2 Experimental methods

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Materials and Methods 3.2.1 Analytical methods 3.2.1.1 Particle size distribution The particle size distribution of ESM was analysed using the Coulter LS 230 particle size analyzer. The material was wetted with ultrapure water and loaded into the instrument which can analyze particle size distribution up to 2000 m. The light scattering particle size analyzer consists of 116 channels spaced logarithmically, and is capable of detecting a specified particle size at each channel. Particle size distribution is reported in volume percent. 3.2.1.2 Specific Surface Area A BrunauerEmmettTeller (BET) surface area analyzer (Quantachrome, Nova 3000 ver 6.07) was used in determining the specific surface area of ESM. Sample weights at 0.200 0.005 g were degassed overnight at 100C using nitrogen gas as the absorbent. The sample was immersed in liquid nitrogen at a pressure of 770 mm Hg and a temperature of 77.40K. The specific surface area was then calculated based on the BET equation. 3.2.1.3 Acid digestion Ten samples with weight 1.0000 g 0.0005 g were added in 250-ml Erlenmeyer flasks. Each sample was digested by three batches of acids (two batches of 40 ml of concentrated nitric acid, followed by one batch of 40 ml of aqua regia). After each batch of acid was added, the mixture was left for one day, then centrifuged at 5000 rpm, in 15

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Materials and Methods minutes and the liquor was collected. Ultra-pure water was used for washing the residue after aqua regia digestion. The liquor and the washing solution were added into a 200-ml volumetric flask, and topped up using ultra-pure water and then kept at 4oC prior to metal analysis by ICPOES. Metal compositions of the ESM were calculated as shown in Appendix A.1. The residue was weighed until constant mass and kept in a dry box for qualitatively elemental analysis by SEM/EDX (see 3.2.1.5). 3.2.1.4 Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) Equipment Metal analysis for aqueous sample was performed using inductively coupled plasma optical emission spectrometer (ICP-OES, Perkin-Elmer, Optima 3000V) with the following settings: Table 3.1 Operating conditions of ICP-OES Parameter RF Power (W) Argon Gas Flow rate (l/min) Plasma Auxiliary Nebulizer Pump Speed (rpm) Sample uptake (ml/min) Plasma _ Value_______________ 1300 15 0.5 0.8 18.48 1.0 Radial

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Materials and Methods Table 3.2 The wavelengths (nm) used for metal analysis by ICP-OES Element Gold Platinum Silver Aluminum Camidium Cobalt Cromium Copper Iron Lithium Niken Lead Zinc Symbol Au Pt Ag Al Cd Co Cr Cu Fe Li Ni Pb Zn Wavelength (nm) 242.795 214.423 328.068 308.215 214.438 228.616 205.560 324.754 238.204 670.781 232.003 220.353 213.856

Calibration Calibration standards (10, 20, 50, 100 ppm) for the analysis all of the metals above (except gold and platinum) was prepared using an ICP multi-element standard IV (Merck) with 1000 mg /l by diluting with 5% w/w nitric acid. Gold calibration standards (0.1025, 0.5125, 1.025, 2.05 and 10.25 ppm) was prepared using AAS Gold solution (1025 0.5 mg/l) by diluting with a solution of 5% w/w nitric acid and 2% w/w hydrochloric. Blanks for calibration were solutions used to prepare standards. The calibration was accepted when the error coefficient of all metal analyzed were 0.9990 at least.

31

Materials and Methods Sample preparation Samples were filtered via nylon membranes, 0.45 m, 25 mm (Whatman) after collection, acidified with concentrated nitric acid at 5% and stored at 40C. Stored samples were diluted by 5% w/w nitric acid to metal concentration up to 100 ppm before injected to ICP system. 3.2.1.5 Scanning Electron Microscopy/Energy Dispersive X-Ray Analysis Scanning Electron Microscopy/Energy Dispersive X-Ray (SEM/EDX) Analysis (Joel JSM-5600LV) was used to observe the morphology as well as to determine the elemental compositions of the solid samples. Samples were loaded on copper studs using carbon tape and then coated with 10nm platinum particles under vacuum condition at 30 mA over 30 seconds, using Jeol JFC1300 auto fine coater. Images of the ESM at different magnifications were then captured using the Jeol JSM-5600 LV scanning electron microscope, at acceleration voltage 15kV and spot size 20 mm. The smallest magnification (50x) was used in the EDX analysis for determination of the elemental compositions. At lower magnification, a broader field over a larger area would give a better and more representative of the results. 3.2.1.6 Toxicity Characteristic Leaching Procedure Toxicity Characteristic Leaching Procedure was conducted with US EPA SW 846 method 1311, followed by comparing metal concentration of TCLP extracts with regulatory levels stated in this method to determine the toxicity of the solids. In general, the solid material is extracted with an amount of extraction fluid equal to 20 times the

32

Materials and Methods weight of the solid phase. The extraction fluid employed is a function of the alkalinity of the solid phase of the waste. Extraction fluids Extraction fluid # 1 was prepared as follows: Add 5.7 ml glacial CH3CH2OOH to 500 ml of ultrapure water, add 64.3 ml of 1N NaOH, and dilute to a volume of 1 liter (pH of the fluid should be 4.93 0.05). Extraction fluid # 2 was prepared as follows: Dilute 5.7 ml glacial CH3CH2OOH with ultrapure water to a volume of 1 liter (pH of the fluid should be 2.88 0.05). Determine extraction fluid 96.5 ml of ultrapure water was added to 5.0 g of ESM and stirred vigorously for 5 minutes using a magnetic stirrer. The pH was measured and recorded. - If the pH was <5.0, extraction fluid #1 was used. - If the pH was >5.0, 3.5 ml 1N HCl was added, stirred briefly, then heated to 500C, and held at 500C for 10 minutes. The solution was cooled to room temperature and the pH was noted. If the pH was <5.0, the extraction fluid #1 was used. If the pH is >5.0, the extraction fluid #2 was used. Extraction 100 ml of the appropriate extraction fluid was added to 5 g of material. The mixture was shaken for 182 hours at 230C. After that, the mixture was filtered via a glass fiber to

33

Materials and Methods collect the TCLP extract. The extract was acidified using concentrated nitric acid at 5% and stored at 40C for metal analysis using ICP-OES. The concentration of metals in the TCLP extract was compared to the regulatory level of US EPA. 3.2.1.7 pH measurement The pH of all the samples was measured using a Mettler Toledo 320 pH meter and Orion 9156BNWP pH electrode. Two-point calibration (4 and 7, or 7 and 10) was conducted before measurement. 3.2.1.8 Optical Density Optical density was measured with a UV-Spectrophotometer using plastic cuvette of beam path of 10mm. Optical density of cultures of C. violaceum and P. fluorescens were measured at wavelength 600 nm and 450 nm respectively (following the protocol of Faramarzi et al., 2004). 3.2.1.9 Free Cyanide Concentration Free cyanide concentration measurement was conducted following the Colorimetric Method (Eaton et al., 1995). Reagents Chloramine-T solution was prepared by adding 0.2 g Chloramine-T in 20 ml ultrapure water. This solution was kept in refrigerator for up to one week. Pyridine-barbituric acid was prepared as follows: for 50 ml, 3 g of barbituric acid was placed in a 50 ml amber bottles. 15 ml of pyridine and 3 ml of concentrated hydrochloric 34

Materials and Methods acid were added in fumehood. The mixture was stirred and cooled to room temperature before it was topped up to 50 ml using ultrapure water. This reagent was kept in a refrigerator for up to 6 months and discarded if precipitation develops. Acetate buffer was prepared as follows: for 10 ml, 4.1 g sodium acetate trihydrate (NaC2H3O2.3H2O) was dissolved in 5 ml water. Then 5 ml glacial acetic acid was added to adjust to pH 4.5 and to top up to 50 ml. Sample storage After collection, samples were filtered via nylon membrane, 0.45 m, 25 mm (Whatman). The filtered samples were preserved by adding 1% NaOH 10M and kept in plastic containers at 40C until analysis (up to 14 days after collection). Calibration 1000 ppm Cyanide Ion Chromatography Standard Solution (Fluka) was used to prepare a series of cyanide standards at concentrations of: 0, 0.02, 0.04, 0.08, 0.12, 0.16, 0.2 ppm in NaOH 0.04M. Fresh standards were prepared daily. Procedure Samples were diluted to final NaOH concentration of 0.04M just before analysis. Mixture of 3 ml sample, 150 l chloramine-T solution and 75 l acetate buffer are mixed and left to stand for two minutes exactly. Then, 375 l pyridine-barbituric acid was added, mixed thoroughly, and left to stand for eight minutes. After that, absorbance at 578 nm was measured using a spectrophotometer at 578nm. The sample was diluted further and the

35

Materials and Methods procedure was repeated if the absorbance was higher than the absorbance of the 0.2 ppm standard. The calibration curve and cyanide concentration were calculated as shown in Appendix A.2. 3.2.2 Bacterial culture All bacterial cultures were kept frozen in a deep freezer in their medium supplemented with 15% glycerol. Before inoculation, they were activated by subculturing in fresh medium. The activated cultures were inoculated in a fresh medium to obtain bacterial culture at log phase for use as an inoculum for experimental cultures. Chromobacterium violaceum and Pseudomonas fluorescens C. violaceum and P. fluorescens were cultivated in LB medium (Bertani, 1951) in a batch culture (in 250-ml Erlenmeyer flasks, or a bioreactor), with inoculum size at 1%v/v, maintained at 300C and shaken/stirred at 200 rpm. The LB medium consists of 10 g/l bacto tryptone, 5 g/l yeast extract and 10 g/l NaCl. The medium was autoclaved at 121oC in 20 minutes, cooled to room temperature before inoculation. Plate count method was used to determine concentration of bacteria in the culture. The culture was diluted serially and then spread on LB agar plates; results were based on Petri dishes with 20-200 CFUs (colony forming unit) and CFU/ml of the original culture was calculated based on the dilution used.

36

Materials and Methods Acidithiobacillus ferrooxidans At. ferrooxidans was cultivated in Silverman and Lundgrens 9K medium (Silverman and Lundgren, 1959), in a batch culture (in 250-ml Erlenmeyer flasks, or a bioreactor), with inoculum size at 10%v/v, and maintained at 280C and stirred at 200 rpm. One liter of 9K medium was prepared by mixing an iron solution and a basal salts solution. For the iron solution, 44.1 g FeSO4.7H2O was added in 300 ml water acidified with 1ml H2SO4 10N. This solution was filtered sterilised via a Whatman cellulose acetate membrane (0.2m, 47mm). The basal salts solution consisted of 3.0 g (NH4)2SO4, 0.1 g KCl, 0.5 g K2HPO4, 0.5 g MgSO4.7H2O and 0.01 g Ca(NO3)2 in 700 ml DI water. This solution was autoclaved at 1210C in 20 minutes. 3.2.3 Bioleaching experiments 3.2.3.1 Shake flask bioleaching Bioleaching was conducted in 250-ml Erlenmeyer flasks with 100 ml of LB medium, at five pulp densities: 0.5%, 1%, 2%, 4% and 8% w/v. The cultures were kept at 300C, 200 rpm, over seven days after addition of ESM. 10 ml of each sample was withdrawn daily for following analysis: pH, free cyanide concentration, and metal concentration. All experiments were conducted in duplicate. Autoclaved medium with ESM but without bacteria were used as controls. One-step bioleaching

37

Materials and Methods One-step bioleaching was conducted following the method in Bosshard et al. (1996). Flasks containing ESM and LB medium were autoclaved. Activated bacteria at the log phase were inoculated at 1%v/v. Two-step bioleaching In two-step bioleaching, ESM was autoclaved separately and added to bacterial culture after one day of inoculation, when the culture obtained significant amount of cell density and free cyanide concentration. 3.2.3.2 Bioleaching in bioreactor Equipment A bioreactor (LiFlus GS, Biotron) (see Figure 3.1) with a working volume of 800 ml (inner diameter D=190 mm; inner height H=367 mm) was used. The temperature of medium in the vessel was monitored by a platinum probe and controlled by circulating water from a water bath into the double-jacketed vessel. Gas exhausts via a condenser on top of the bioreactor was cooled using cooling water from the water bath to prevent loss of medium via vapourization. Dissolved oxygen (DO) value was controlled in the range of 0-100% and sensed using a Hamilton electrode. Calibration of 0% and 100% was achieved by flushing pure nitrogen gas and air in water until saturation. pH was monitored over the range 2.0-12.0 and measured using a Melter Toledo electrode. Threepoint calibration was conducted with pH standard solutions of pH 4, 7 and 10. Air was supplied by a pump via a 0.2 m PTFE sterilized membrane. The air flow rate was set

38

Materials and Methods manually via a control valve on a flow meter. Liquid samples were collected by applying pressure through the sampling line. The operating parameters DO, pH and temperature were monitored and controlled online by the LiFlus GS software.

39

Materials and Methods Set-up

7 4

3 2

8 1

Figure 3.1 Experimental set up of bioreactor system 1-Double jacketed vessel; 2-pH probe; 3-DO probe; 4-motor; 5-condenser; 6-control panel; 7-peristaltic pump; 8-air filter

The medium used in bioleaching in the bioreactor was prepared in the same way as the shake flask bioleaching experiments. Calibration of DO probe and pH probe were conducted before being autoclaving. The bioreactor (with medium and reagents) was autoclaved at 1210C, for 30 minutes and cooled to room temperature before inoculation. Bioleaching was carried out at 300C, 200 rpm, with air flow rate at 1 vvm (or without aeration). The DO, pH and temperature were recorded every minute. Sample was

40

Materials and Methods withdrawn daily for analysis of the free cyanide concentration and the metal ion concentration. 3.2.4 Bio-oxidation experiments Bio-oxidation was conducted in the bioreactor (as discussed in 3.2.3.2) but using 800 ml of 9K medium supplemented with ferrous ion (9 g/l), at 280C, a stirring speed of 200 rpm, and air flow rate of 1 vvm. 1%w/v autoclaved ESM was added seven days after inoculation. In the control batch, the medium was added with 10% sterilized medium instead of bacterial culture. Sample was then withdrawn daily for analysis of the metal ion concentration. After that, the culture was centrifuged at 40C, 10,000 rpm for 10 minutes to separate the biooxidized ESM. The recovered ESM was then washed by DI water. The procedure was repeated twice to ensure complete removal of the medium. Finally, the biooxidized ESM was dried for bioleaching experiments. 3.2.5 Chemical leaching ESM were leached using commercial reagents. These include common inorganic acids (nitric acid, and sulphuric acid), organic acids which are commonly produced by fungal cultures (citric, oxalic, and gluconic acids), and ferric sulphate (i.e. ferric ion produced from the oxidation of ferrous ions by At. ferrooxidans). Acid leaching

41

Materials and Methods 100 ml of acid solutions at different concentrations (10 mM, 50 mM, 100 mM and 200 mM) in DI water were prepared in 250-ml Erlenmeyer flasks. Each flask was added with 1 g ESM, covered with parafilm, shaken at 300C, and 200 rpm in an incubator. Control was run with 1 g ESM in 100 ml DI water. After seven days, samples were collected and filtered through 0.45 m, 25 mm membranes (Whatman). Samples were then acidified by 5% HNO3 and stored at 40C until analysis. All experiments were conducted in duplicates. Ferric ion leaching Ferric ion leaching was conducted in 250-ml Erlenmeyer flasks. 100 ml ferric chloride solution (with or without 5 mM sulphuric acid) at the same concentration as the initial ferrous concentration in the 9K medium- 9 g/l was added in each flask. To each flask was added 1g ESM, covered with parafilm, shaken at 300C, and at 200 rpm in an incubator. Control was run with 1 g ESM in 100 ml DI water. After one week and two weeks, all samples were collected and filtered via 0.45 m, 25 mm membranes (Whatman) after collection. Samples were then acidified by 5% HNO3 and stored at 40C until further analysis. All experiments were conducted in duplicates.

42

Results and Discussion CHAPTER 4: RESULTS AND DISCUSSION 4.1 Characterization of ESM 4.1.1 Particle size distribution The size range and corresponding percentage weight distribution of the as-received ESM are shown in Table B.1. Although the as-received ESM showed a quite fine particle size (nearly 50% smaller than 150 m), grinding and sieving of the as-received ESM were applied to obtain more homogenous particles for experiments. The particle size distribution of the <75m ESM is shown in Figure 4.1; the particle had a mean size of 36.56 m. Full results of the particle size analysis can be found in Appendix B.1.2.1.

Figure 4.1 Particle size distribution of <75m ESM

4.1.2 Specific surface area In comparison with the ESM reported in Ten and Ting (2003), this fine ESM had a similar size range but an even lower specific surface area (0.6136 m2/g compared with

42

Results and Discussion 1.95 m2/g). Both of the ESM (from different sources) showed a much lower specific surface area than powdered activated carbon (500-1500 m2/g) with the same range of particle size (1-150 m) (Norit Activated Carbon, 2008, http://www.norit-

ac.com/english/activatedcarbon.asp?submenuCat=introduction). This is most probably due to its non-porous structure and smooth particle surfaces as can be seen in the morphology of ESM in Figure 4.2 (a-d).

4.1.3 ESM elemental composition Metal composition of the liquor after acid digestion is shown in Table 4.1. In SEM/EDX, none of the elements above was detected in the residues after acid digestion (see c d Appendix B1.4.2). Thus the metal composition in the liquor after acid digestion reflects the actual metal content in ESM. The concentration of each of the metal ions after digestion is thus used in the calculation of the percentage recovery in bioleaching as the maximum amount of that metal can be recovered from this material (i.e.100% recovery). Table 4.1 also shows that the liquors from the as-received ESM and the <75um ESM are Figure 4.2 Images of <75m ESM under Scanning Electron Microscope (SEM) (at significantly different, thus confirming that the particles are heterogeneous, and that the different magnifications: (a) 50X; (b) 250X; (c) 500X; (d) 2000X) 4.1.3 ESM elemental composition The metal composition of the liquor after acid digestion is shown in Table 4.1. In SEM/EDX, none of these elements was detected in the residues after acid digestion (see

43

Results and Discussion Appendix B.1.4.2). Thus the metal composition in the liquor after acid digestion reflects the actual metal content in ESM. The concentration of each of the metal ions after digestion is thus used in the calculation of the percentage recovery in bioleaching as the maximum amount of that metal can be recovered from this material (i.e.100% recovery). Table 4.1: Metal composition of the liquor after acid digestion Content (g/kg ESM) Metal Au Ag Al Cu Fe Ni Pb nd: not detected Table 4.1 also shows that the digestates from the as-received ESM and the <75m ESM are significantly different, thus confirming that the particles are heterogeneous, and that the various metal ions are not homogeneously distributed throughout the particles. (For instance, it was noted that the <75m ESM showed copper content of only 11% of the asreceived ESM). Gold content in this ESM was also much higher compared with other ESM reported in Brandl et al. (2001), Ten and Ting (2003), and Cui and Zhang (2008). The high copper content in the as-received ESM is comparable with ESM in Cui and Zhang (2008), but is much higher than ESM in Brandl et al. (2001) and Ten and Ting (2003). As-received ESM 2.4320.502 3.4360.158 2.8250.053 253.92.3 17.730.52 7.9510.517 2.3410.041 <75m ESM 4.8720.115 nd 2.8800.048 28.320.33 20.590.49 5.8060.207 1.0050.036

44

Results and Discussion Besides, SEM/EDX spectra of ESM at different positions (Appendix B.1.4.1) showed the abundance of silica in ESM which accounts for the high percentage residue weight remaining after acid digestion (more than 90% weight). 4.1.4 Toxicity Characteristic Leaching Procedure Toxicity Characteristic Leaching Procedure (TCLP) tests were conducted using US EPA SW 846 method 1311. A comparison of the metal concentration in the TCLP extracts against the regulatory levels stated allows one to characterize the hazardous nature of the ESM. To select a suitable extraction fluid, the pH of the mixtures from reagent water and ESM were determined. The value were 7.950.05 and 8.450.05, for as-received ESM and the fine ESM accordingly. Following the procedure, the mixtures were treated with hydrochloric acid and heat. With the resultant pH of 1.050.05 and 1.200.05

respectively, extraction fluid #1 was used for the TCLP test. As can be seen in Table 4.2, the concentration of lead in the TCLP extracts of both ESM (<75m and as-received) were higher than the regulatory level stated in CFR (Code of Federal Regulations) 261.24 Toxicity characteristic by US EPA (Environmental Protection Agency). Hence, the ESM is characterized as a hazardous waste. The

concentration of nickel and lead in the extracts from the TCLP tests of both ESM were much higher than the regulatory level of hazardous waste for land disposal (CFR 268.40) by US EPA. Under the Land Disposal Restriction (LDR) regulations, the concentration of these metals must be reduced to lower than the regulatory level before land disposal.

45

Results and Discussion Table 4.2 Metal concentration of the TCLP extract compared with Regulatory levels by US EPA TCLP extract (mg/l TCLP) <75m ESM 1.6730.030 450.810.1 10.450.106 130.32.298 nd nd 40.750.778 4.1080.274 Regulatory level (mg/l TCLP) Hazardous waste Metal Al Cu Fe Ni Co Ag Pb Zn As-received ESM 2.090.088 282.315.03 10.650.318 93.5.354 nd nd 61.689.776 nd for land disposala ns ns ns 11 ns 0.14 0.75 4.3 ns ns ns ns ns 5 5 ns Toxic wasteb

ns: not stated; nd: not detected a 268.40 Applicability of treatment standards. U.S Code of Federal Regulations (CFR), Title 40, Part 268: Land Disposal Restriction (LDR). Subpart D: Treatment Standards. http://ecfr.gpoaccess.gov/cgi/t/text/textidx?c=ecfr&sid=58204e14b6f985f03d09f71e683c830c&rgn=div5&view=text&node=40: 26.0.1.1.3&idno=40#40:26.0.1.1.3.4.27.1 b 261.24 Toxicity characteristic. U.S Code of Federal Regulations, Title 40, Part 261: Identification and Listing of Hazardous Waste. Subpart C: Characteristics of Hazardous Waste. http://ecfr.gpoaccess.gov/cgi/t/text/textidx?c=ecfr&sid=385ea9ac5d6e0fb83be2351f0836eb09&rgn=div5&view=text&node=40: 25.0.1.1.2&idno=40#40:25.0.1.1.2.3.1.5 4.2 Pretreatment ESM 4.2.1 Chemical leaching ESM were leached using commercial reagents. These include common inorganic acids (nitric and sulphuric acid), organic acids which are commonly produced by fungal cultures (citric, oxalic, and gluconic acids), and ferric sulphate (i.e. ferric ion produced from the oxidation of ferrous ions by At. ferrooxidans).

46

Results and Discussion 4.2.1.1 Acid leaching In acid leaching, 1% w/v ESM was added to various acid solutions (mentioned above) at 10-200 mM. Leaching of some of the major metal ions from ESM in these solution is shown in Figures 4.3 (a, b, c, d, and e). More details in the removal other metals can be found in Appendix B.6. As can be seen in Figures 4.3 (a, b, c, d, and e), at the concentration range from 10-200 mM, these acids (except oxalic acid) showed good removal of copper and nickel. The poor dissolution of copper and nickel by oxalic acid (see Figure 4.3 (d)) can be explained by the low solubility product of copper oxalate (CuC2O4, Ksp=2.3x10-8) and nickel oxalate (CuC2O4, Ksp=4.0x10-10) (Patnaik, 2004). The data reported may provide some useful information if pretreatment of ESM (by chemical or biological means) is considered prior to its bioleaching, as a strategy for the recovery of gold. For instance, in gold bioleaching where the co-existence of copper may interfere and compete with gold for the lixiviant and hence reduce the gold recovery, the use of any of the acids (except oxalic acid; see Fig. 4.3 (d)) result in the removal of copper, and thus enhance gold recovery.

47

Results and Discussion

Metal removal by nitric acid 100 90 80 Metal removal (%) 70 60 50 40 30 20 10 0 10mM 50mM 100mM 200mM Al Ni Fe Cu

Figure 4.3 (a) Removal of metals from <75m ESM by nitric acid

Metal removal by sulphuric acid

100 90 80 Metal removal (%) 70 60 50 40

Al Ni Fe Cu

30 20 10 0 10mM 50mM 100mM 200mM

Figure 4.3 (b) Removal of metals from <75m ESM by sulphuric acid

48

Results and Discussion

Metal removal by citric acid

100 90 80 Metal removal (%) 70 60 50 40

Al Ni Fe Cu

30 20 10 0 10mM 50mM 100mM 200mM

Figure 4.3 (c) Removal of metals from <75m ESM by citric acid

Metal removal by oxalic acid

100 90 80 Metal removal (%) 70 60 50 40

Al Ni Fe Cu

30 20 10 0 10mM 50mM 100mM 200mM

Figure 4.3 (d) Removal of metals from <75m ESM by oxalic acid

49

Results and Discussion

Metal removal by gluconic acid

100 90 80 Metal removal (%) 70 60 50 40 30 20 10 0 10mM 50mM 100mM 200mM

Al Ni Fe Cu

Figure 4.3 (e) Removal of metals from <75m ESM by gluconic acid

4.2.1.2 Ferric leaching In ferric leaching, 1% w/v ESM was added in the ferric solution with the same iron concentration (i.e. 9 g/l) with the initial ferrous concentration in the 9K medium for Acidithiobacillus ferrooxidans (At. ferrooxidans). The role of ferric ion in the dissolution of metal from ESM is shown in Table 4.3, in comparison with two controls (i.e. ferric solutions acidified by sulphuric acid, and water). As can be seen in Table 4.3, the ferric solution removed about 55% of copper in ESM. Sulphuric acid added to adjust the pH to the initial pH of the 9K medium (which is suitable for the growth of At. ferrooxidans) did not increase the removal of copper, nickel and aluminum significantly. Full details in metal removal by ferric solution can be found in Appendix B.6.

50

Results and Discussion Table 4.3 Metal removal of ESM by ferric solution Fe3+ No 1 2 3
*

H2SO4 5 mM
*

Metal removal (%) Cu 0.005 Ni 1.206 97.40 99.55 Al 0.156 47.40 48.35

water 9 9 9

(9 g/l) 9 9

55.14 9 54.87

5 mM H2SO4 equals to the concentration of sulphuric acid in the 9K medium before autoclave.

4.2.2 Bio-oxidation by Acidobacillus ferrooxidans Bio-oxidation by At. ferrooxidans was conducted at 280C and at 200 rpm, with an air flow rate of 1v/v/m in a bioreactor. 1% w/v ESM was added to the culture after seven days of inoculation to ensure the cell population was high enough to be maintained in the presence of the ESM, as well as a high concentration of ferric ion. In the control batch, the medium was added with 10% sterilized medium instead of the bacterial culture. The pH profiles and removal of the three major metals (copper, nickel, and aluminum) in the experimental culture (with bacteria) and the control (no bacteria) are shown in Figure 4.4 (a) and (b) respectively. Oxygen dissolved in bio-oxidation is not shown since oxygen was kept in the 9K medium at too low concentration to be detected that may be caused by its acidic property. The conversion of ferrous ion by At. ferrooxidans, as an energy source, to ferric ions is shown in Equation 4.1 (Rossi, 1990): 2Fe2+ +2H+ +1/2O2 At. ferrooxidans 2Fe3+ + H2O (4.1)

51

Results and Discussion The consumption of H+ contributed to the increase in pH in the At. ferrooxidans culture before the addition of ESM (Figure 4.4 (a)). This did not occur in the control (without bacteria- Figure 4.4 (b)).

4 3.5 3 2.5 pH 2 1.5 1 0.5 0 0 2 4 6 8 10 12 14 Time (days)

90

(a)

80 Metal removal (%) 70 60 50 40 30 20 10 0

pH Copper Aluminum Nickel

Figure 4.4(a) Bio-oxidation of ESM by At. ferrooxidans (ESM was added on day 7)

4 3.5 3 2.5 pH 2 1.5 1 0.5 0 0 2 4 6 8 10 12 14 Time (days)

90

(b)

80 60 50 40 30 20 10 0 Metal removal (%) 70

pH Copper Aluminum Nickel

Figure 4.4(b) Control of bio-oxidation of ESM (ESM was added on day 7)

After adding ESM, leaching of metals was recorded daily. In the experimental culture, metal ions may be leached by ferric iron produced by At. ferrooxidans. For example, copper may be leached following Equation 4.2: 52

Results and Discussion 2Fe3+ + Cu0 Cu2+ + 2Fe2+ (4.2)

Meanwhile, it should be noted that a significant amount of metals was dissolved in the control. This is due to the presence of ferric ion arising from oxidation of ferrous ion in the medium rather than bio-oxidation by the bacteria. Moreover, sulphuric acid which is added at concentration of 5 mM to adjust pH of the medium to optimal pH for growth of At. ferrooxidans may also contribute to dissolution of metals from ESM. The dissolution of metals in acidic solution can generalized in Equation 4.3: 2M0 + 2nH+ 2Mn+ + H2 (4.3)

After one day of addition of ESM, copper, aluminum and nickel were detected in the experimental culture (in presence of At. ferrooxidans) at quite high concentration, with metal removals at 69.6%, 50.2% and 17.7% respectively. Meanwhile, copper and aluminum were also detected in the control at lower concentration (at 47.1% and 35.4% removal respectively). Nickel was only detected in the control after two days of adding ESM, with lower concentration than in the experimental culture. Seven days after the addition of ESM, At. ferrooxidans removed more than 80% copper, nearly 65% aluminum and nearly 70% nickel in ESM. In comparison, metal removals in the control after seven days were about 55% copper, 50% aluminum and 40% nickel. Higher metal removals were reported by Brandl et al., 2001; a mixture of At. ferrooxidans and At. thiooxidans removed more than 90% of copper, nickel and aluminum from 1% w/v ESM. As can be seen in Table 4.4, the composition of the three major-content metals in ESM (copper, nickel, and aluminum) decreased after bio-oxidation. After bio-oxidation, more iron from the culture was deposited (see Table 4.4) and bacterial biomass is expected to 53

Results and Discussion deposited in ESM; thus even if the mass of gold is not lost after bio-oxidation, the gold content will reduced obviously. As the result, gold content in bio-oxidized ESM was more than five times lower than non-bio-oxidized ESM. Overall, the pretreatment by At. ferrooxidans resulted in a reduced the copper/gold ratio in ESM from 5.8 to 3.1. Table 4.4 Comparison of metal composition in ESM before and after bio-oxidation by At. ferrooxidans Metal content (g/kg ESM) Metal Au Al Cu Fe Ni Before bio-oxidation 4.8720.115 2.8800.048 28.320.33 20.590.49 5.8060.207 After bio-oxidation 0.9310.081 0.4710.025 2.8360.002 119.40.6 0.9600.03

Compared with chemical pretreatment, it was found that At. ferrooxidans (in 5mM sulphuric acid) removed copper better than chemical pretreatment, with more than 80%, compared with about 55% in ferric leaching (section 4.2.1.2), and 78.6% in sulphuric acid leaching at higher acid concentration - 10 mM (Fig 4.3 (b)). 4.3 Culture Chromobacterium violaceum and Pseudomonas fluorescens in fresh medium in flasks The growth of C. violaceum and P. fluorescens, when cultured in LB medium at 300C, 200 rpm, in the absence of ESM, was determined by optical density measurement and plate count method. The growth is plotted in Figure 4.5 (a) for C. violaceum and Figure 4.6 (a) for P. fluorescens. The pH and free cyanide concentration of the cultures were also monitored and shown in Figure 4.5 (b) for C. violaceum and Figure 4.6 (b) for P.

54

Results and Discussion fluorescens. Full details in growths, pH profiles and cyanide production can be found in Table B.25 for C. violeacum and Table B.26 for P. fluorescens. Batch growth curves of both bacteria showed good agreement between the optical density measurement and the plate count method in lag phase, exponential phase and stationary phase. Thus in monitoring growth of these bacteria in the absence of ESM, the optical density measurement was applied with the advantage of faster response (immediately) compared with plate count method (requiring one day of incubation). In the presence of ESM, where the turbidity resulting from ESM in the culture affected the cell density analysis, the plate count method was used. It was found that in LB medium, both optical density and viable cell counts of C. violaceum were higher than of P. fluorescens. The calculated maximum growth rate of these bacteria was
C.violaceum=

2.69 hour-1 and

P.fluorescen

= 1.23 hour-1. The doubling

time was d.C.violaceum= 0.26 hour and d P.fluorescens= 0.56 hour. (Calculation can be found in Appendix A.5). Although it took a longer time for C. violaceum to adapt to a fresh medium (i.e. longer lag phase, see Figures 4.5 (a) and 4.6 (a)) than P. fluorescens; the higher maximum growth rate of C. violaceum compared with of P. fluorescens means that, under experimental conditions, C. violaceum grews faster than P. fluorescens in the exponential phase. As can be seen in Figures 4.5 (b) and 4.6 (b), the pH was lower for the culture of C. violaceum than P. fluorescens. It was also shown that pH of the medium increased after the lag phase. Thus, increasing pH value may be considered as an indicator of growth of

55

Results and Discussion bacteria. However, it was noted that the pH of culture continued to increase after the stationary phase, due to the release of cellular substances during cell lysis. It is evident from Figures 4.5 (a and b) and 4.6 (a and b) that no cyanide production occurred during the lag phase. Only a low concentration of cyanide was produced from the late exponential phase to the onset of the stationary phase. This observation corroborates published reports which showed that the maximum cyanide concentration was reached at the early stationary phase (Knowles, 1976; Rodgers, 1978; Lawson et al., 1999; Kita et al., 2006). Maximum free cyanide concentration observed in culture of C. violaceum were found to be about 50 mg/l; this is comparable with the maximum free cyanide concentration of 45 mg/l produced by C. violaceum ( Lawson et al., 1999). In LB medium, cyanide production by P. fluorescens was lower than C. violaceum when its maximum free cyanide concentration was just 18 mg/l. After reaching a peak, the free cyanide concentration decreased rapidly, due to vaporization in the shake flask or consumption by bacteria. For instance, C. violaceum can convert cyanide into cyanoalanine and -cyano--aminobutyric acid during the stationary phase and death phase (Knowles, 1976; Rodgers, 1978; Kita et al., 2006); and P. fluorescens can use cyanide as a nitrogen source after conversion of cyanide to ammonia (Harris and Knowles, 1983; Rollison et al., 1987): HCN + O2 + NADH + H+ NH3 + CO2 + NAD+ (4.5)

56

Results and Discussion

OD 10

Viable cell 1.E+11

(a)

1.E+10 1.E+09 1.E+08 1.E+07 1.E+06 Viable Cell Counts/ml

1 OD600nm 0.1 0.01 0 5 10 15 Time (hours) 20 25

1.E+05

Figure 4.5 (a) Growth of C. violaceum in the shake flask in the absence of ESM

pH 9

Cyanide 60

(b)
8.5

50 40 Cyanide concentration (mg/l)

pH

30 20

7.5 10 7 0 5 10 15 20 25 Time (hours) 0

Figure 4.5 (b) pH profile and cyanide production of C. violaceum in the flask in the absence of ESM

57

Results and Discussion

OD 10

Viable Cell 1.E+11

(a)
1 OD450nm

1.E+10 1.E+09 Viable Cell Counts/ml

1.E+08 1.E+07 1.E+06

0.1

0.01 0 5 10 15 Time (hours) 20 25

1.E+05

Figure 4.6 (a) Growth of P. fluorescens in the flask in the absence of ESM

pH 9

Cyanide 20

(b)
Cyanide concentration (mg/l) 8.5 15

pH

10

7.5

7 0 5 10 15 Time (hours) 20 25

Figure 4.6 (b) pH profile and cyanide production of P. fluorescens in the flask in the absence of ESM

58

Results and Discussion 4.4 Bioleaching of non-pretreated ESM 4.4.1 One-step bioleaching In one-step bioleaching, ESM was added to LB medium before it was autoclaved, and a bacterial culture at the exponential phase was inoculated at 1%v/v. Samples were withdrawn at intervals during bioleaching period of seven days after inoculation. 4.4.1.1 pH profiles pH of the cultures in one-step bioleaching by C. violaceum and P. fluorescens, at different pulp densities were monitored and are shown in Figures 4.7 (a, b, c, d, and e). Full details of the pH profiles can be found in Table B.11. It can be seen from Figures 4.7 (a, b, c, d, and e) that the pH of all the control samples in one-step bioleaching was nearly constant. This suggests that ESM and any metal leached during the experiment (which will be discussed in Section 4.4.1.4) do not significantly affect the pH of the medium. In contrast, the pH of the two bacterial cultures increased after inoculation and then fluctuated slightly. A higher pH value was found in the culture of P. fluorescens compared to C. violaceum. This observation corroborates the results of Brandl et al, 2008. Cyanogenic bacteria grow under alkaline conditions, in contrasts with the acidic conditions produced by oxidizing bacteria such as A. thiooxidans, A. ferrooxidans, or fungi such as A. niger, P. simplicissimum (Aung and Ting, 2008; Brandl et al., 2008). Figure 4.8 shows the influence of pulp density on pH in one-step bioleaching. Both bacteria exhibited lower pH at higher pulp density, suggesting inhibition effect of the

59

Results and Discussion ESM. At lower pulp densities (0.5% and 1%), the highest pH for P. fluorescens varied from 9 to 9.5 while that for C. violaceum varied from 8.5 to 9. At higher pulp densities, the pH was even lower for C. violaceum. The higher pH of P. fluorescens culture than of C. violaceum occurred even in the absence of ESM (see Section 4.3), but the difference was only about 0.2. The greater difference in pH between the two bacteria cultures at higher pulp densities can also be explained by two factors: (1) P. fluorescens is better able to tolerate the toxic effects of ESM than C. violaceum, and thus the former grew better than the latter in the presence of ESM, causing the pH to increase; (2) The presence of ESM may even enhance the growth of P. fluorescens in some aspects (since among cultures of P. fluorescens, pH of the culture at 1% pulp density was higher than of the culture at 0.5% pulp density, see Figure 4.8).

control 10

C. violaceum

P.fluorescens

(a)
9

pH 8 7 6 0 1 2 3 4 5 6 7 Time (days)

Figure 4.7(a) pH profile in one-step bioleaching with 0.5% pulp density

60

Results and Discussion

control 10

C. violaceum

P.fluorescens

(b)
9

pH 8 7 6 0 1 2 3 4 5 6 7 Time (days)

Figure 4.7(b) pH profile in one-step bioleaching with 1% pulp density

control 10

C. violaceum

P.fluorescens

(c)
9

pH 8 7 6 0 1 2 3 4 5 6 7 Time (days)

Figure 4.7(c) pH profile in one-step bioleaching with 2% pulp density

61

Results and Discussion

control 10 C. violaceum P.fluorescens

(d)
9

pH 8 7 6 0 1 2 3 4 Time (days) 5 6 7

Figure 4.7(d) pH profile in one-step bioleaching with 4% pulp density

control 10

C. violaceum

P.fluorescens

(e)
9

pH 8 7 6 0 1 2 3 4 Time (days) 5 6 7

Figure 4.7 (e) pH profile in one-step bioleaching with 8% pulp density

62

Results and Discussion

C. violaceum 10

P. fluorescens

pH 8 7 0.5 1 2 Pulp density (%) 4 8

Figure 4.8 Influence of pulp density on pH value (in the end - seven days after inoculation) of one-step bioleaching of non-treated ESM 4.4.1.2 Free cyanide concentration Free cyanide concentration in the cultures in one-step bioleaching ESM by C. violaceum and P. fluorescens, at different pulp densities were monitored and are shown in Figures 4.9 (a, b, c, d, e and f). Full details of these cyanide profiles can be found in Table B.14. In the presence of ESM, the free cyanide concentration in bacterial cultures was found to be much lower than in absence of ESM. The maximum cyanide concentration detected in cultures of P. fluorescens at 0.5% w/v ESM was about 13 ppm, a value lower than cyanide concentration of 18 ppm in the absence of ESM (see Section 4.3). Compared with P. fluorescens, the cyanide production of C. violaceum decreased more significantly when maximum cyanide concentration found in cultures of C. violaceum at 0.5% w/v ESM was about 18 ppm, much lower than the concentration of 50 ppm in the absence of 63

Results and Discussion ESM (see Section 4.3). Moreover, as can be seen in Figures 4.9 (a, b, c, d and e), at higher pulp densities (1, 2, 4, and 8% w/v), free cyanide concentration detected in cultures of both bacteria was much lower than at 0.5% pulp density. Free cyanide concentration in cultures of cyanogenic bacteria (such as C. violaceum and P. fluorescens) depends on several factors: the cyanide production by bacteria (Castric, 1981; Askeland and Morrison, 1983; Knowles and Bunch, 1986); cyanide consumption by bacteria (Knowles, 1976; Rodgers, 1978; Harris and Knowles, 1983; Rollison et al., 1987; Kita et al., 2006); reactions of cyanide (such as complexation with metals); and loss via vaporization (i.e. depending on environmental conditions such as aeration and temperature). The decrease in free cyanide concentration (in presence of ESM compared with in absence of ESM) may be due to a reduction in cyanide production by the bacteria under the toxic effects of ESM; and/or the complexation of cyanide with metals (thus leading to a higher cyanide concentration in the cultures than the free cyanide concentration detected). It is clearly shown from Figures 4.9 (a, b, c, d, and e) that the peaks of free cyanide concentration in the presence of ESM occurred later than in the absence of ESM. In the absence of ESM, the maximum cyanide concentration was observed about ten hours after inoculation; at 1% w/v and 2% w/v ESM, the peak occurred after two days and three days respectively. At higher densities of 4% w/v and 8% w/v, the delay was even to six days after inoculation. Because cyanide production is accelerated in early stationary phase (Knowles, 1976; Rodgers, 1978; Lawson et al., 1999; Kita et al., 2006), this delay may indicate the delay of stationary phase which implies the lengthened lag phase.

64

Results and Discussion The free cyanide concentration in cultures was affected by pulp density. As is evident in Figures 4.9 (a and b), at low pulp density (0.5-1% w/v), C. violaceum produced more free cyanide than P. fluorescens. At higher pulp density (4 and 8%) however, the free cyanide concentration detected in both cultures was insignificant. At the lowest pulp density (0.5% w/v), the free cyanide concentration of both bacterial culture decreased gradually after reaching a peak; for instance, one day after the peaks were observed, the concentration was less than the maximum value about 5% for C. violaceum and 8% for P. fluorescens. A similarly slight decrease was observed in Brandl, et al., 2008. However, at higher pulp densities of 1% and 2%, the decrease was significant, about 22% and 55% for C. violaceum (at 1% and 2% respectively), and more than 30% for P. fluorescens. The decrease of free cyanide concentration can be explained as above, i.e. cyanide consumption by bacteria, reactions of cyanide with metals in the cultures, and loss via vaporization. Among of them, reactions of cyanide with metals are possibly the most important reason for the reduction of free cyanide concentration in cultures of high pulp densities. At pulp density of 1-2% w/v, free cyanide concentration reached another peak after the first. This phenomenon was shown in Campbell et al., 2001. It is most likely due to the recovered bacterial growth which will be discussed in Section 4.6.2.

65

Results and Discussion

control 20 (a) Cyanide concentration (mg/l) 16 C. violaceum P. fluorescens

12

0 0 1 2 3 4 Time (days) 5 6 7

Figure 4.9 (a) Cyanide profile in one-step bioleaching with 0.5% pulp density

control 20 (b) Cyanide concentration (mg/l) 16

C. violaceum

P. fluorescens

12

0 0 1 2 3 4 Time (days) 5 6 7

Figure 4.9 (b) Cyanide profile in one-step bioleaching with 1% pulp density

66

Results and Discussion

control 20 (c) Cyanide concentration (mg/l) 16 C. violaceum P. fluorescens

12

0 0 1 2 3 4 Time (days) 5 6 7

Figure 4.9 (c) Cyanide profile in one-step bioleaching with 2% pulp density

control 20 (d) Cyanide concentration (mg/l) 16

C. violaceum

P. fluorescens

12

0 0 1 2 3 4 Time (days) 5 6 7

Figure 4.9 (d) Cyanide profile in one-step bioleaching with 4% pulp density

67

Results and Discussion

control 20 (e) Cyanide concentration (mg/l) 16 C. violaceum P. fluorescens

12

0 0 1 2 3 4 Time (days) 5 6 7

Figure 4.9 (e) Cyanide profile in one-step bioleaching with 8% pulp density

4.4.1.3 Gold leaching profiles In one-step bioleaching, ESM was added into the LB medium before autoclave and gold concentration in cultures was measured using ICP-OES. Figures 4.10 (a, b, c, d and e) show the concentration of gold leached in the cultures of C. violaceum and P. fluorescens in one-step bioleaching. Full details of these gold leaching profiles can be found in Table B.17. As can be seen in Figures 4.10 (a, b, c, d and e), at the end of the leaching period, P. fluorescens leached more gold than C. violaceum over the pulp density range from 0.58% w/v. The figures clearly show that the bacteria play a role in leaching gold since the amount of gold leached in the control samples (without bacteria) was insignificant. Gold was leached from ESM in bacterial cultures by the cyanide produced by C. violaceum and P. fluorescens (which is discussed in Section 4.4.1.2). Faramarzi and Brandl, 2006 have 68

Results and Discussion confirmed using HPLC that gold was dissolved in bioleaching by cyanogenic bacteria in the form of dicyanoaurate [Au(CN)2]-. It should be noted that after reaching a peak, gold concentration decreased. There are several possibilities for this phenomenon: (i) loss of cyanide by bacteria consumption or vaporization that reduces the amount of cyanide complexes, (ii) gold sorption by the bacterial biomass (Patil and Paknikar, 1999), (iii) gold cyanide complexes stability in competition with other metals, and (iv) changes in the properties of the culture that reduce the stability of dicyanoaurate. The bioleaching of metals could be affected by material properties. Gold leaching by C. violaceum has been reported to be as high as 30-40 mg/l from gold coated glass slides, but as low as 0.25-0.34 mg/l from ore concentrate in the same report of Campbell et al., 2001. The concentration of gold leached in our study was much higher when compared with two other strains of P. fluorescens and C. violaceum with the same range of ESM pulp density (lower than 8%) in the same LB medium (Brandl et al, 2008) (which reported gold concentration of about 0.6 mg/l for C. violaceum and 0.2 mg/l for P. fluorescens). The influence of pulp density on the leaching gold in one-step bioleaching by C. violaceum and P. fluorescens is shown in Figure 4.11. The gold concentration shown in these figures refer to maximum gold concentration during the leaching period. An increase in pulp density led to a corresponding decrease in dissolved gold concentration in the culture, and hence a decreased recovery efficiency. It may due to the increase in

69

Results and Discussion toxicity of heavy metals, mainly copper in this case, when pulp density is increased. (This will be discussed in Section 4.4.1.4). Results from the bioleaching of minor-content metals (such as gold) are very different from that of major-content metals (such as copper) in ESM. For minor-content metals, our study shows that a higher pulp density resulted in a lower gold concentration in the leaching solution; the maximum gold concentration at 0.5, 1, 2, 4 and 8% pulp density was 2.31, 1.53, 0.21, 0.27, and 0.01 mg/l respectively for C. violaceum and 2.49, 1.95, 1.35, 0.33 and 0.11 mg/l for P. fluorescens. For major-content metals, our study shows that a higher pulp density led to higher copper concentration (details will be provided in Section 4.4.1.4).

control 3 (a) Gold concentration (mg/l) 2.5 2 1.5 1 0.5 0 0 1 2

C. violaceum

P. fluorescens

3 4 Time (days)

Figure 4.10 (a) Gold leaching in one-step bioleaching with 0.5% pulp density

70

Results and Discussion

control 3 (b) Gold concentration (mg/l) 2.5 2 1.5 1 0.5 0 0 1 2

C. violaceum

P. fluorescens

Time (days)

Figure 4.10 (b) Gold leaching in one-step bioleaching with 1% pulp density

control 3 (c) Gold concentration (mg/l) 2.5 2 1.5 1 0.5 0 0 1 2

C. violaceum

P. fluorescens

Time (days)

Figure 4.10 (c) Gold leaching in one-step bioleaching with 2% pulp density

71

Results and Discussion

control
3

C. violaceum

P. fluorescens

(d)
Gold concentration (mg/l) 2.5

1.5

0.5

0 0 1 2 3 4 5 6 7 Tim e (days)

Figure 4.10 (d) Gold leaching in one-step bioleaching with 4% pulp density

control
3

C. violaceum

P. fluorescens

(e)
Gold concentration (mg/l) 2.5

1.5

0.5

0 0 1 2 3 4 5 6 7 Tim e (days)

Figure 4.10 (e) Gold leaching in one-step bioleaching with 8% pulp density

72

Results and Discussion

control 3

C. violaceum

P. fluorescens

2.5 Gold concentration (mg/l)

1.5

0.5

0 0.5 1 2 Pulp density (%) 4 8

Figure 4.11: Influence of pulp density on the leaching of gold in one-step bioleaching

4.4.1.4 Copper leaching profiles In one-step bioleaching, ESM was added into the LB medium before autoclave and the copper concentration in the cultures was measured using ICP-OES. Figures 4.12 (a, b, c, d, and e) show the concentration of copper leached in the culture of C. violaceum and P. fluorescens in one-step bioleaching. Full details of copper leaching profiles in one-step bioleaching can be found in Table B.21. Figures 4.12 (a, b, c, d and e) show that copper leaching by P. fluorescens and C. violaceum in one-step bioleaching showed similar profiles. In contrast with gold, significant amount of copper was leached from the controls (i.e. compare with Figures 4.10 (a, b, c, d, and e); the copper concentration increased up to two days, and generally

73

Results and Discussion stabilized over time. Parallel experiments with de-ionized water (DI water) showed minor leaching of copper. It is likely that some chemical components present in the LB medium are responsible for the leaching of copper from ESM. Although other reports (Brandl et al., 2008) also showed that significant amount of copper was detected in the controls of bioleaching experiments, this phenomenon was unfortunately not discussed. It was shown that copper recovery was not much different among experimental culture (in presence of bacteria) and control (in absence of bacteria) at the same pulp density. It is likely that the cyanide produced by C. violaceum and P. fluorescens is too low, when compared with amount of copper from ESM, to make a significant difference. Besides, changes in the properties of the growth medium during bacterial growth may result in reprecipitating the dissolved copper. This may explain the observation in Figure 4.12 (a) where, at pulp density of 0.5%, copper concentration in cultures of P. fluorescens was higher (than the control) after one day of inoculation, but decreased and was lower than the control subsequently. The effect of pulp density on the leaching of copper by C. violaceum and P. fluorescens using one-step bioleaching is shown in Figure 4.13. The copper concentration shown in these figures refer to maximum copper concentration during the leaching period. It is evident that an increase in pulp density leds to an increase in the copper concentration in the leachate, both for the control as well as the bacterial cultures. This phenomenon is in direct contrast with the decrease of gold concentration (see Figure 4.11). While inhibition of ESM on growth and cyanide production of bacteria is a the reason for the decrease of gold concentration (since cyanide is the only lixiviant capable of dissolving gold in the

74

Results and Discussion growth medium), the increase in copper concentration may be due to some leaching reagents in the medium as discussed above.

control 150 (a) Copper concentration (mg/l) 120

C. violaceum

P. fluorescens

90

60

30

0 0 1 2 3 Time (days) 4 5 6 7

Figure 4.12 (a) Copper leaching in one-step bioleaching with 0.5% pulp density

75

Results and Discussion

control 250 (b) Copper concentration (mg/l) 200

C. violaceum

P. fluorescens

150

100

50

0 0 1 2 3 Time (days) 4 5 6 7

Figure 4.12 (b) Copper leaching in one-step bioleaching with 1% pulp density

control 400 (c) Copper concentration (mg/l)

C. violaceum

P. fluorescens

300

200

100

0 0 1 2 3 Time (days) 4 5 6 7

Figure 4.12 (c) Copper leaching in one-step bioleaching with 2% pulp density

76

Results and Discussion

control 600 (d) Copper concentration (mg/l) 500 400 300 200 100 0 0 1 2

C. violaceum

P. fluorescens

3 Time (days)

Figure 4.12 (d) Copper leaching in one-step bioleaching with 4% pulp density

control 1600 (e) 1400 Copper concentration (mg/l) 1200 1000 800 600 400 200 0 0 1 2

C. violaceum

P. fluorescens

3 Time (days)

Figure 4.12 (e) Copper leaching in one-step bioleaching with 8% pulp density

77

Results and Discussion

Control 1600 1400 Copper concentration (mg/l) 1200 1000 800 600 400 200 0 0.5 1

C. violaceum

P. fluorescens

2 Pulp density (%)

Figure 4.13 Influence of pulp density on the leaching of copper in one-step bioleaching

4.4.2 Two-step bioleaching In two-step bioleaching, bacteria were initially cultured in LB medium in the absence of the ESM. Sterilized ESM was added one day after inoculation when the cultures attained significant cell density and free cyanide concentration. Samples were withdrawn and analyzed at intervals during the seven days bioleaching period after the addition of ESM. 4.4.2.1 pH profiles pH of the cultures in two-step bioleaching ESM by C. violaceum and P. fluorescens, at different pulp densities were monitored and are shown in Figures 4.14 (a, b, c, d, and e). Full details of the pH profile can be found in Table B.12.

78

Results and Discussion Similar to one-step bioleaching, the pH increase in P. fluorescens culture was greater than in the C. violaceum culture. The pH difference was more significant at higher pulp density. One day after inoculation, the pH of both cultures in the two-step bioleaching (without ESM) was higher than in one-step bioleaching (with ESM). The pH of bacterial cultures in two-step bioleaching was in range of 8.2-8.8 while the pH in one-step bioleaching was not higher than 8.0. The higher pH clearly shows a better growth of both bacteria in the absence of ESM. This suggests that the inhibitory effect of ESM on the growth of both bacteria is reduced in two-step bioleaching and the more favourable bacterial growth during this period may also affect the leaching later.

control 10

C.violaceum

P.fluorescens

(a)
9

pH

6 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.14 (a) pH profile in two-step bioleaching with 0.5% pulp density

79

Results and Discussion

control 10

C.violaceum

P.fluorescens

(b)
9

pH

6 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.14 (b) pH profile in two-step bioleaching with 1% pulp density

control 10

C.violaceum

P.fluorescens

(c)
9

pH

8
y

6 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.14 (c) pH profile in two-step bioleaching with 2% pulp density

80

Results and Discussion

control 10

C.violaceum

P.fluorescens

(d)
9

pH

6 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.14 (d) pH profile in two-step bioleaching with 4% pulp density

control 10

C.violaceum

P.fluorescens

(e)
9

pH

6 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.14 (e) pH profile in two-step bioleaching with 8% pulp density

81

Results and Discussion 4.4.2.2 Free cyanide concentration Free cyanide concentration in the cultures in the two-step bioleaching of ESM by C. violaceum and P. fluorescens, at different pulp densities is shown in Figures 4.15 (a, b, c, d, and e). Full details of these cyanide profiles can be found in Table B.15. Figures 4.15 (a, b, c, d, and e) show that one day after inoculation, C. violaceum produced much more cyanide than P. fluorescens in the absence of ESM. (No cyanide was detected in uninoculated controls during two-step bioleaching all pulp densities). The free cyanide concentration detected was about 55-60 mg/l in C. violaceum, but below 20 mg/l in P. fluorescens. In contrast, the free cyanide concentration one day after inoculation in one-step bioleaching was much lower; at 0.5% pulp density the concentration was about 16 mg/l for C. violaceum and 14 mg/l for P. fluorescens. At 1% pulp density, the free cyanide concentration was lower than 3 mg/l for cultures of both bacteria. At higher pulp densities, the free cyanide concentration was even lower than 1 mg/l. The higher concentration of free cyanide in two-step bioleaching compared with one-step bioleaching can be explained by the advantages for better growth of bacteria (and hence better cyanide production) in the absence of ESM during the first day in twostep bioleaching. The higher concentration of free cyanide produced before the addition of ESM enabled C. violaceum to leach more copper than P. fluorescens at the low pulp density (0.5 and 1%), but at higher pulp densities, P. fluorescens still gave better leaching. (Copper leaching in two-step bioleaching will be discussed in Section 4.4.2.4). After the addition of ESM, the free cyanide concentration reduced rapidly due to the complexation of cyanide and metals from ESM. One day after this addition (Day 2), the

82

Results and Discussion free cyanide concentration in P. fluorescens cultures reduced 2.1, 3.1, 6.4, 7.8, and 10.4 times at 0.5, 1, 2, 4 and 8% correspondingly. Free cyanide concentration in C. violaceum cultures reduced even more rapidly 3.5, 5.7, 12.5, 23.4, and 26.6 times at 0.5, 1, 2, 4 and 8% correspondingly. As can be seen in Figures 4.15 (a, b, c, and d), the low free cyanide concentration from Day 2 to Day 6 in two-step bioleaching suggests that, at pulp density from 0.5-4%, the bacteria continue to grow in this period. The cyanide profiles at pulp density 0.5-4% was similar to the cyanide profile in two-step bioleaching in a previous study (Tan, 2006) by the same bacteria at 5% w/v of another batch of ESM. In this report, the free cyanide concentration was maximal on Day 1 at 45 mg/l for C. violaceum and 20 mg/l for P. fluorescens. After the addition of ESM, the free cyanide concentration also reduced rapidly and maintained at low level in some days before the depletion. In contrast, at 8% pulp density, free cyanide was not detected from Day 3 onwards (see Figure 4.15 (e)); this was due to the strong inhibition of ESM on the bacterial growth and/or the strong complexation with metals in ESM at this high pulp density.

83

Results and Discussion

control 70 (a) Cyanide concentration (mg/l) 60 50 40 30 20 10 0 0 1 2 3

C.violaceum

P.fluorescens

ESM added

4 Time (days)

Figure 4.15 (a) Cyanide production in two-step bioleaching with 0.5% pulp density

control 70 (b) Cyanide concentration (mg/l) 60 50 40 30 20 10 0 0 1 2 3

C.violaceum

P.fluorescens

ESM added

4 Tme (days)

Figure 4.15 (b) Cyanide production in two-step bioleaching with 1% pulp density

84

Results and Discussion

control 70 (c) Cyanide concentration (mg/l) 60 50 40 30 20 10 0 0 1 2 3

C.violaceum

P.fluorescens

ESM added

4 Time (days)

Figure 4.15 (c) Cyanide production in two-step bioleaching with 2% pulp density

control 70 (d) Cyanide concentration (mg/l) 60 50 40 30 20 10 0 0 1 2 3

C.violaceum

P.fluorescens

ESM added

4 5 Time (days)

Figure 4.15 (d) Cyanide production in two-step bioleaching with 4% pulp density

85

Results and Discussion

control 70 (e) Cyanide concentration (mg/l) 60 50 40 30 20 10 0 0 1 2 3

C.violaceum

P.fluorescens

ESM added

4 5 Time (days)

Figure 4.15 (e) Cyanide production in two-step bioleaching with 8% pulp density

4.4.2.3 Gold leaching profiles In two-step bioleaching, the ESM was added on Day 1 (i.e. one day after inoculation) and gold concentration in the cultures was measured from Day 2 onwards. Gold leaching from ESM in two-step bioleaching by C. violaceum and P. fluorescens, at different pulp densities was monitored and is shown in Figures 4.16 (a, b, c, d and e). Full details of these gold leaching profiles can be found in Table B.18. As can be seen in Figures 4.16 (a, b, c, d, and e), gold was not detected in all the uninoculated controls. This confirms the role of the cyanogenic bacteria in gold leaching. Figure 4.14 (a) shows that at 0.5% pulp density, the leaching of gold increased gradually with time for both of the bacteria, with higher gold leaching observed for C. violaceum than P. fluorescens from Day 5 onwards. At higher pulp densities of 1, 2 and 4%, more

86

Results and Discussion rapid leaching was observed; and leaching by P. fluorescens was greater than C. violaceum. In the range of pulp density from 1-4%, peaks of gold leaching were detected at Day 4 and the significant difference between the two bacterial cultures increased with pulp density. For instance, gold concentration in P. fluorescens culture was 1.7, 7.4, and 14.0 time higher than in C. violaceum culture at 1, 2, and 4% w/v ESM correspondingly. At 8% pulp density, very poor leaching of gold was observed due to the strong inhibition of ESM on the bacterial growth and hence on cyanide production (as discussed in Sections 4.4.2.1 and 4.4.2.2). The influence of pulp density on gold leaching in two-step bioleaching is shown in Figure 4.17. The maximum gold concentration (higher than 3.7 mg/l) was obtained with P. fluorescens at 2% pulp density. (Thus this pulp density would be used later in bioleaching experiments in a bioreactor). Compared with the culture in one-step bioleaching, ESM in two-step bioleaching was added in bacterial cultures containing 55-60 mg/l cyanide. This is a reason for the higher gold leaching in two-step than in one-step bioleaching. Although the gold concentration was higher in two-step bioleaching, ESM strongly inhibited gold bioleaching by C. violaceum as evident in the decrease in gold concentration with increasing pulp density.

87

Results and Discussion

control 4 (a) 3.5 Gold concentration (mg/l) 3 2.5 2 1.5 1 0.5 0 1 2 3

C. violaceum

P. fluorescens

4 Time (days)

Figure 4.16 (a) Gold leaching in two-step bioleaching with 0.5% pulp density

control 4 (b) 3.5 Gold concentration (mg/l) 3 2.5 2 1.5 1 0.5 0 1 2 3

C. violaceum

P. fluorescens

4 Time (days)

Figure 4.16 (b) Gold leaching in two-step bioleaching with 1% pulp density

88

Results and Discussion

control 4 (c) 3.5 Gold concentration (mg/l) 3 2.5 2 1.5 1 0.5 0 1 2 3 4 Time (days) 5 6 7 8 C. violaceum P. fluorescens

Figure 4.16 (c) Gold leaching in two-step bioleaching with 2% pulp density

control 4 (d) 3.5 Gold concentration (mg/l) 3 2.5 2 1.5 1 0.5 0 1 2 3

C. violaceum

P. fluorescens

4 Time (days)

Figure 4.16 (d) Gold leaching in two-step bioleaching with 4% pulp density

89

Results and Discussion

control 4 (e) 3.5 Gold concentration (mg/l) 3 2.5 2 1.5 1 0.5 0 1 2 3

C. violaceum

P. fluorescens

4 Time (days)

Figure 4.16 (e) Gold leaching in two-step bioleaching with 8% pulp density

control 4.5 4 Gold concentration (mg/l) 3.5 3 2.5 2 1.5 1 0.5 0 0.5 1

C. violaceum

P. fluorescens

2 Pulp density (%)

Figure 4.17 Influence of pulp density on gold leaching in two-step bioleaching

90

Results and Discussion 4.4.2.4 Copper leaching profiles In two-step bioleaching, ESM was added on Day 1 (i.e. after one day of inoculation) and copper concentration in cultures was measured from Day 2 onwards. Figures 18 (a, b, c, d, and e) show the concentration of copper leached in the culture of C. violaceum and P. fluorescens in two-step bioleaching. Full details of these copper leaching profiles in twostep bioleaching can be found in Table B.22. As can be seen in Figures 4.18 (a, b, c, d, and e), copper was detected in uninoculated controls in two-step bioleaching in a similar range of concentration to that in one-step bioleaching (about 70 mg/l, 150 mg/l, 250 mg/l, 450mg/l and 900 mg/l copper at 0.5%, 1%, 2%, 4% and 8% pulp density). Significant amount of copper was leached in the controls by some leaching reagents in the LB medium as discussed in Section 4.4.1.4. Figures 4.18 (a and b) show that at low pulp density of 0.5% and 1%, C. violaceum leached more copper than P. fluorescens. However, at higher pulp density, P. fluorescens again had advantage in leaching copper. It suggests the better metal resistance of P. fluorescens than C. violaceum.

91

Results and Discussion

control 160 (a) 140 Copper concentration (mg/l) 120 100 80 60 40 20 0 1 2 3

C. violaceum

P. fluorescens

4 5 Tme (days)

Figure 4.18 (a) Copper leaching in two-step bioleaching with 0.5% pulp density

control 300 (b) Copper concentration (mg/l) 250

C. violaceum

P. fluorescens

200

150

100

50

0 1 2 3 4 5 Time (days) 6 7 8

Figure 4.18 (b) Copper leaching in two-step bioleaching with 1% pulp density

92

Results and Discussion

control 450 (c) 400 Copper concentration (mg/l) 350 300 250 200 150 100 50 0 1 2 3

C. violaceum

P. fluorescens

4 5 Time (days)

Figure 4.18 (c) Copper leaching in two-step bioleaching with 2% pulp density

control 700 (d) 600 Copper concentration (mg/l) 500 400 300 200 100 0 1 2 3

C. violaceum

P. fluorescens

4 5 Time (days)

Figure 4.18 (d) Copper leaching in two-step bioleaching with 4% pulp density

93

Results and Discussion

control 1800 (e) 1600 Copper concentration (mg/l) 1400 1200 1000 800 600 400 200 0 1 2 3

C. violaceum

P. fluorescens

4 5 Time (days)

Figure 4.18 (e) Copper leaching in two-step bioleaching with 8% pulp density

4.4.3 Comparison of Chromobacterium violaceum and Pseudomonas fluorescens, one-step and two-step in bioleaching non-biooxidized ESM Gold recovery Comparison of gold leaching leaching by C. violaceum and P. fluorescens in one-step and two-step bioleaching of non-biooxidized ESM is shown in Figure 4.19.

94

Results and Discussion

C. violaceum one-step P. fluorescens one-step 12 10 Gold recovery (%) 8 6 4 2 0 0.5% 1% 2% Pulp density 4% 8% C. violaceum two-step P. fluorescens two-step

Figure 4.19 Comparison of gold recovery in one-step and two-step bioleaching In almost all instances, P. fluorescens was better than C. violaceum in gold bioleaching from non-biooxidized ESM over the range of pulp density from 0.5-8% w/v. As discussed in Section 4.4.2.2, much higher free cyanide concentration was observed in two-step than in one-step bioleaching. Two-step bioleaching improves metal leaching over one-step bioleaching because the microorganisms are not affected by the toxicity of the materials and hence produce a higher concentration of the metabolites or lixiviants needed for the leaching process. Higher gold recovery in two-step than in one-step bioleaching was observed in Figure 4.19, except for the leaching at 0.5% w/v, where gold recovery in two-step bioleaching is lower than in one-step bioleaching. This worse gold recovery might be explained that, in two-step bioleaching, the bacteria grew fast and significant amount of cyanide was lost via ventilation before adding ESM. In one-step bioleaching, if the inhibition of such low pulp density as 0.5% to cyanide production was

95

Results and Discussion smaller than more amount of cyanide kept in the culture by the presence of ESM, cyanide concentration in culture should be higher than in two-step bioleaching. Copper recovery A comparison of copper leaching by C. violaceum and P. fluorescens in one-step and two-step bioleaching non-biooxidized ESM is shown in Figure 4.20.

C.violaceum one-step P. fluorescens one-step 100 90 80 Copper recovery (%) 70 60 50 40 30 20 10 0 0.5% 1%

C. violaceum two-step P. fluorescens two-step

2% Pulp density

4%

8%

Figure 4.20 Comparison of copper recovery in one-step and two-step bioleaching

At the pulp density 0.5 and 1% w/v, C. violaceum leached marginally more copper than P. fluorescens in both one-step and two-step bioleaching. At higher pulp densities, however, P. fluorescens showed better copper leaching. This is probably because at low pulp density, C. violaceum gave higher cyanide production; at high pulp density, the cyanide production was inhibited strongly. For instance, as shown in Section 4.4.1.2, in

96

Results and Discussion one-step bioleaching, C. violaceum produced more free cyanide than P. fluorescens at 0.5-1% pulp density, and this comparison reversed at 8% pulp density. Compared with other studies, at 1% pulp density, copper recovery by P. fluorescens and C. violaceum was lower than by a mixed culture of At. ferrooxidans and At. thiooxidans (more than 90%), but higher than A. niger (about 40%) and P. simplicilicum (less than 20%) reported in Brandl et al., 2001. Copper recovery by P. fluorescens and C. violaceum was higher than by At. ferrooxidans (maximum was 37%) with sulfur as the energy source, as reported by Choi et al., 2005. 4.5 Bioleaching bio-oxidized ESM Although two-step bioleaching usually results in higher metal recovery than one-step bioleaching (Brandl et al., 2001), one-step is conducted more frequently. This is because it is easier to compare among one-step bioleaching studies where initial conditions can be considered similar. In two-step bioleaching, however, the slightly differences in growth conditions (such as medium components, innoculum, activation status of

microorganisms, etc.) can significantly affect the time for the addition of the solid waste and hence may cause considerable differences in metal recovery from batch to batch. For this reason, one-step bioleaching was conducted with bio-oxidized ESM to examine the effects of bio-oxidation on the bioleaching of gold from ESM by C. violaceum and P. fluorescens. After bio-oxidation, the ESM was separated and dried for bioleaching experiments. Biooxidized ESM was added to LB medium before autoclave and bacterial cultures at the

97

Results and Discussion exponential phase were inoculated at 1%v/v. Samples were withdrawn at intervals during the bioleaching period of seven days after inoculation. 4.5.1 pH profiles Figures 4.21 (a, b, c, d, and e) show the pH profiles in bioleaching bio-oxidized ESM by C. violaceum and P. fluorescens. Full details of the pH profiles can be found in Table B.13. It can be seen from Figures 4.21 (a, b, c, d, and e) that in contrast with the experimental cultures, the pH of all uninoculated controls in bioleaching of bio-oxidized ESM was nearly constant. pH changes were observed during bioleaching. The pH value of the uninoculated controls was lower than the experimental cultures, possibly due to the acidic property of ESM after bio-oxidation. In contrast with the constant pH profiles of the control samples, the pH of the two bacterial cultures increased after inoculation and then fluctuated slightly. Compared with the bioleaching of non-bio-oxidized ESM by C. violaceum, although the addition of biooxidized ESM resulted in a lower pH in the beginning, pH of the latter became higher at the end (see Table 4.5). This suggests the better growth of C. violaceum due to lower toxicity from the heavy metals in the presence of bio-oxidized ESM, compared with nonbiooxidized ESM. It is also noted that the pH values in cultures of P. fluorescens were similar in both cases.

98

Results and Discussion

control 10

C. violaceum

P.fluorescens

(a)
9

pH 8 7 6 0 1 2 3 4 Time (days) 5 6 7

Figure 4.21 (a) pH profile in bioleaching bio-oxidized ESM with 0.5% pulp density

control 10

C. violaceum

P.fluorescens

(b)
9

pH 8 7 6 0 1 2 3 4 Time (days) 5 6 7

Figure 4.21 (b) pH profile in bioleaching bio-oxidized ESM with 1% pulp density

99

Results and Discussion

control 10

C. violaceum

P.fluorescens

(c)
9

pH 8 7 6 0 1 2 3 4 Time (days) 5 6 7

Figure 4.21 (c) pH profile in bioleaching bio-oxidized ESM with 2% pulp density

control 10

C. violaceum

P.fluorescens

(d)
9

pH 8 7 6 0 1 2 3 4 Time (days) 5 6 7

Figure 4.21 (d) pH profile in bioleaching bio-oxidized ESM with 4% pulp density

100

Results and Discussion

control 10

C. violaceum

P.fluorescens

(e)
9

pH 8 7 6 0 1 2 3 4 Time (days) 5 6 7

Figure 4.21 (e) pH profile in bioleaching bio-oxidized ESM with 8% pulp density

Table 4.5 Comparison of pH in bioleaching non-biooxidized and bio-oxidized ESM C. violaceum Day 0 Non-biooxidized Day 7 Non-biooxidized P. fluorescens Day 0 Non-biooxidized Day 7 Non-biooxidized 9.05 9.19 8.79 8.83 8.81

Bio-oxidized

Bio-oxidized

Bio-oxidized

Pulp density (%) 0.5 1 2 4 8

7.20 7.20 7.20 7.20 7.20

6.95 6.90 6.83 6.64 6.33

8.75 8.43 8.10 8.10 7.65

8.94 8.95 8.87 8.88 8.80

7.20 7.20 7.20 7.20 7.20

6.95 6.91 6.83 6.64 6.33

9.01 9.01 8.95 8.93 8.80

Bio-oxidized

101

Results and Discussion 4.5.2 Free cyanide concentration Free cyanide concentration in the cultures in bioleaching bio-oxidized ESM by C. violaceum and P. fluorescens, at pulp densities from 0.5-8% was monitored and is shown in Figures 4.24 (a, b, c, d, and e). Full details of these cyanide profiles can be found in Table B.16. In general, in the presence of bio-oxidized ESM at 0.5-8% pulp density, the free cyanide concentration in bacterial cultures was still lower than in absence of ESM (where the maximum free cyanide concentration was 18 mg/l for P. fluorescens and 50 mg/l for C. violaceum). However, the maximum concentration of free cyanide in the presence of biooxidized ESM was higher than of non-biooxidized ESM. For instance, in bioleaching with 2% pulp density by C. violaceum, it was about 6 mg/l for bio-oxidized ESM (see Figure 4.22 (c)) and only 3.5 mg/l for non-biooxidized ESM (see Figure 4.9 (c)). It may be due to higher cyanide production by bacteria and/or lower cyanide concentration with complexation with metals after bio-oxidation of ESM. As can be seen in Figures 4.24 (a, b, c, d, and e), the peaks of free cyanide concentration were observed at Day 1 (one day after inoculation) at all pulp densities. In contrast with the delay in bioleaching non-biooxidized ESM (Section 4.4.1.2), the earlier observation of these peaks suggests the better adaptation of bacteria with bio-oxidized ESM than nonbiooxidized ESM.

102

Results and Discussion

control 45 40
Cyanide concentration (mg/l)

C.violaceum

P.fluorescens

(a)

35 30 25 20 15 10 5 0 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.22 (a) Cyanide production in bioleaching bio-oxidized ESM with 0.5% pulp density

control 45 40
Cyanide concentration (mg/l)

C.violaceum

P.fluorescens

(b)

35 30 25 20 15 10 5 0 0 1 2 3 4 5 Time (days) 6 7 8

Figure 4.22 (b) Cyanide production in bioleaching bio-oxidized ESM with 1% pulp density

103

Results and Discussion

control
45

C.violaceum

P.fluorescens

(c)
40 Cyanide concentration (mg/l) 35 30 25 20 15 10 5 0 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.22 (c) Cyanide production in bioleaching bio-oxidized ESM with 2% pulp density

control 45 40
Cyanide concentration (mg/l)

C.violaceum

P.fluorescens

(d)

35 30 25 20 15 10 5 0 0 1 2 3 4 5 Time (days) 6 7 8

Figure 4.22 (d) Cyanide production in bioleaching bio-oxidized ESM with 4% pulp density

104

Results and Discussion

control
45

C.violaceum

P.fluorescens

(e)
40 Cyanide concentration (mg/l) 35 30 25 20 15 10 5 0 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.22 (e) Cyanide production in bioleaching bio-oxidized ESM with 8% pulp density

4.5.3 Gold leaching profiles In the bioleaching of bio-oxidized ESM, the ESM was added into the LB medium before it was autoclaved, and gold concentration in the cultures was analysed daily. Figures 4.23 (a, b, c, d and e) show the concentration of gold in the bioleaching bio-oxidized ESM. Full details of these gold leaching profiles can be found in Table B.19. As can be seen in Figures 4.23 (a, b, c, d and e), after the bio-oxidation of ESM, C. violaceum leached more gold than P. fluorescens over the entire range of pulp density (0.5-8% w/v). A maximum gold concentration of 3 mg/l was observed in C. violaceum with 4% w/v bio-oxidized ESM. The better performance of C. violaceum over P. fluorescens in gold bioleaching can be explained by the higher cyanide production of the former over the latter (Section 4.5.2).

105

Results and Discussion In term of gold recovery (i.e. the percentage of leached gold from the total amount of leachable gold in the material), bio-oxidation increased gold recovery by C. violaceum at all pulp densities (see Table B.20), particularly at high pulp densities (2-8%) when the bacteria was strongly inhibited by heavy metals at high concentration. Gold recovery by C. violaceum at 2-4% pulp density improved from below 0.5% for non-biooxidized ESM to more than 8% for bio-oxidized ESM. Insignificant gold recovery at 8% pulp density improved to about 3.6% after bioxidation. This showed the reduction of inhibition of ESM on the growth and cyanide production of C. violaceum. In contrast, gold recovery by P. fluorescens in bio-oxidised ESM at low pulp density (0.5% and 1%) was lower than in the system without bio-oxidation (See Table B20). The reason for this unexpected result is not clear. It is speculated that this could have occurred due to two reasons: (i) adaptation of P. fluorescens in the presence of copper during bioleaching may lead to some beneficial leaching properties which were limited when copper was removed after bio-oxidation; (ii) changes in properties of ESM after bio-oxidation may have negative effects on growth of P. fluorescens and on gold dissolution.

106

Results and Discussion

control 3.5 (a) 3

Gold concentration (mg/l)

C.violaceum

P.fluorescens

2.5 2 1.5 1 0.5 0 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.23 (a) Bioleaching gold from 0.5% w/v bio-oxidized ESM

control 3.5 (b) 3

Gold concentration (mg/l)

C.violaceum

P.fluorescens

2.5 2 1.5 1 0.5 0 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.23 (b) Bioleaching gold from 1% w/v bio-oxidized ESM

107

Results and Discussion

control 3.5 (c) 3

Gold concentration (mg/l)

C.violaceum

P.fluorescens

2.5 2 1.5 1 0.5 0 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.23 (c) Bioleaching gold from 2% w/v bio-oxidized ESM

control 3.5 (d) 3

Gold concentration (mg/l)

C.violaceum

P.fluorescens

2.5 2 1.5 1 0.5 0 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.23 (d) Bioleaching gold from 4% w/v bio-oxidized ESM

108

Results and Discussion

control 3.5 (e) 3

Gold concentration (mg/l)

C.violaceum

P.fluorescens

2.5 2 1.5 1 0.5 0 0 1 2 3 4 Time (days) 5 6 7 8

Figure 4.23 (e) Bioleaching gold from 8% w/v bio-oxidized ESM

4.5.4 Copper leaching profiles In the bioleaching of bio-oxidized ESM, the ESM was added into the LB medium before it was autoclaved, and copper concentration in cultures was analysed daily. Figures 4.24 (a, b, c, d, and e) show the concentration of copper leached in the culture of C. violaceum and P. fluorescens in one-step bioleaching of the bio-oxidized ESM. Full details of these copper leaching profiles can be found in Table B.23. Figures 4.24 (a, b, c, d, and e) show that C. violaceum gave better performance than P. fluorescens in the bioleaching of the bio-oxidized ESM; the concentration of copper in the bioleaching cultures was much lower compared with that in the bioleaching of nonobiooxidized ESM. The maximum copper concentration detected at 8%w/v was only about 75 mg/l in C. violaceum culture with bio-oxidized ESM while it reached 1500 mg/l

109

Results and Discussion in P. fluorescens culture with non-biooxidized ESM. Although compared with nonbiooxidized ESM, gold recovery did not increase over all pulp densities applied, this much lower copper concentration helped to increase the gold/copper ratio in the leachate (as will be discussed in Section 4.5.5) and facilitates gold separation from the leachate.

control 80 (a) 70 Copper concentration (mg/l) 60 50 40 30 20 10 0 0 1 2 3

C. violaceum

P. fluorescens

4 Time (days)

Figure 4.24 (a) Bioleaching copper from 0.5% w/v bio-oxidized ESM

110

Results and Discussion

control 80 (b) 70 Copper concentration (mg/l) 60 50 40 30 20 10 0 0 1 2 3 4 Time (days) 5 6 7 8 C. violaceum P. fluorescens

Figure 4.24 (b) Bioleaching copper from 1% w/v bio-oxidized ESM

control 80 (c) 70 Copper concentration (mg/l) 60 50 40 30 20 10 0 0 1 2 3

C. violaceum

P. fluorescens

4 Time (days)

Figure 4.24 (c) Bioleaching copper from 2% w/v bio-oxidized ESM

111

Results and Discussion

control 80 (d) 70 Copper concentration (mg/l) 60 50 40 30 20 10 0 0 1 2 3 4 Time (days) 5 6 7 8 C. violaceum P. fluorescens

Figure 4.24 (d) Bioleaching copper from 4% w/v bio-oxidized ESM

control 80 (e) 70 Copper concentration (mg/l) 60 50 40 30 20 10 0 0 1 2 3

C. violaceum

P. fluorescens

4 Time (days)

Figure 4.24 (e) Bioleaching copper from 8% w/v bio-oxidized ESM

112

Results and Discussion 4.5.5 Comparison of bioleaching non-biooxidized ESM and bio-oxidized ESM by Chromobacterium violaceum and Pseudomonas fluorescens Comparison of bioleaching gold and copper between non-biooxidized ESM and biooxidized ESM by C. violaceum and P. fluorescens is shown in Figure 4.25 and Figure 4.26. As discussed in Section 4.5.3, gold recovery by C. violaceum improved significantly after bio-oxidation at all pulp densities applied, while gold recovery by P. fluorescens only improved at high pulp densities (i.e. 2, 4, and 8%). At 0.5 and 1%, gold recovery of P. fluorescens decreased unexpectedly. The decrease in copper recovery by both bacteria compared with bioleaching from nonbiooxidized ESM (Figure 4.26) suggests the some negative effects of ESM on metal dissolution. However, it is obvious that as decreasing copper recovery facilitates gold separation from the leachate. Table 4.6 shows that the gold/copper in the leachate increased in bio-oxidation, more significantly, for C. violaceum than P. fluorescens, particularly at high pulp densities. Table 4.6 Increasing of gold/copper ratio (ppm gold/ ppm copper) in leachate by biooxidation Pulp density (%) 0.5 1 2 4 8 Non-biooxidized 1.9E-02 6.8E-03 6.3E-04 5.0E-04 1.3E-05 C. violaceum Biooxidized 7.3E-02 4.2E-02 4.7E-02 5.1E-02 3.5E-02 2.1E-02 9.1E-03 4.0E-03 6.2E-04 7.2E-05 P. fluorescens Non-biooxidized Biooxidized 3.2E-02 2.8E-02 3.0E-02 2.7E-02 2.1E-02 It is necessary to explore the real reason and apply if it does not reduce leaching gold efficiency.

113

Results and Discussion

C. violaceum - non-biooxidized P. fluorescens - non-biooxidized 12 10 Gold recovery (%) 8 6 4 2 0 0.5% 1% 2% Pulp density 4% 8% C. violaceum -bio-oxidized P. fluorescens - bio-oxidized

Figure 4.25 Comparison of bioleaching gold from non-biooxidized and bio-oxidized ESM
C.violaceum - non-biooxidized P. fluorescens - non-biooxidized 100 90 80 Copper recovery (%) 70 60 50 40 30 20 10 0 0.5% 1% 2% Pulp density 4% 8% C. violaceum - bio-oxidized P. fluorescens - bio-oxidized

Figure 4.26 Comparison of bioleaching copper from non-biooxidized and bio-oxidized ESM

114

Results and Discussion 4.6 Bioleaching ESM by P. fluorescens in bioreactor 4.6.1 Growth in fresh medium The growth of P. fluorescens in a stirred tank bioreactor, at 300C, agitation at 200 rpm, and aeration at 1 vvm, is plotted in Figure 4.27. Full details on the growth and cyanide production are given in Table B.27.

Figure 4.27 Monitoring growth of P. fluorescens in bioreactor, at 300C, 200rpm, aeration at 1vvm. As can be seen in Figure 4.27, the lag phase lasted for ten hour after inoculation. During the lag phase, the pH of the culture gradually increased and the oxygen consumption decreased. Virtually no cyanide was produced. The exponential phase (which occurred from about 11 hours after inoculation) was accompanied by an increase in pH, and the dissolved oxygen in medium was rapidly depleted.

115

Results and Discussion The onset of cyanide production occurred in the middle of the exponential phase, and maximum cyanide concentration (around 18 ppm) was observed at the beginning of the stationary phase. Cyanide concentration decreased during the stationary phase. The observed cyanide profile of P. fluorescens in the bioreactor with maximum cyanide concentration detected in the beginning of the stationary phase was in good agreement with literature (Knowles, 1976; and Lawson et al., 1999). There was a period when the pH value decreased towards the end of the exponential phase and into the stationary phase. This may due to loss of cyanide via hydrogen cyanide production which is enhanced with aeration. Except for this period, pH of culture had good agreement with optical density of the culture, thus pH value of culture may be representative to the growth of bacteria in the absence of ESM. 4.6.2 Bioleaching ESM It has earlier been shown in shake flask experiments (in Section 4.4.2.3) that the highest gold concentration was observed at 2% ESM by P. fluorescens (see 4.4.2.3). Thus, bioleaching at 2%ESM by P. fluorescens was further studied in the bioreactor system. Bioleaching at 2% ESM by P. fluorescens was conducted in a stirred tank bioreactor, at 300C, with agitation at 200 rpm, and aeration at 1 vvm. The growth of the bacteria and its bioleaching are plotted in Figure 4.28 and Figure 4.29 correspondingly. Full details in the bacterial growth, cyanide production, and gold and copper leaching can be found in Table B.30.

116

Results and Discussion Figure 4.28 shows that in the presence of ESM, the viable cell count decreased one day after inoculation. The pH of the culture fluctuated around 8.6, and dissolved oxygen in the culture increased until the growth of bacteria recovered (about five days after inoculation). Figure 4.29 shows that cyanide was produced continuously during bioleaching process and free cyanide concentration in the culture was maintained at significant levels that resulted to leaching of gold and copper from the ESM. However, lengthening the bioleaching process did not ensure increasing leached metals concentration, as changes of medium by bacteria may not facilitate dissolution of metals as discussed.

Figure 4.28 Growth of P. fluorescens in bioleaching 2% ESM in bioreactor, with air supply

117

Results and Discussion

Figure 4.29 Bioleaching gold and copper from 2% ESM by P. fluorescens in bioreactor, with air supply

118

Results and Discussion 4.6.3 Effects of aeration to gold leaching efficiency To investigate the effect of oxygen on the growth of the cyanogenic bacteria and gold recovery, a control was set up conducted in a stirred tank bioreactor with P. fluorescens at 2%ESM, at 300C with agitation at 200 rpm, but without air supply. The growth of P. fluorescens and the bioleaching are shown in Figure 4.30 and Figure 4.31 respectively. Full details in the bacterial growth, cyanide production, gold leaching and copper leaching can be found in Table B.31. Figure 4.30 shows without oxygen supply, the viable cell number of P. fluorescens remained generally stable (with ln CFU maintaining between 18 and 20); the bacterium was able to survive at such low oxygen concentration. However, the pH decreased and remained at about 6.5 In the unaerated bioreactor, the free cyanide concentration was very low, thus resulting in a poor leaching of copper (when compared with the aerated system). The impact of aeration on gold bioleaching was marginal, possibly due to its already low leaching efficiency in the presence of copper. The low free cyanide concentration may be due to two reasons: (i) lack of oxygen led to a low cyanide production (Castric, 1975) and (ii) the low pH value enhanced the volatilization of cyanide. The number of living cells from one day of bioleaching onward, in culture with air supplied was lower than in culture without air supplied. That could be explained by the toxicity of cyanide and/or leached copper on bacterial growth.

119

Results and Discussion

Figure 4.30 Growth of P. fluorescens in bioleaching 2% ESM in bioreactor, no air supply

Figure 4.31 Bioleaching gold and copper from 2% ESM by P. fluorescens in bioreactor, no air supply

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Results and Discussion 4.6.4 Comparison of bioleaching in flasks and bioreactor A comparison of bioleaching in shale flasks and in a bioreactor (with aeration at 1 vvm, and without aeration) is shown in Figure 4.32. In bioleaching ESM by P. fluorescens, 25% copper recovery in the absence of aeration increased significantly to 55% with aeration. Meanwhile, gold recovery increased marginally, from 1.5% (without aeration) to 1.9% (with aeration). The figure also shows a higher gold bioleaching performance in the bioreactor; a higher gold recovery and a lower copper recovery were found for the bioreactor (both with and without aeration) than in shaking flask.

Figure 4.32 Comparison of bioleaching in flasks and bioreactor (at 2% pulp density)

121

Conclusions and Recommendations CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions Characterization of ESM The <75m ESM used in this project were fine particles (of mean size 36.56 m), with a low specific surface area of 0.6136 m2/g. Non-metallic components contributed more than 90% in weight of ESM, with the most abundant element being silica. Among the many heavy metals present in ESM, the metals of highest value were gold (4.872 g/kg) and copper (28.32 g/kg). Based on the US EPA and TCLP tests, ESM would be classified as a hazardous waste and must be treated before land disposal. Growth of bacteria and cyanide production in fresh medium In LB medium, both bacteria C. violaceum and P. fluorescens produced cyanide from the mid-log phase, and maximum cyanide concentration was observed during the stationary phase of the batch culture. After reaching a peak, cyanide concentration in the culture rapidly decreased. In fresh medium, C. violaceum grew better and produced more cyanide than P. fluorescens. Bioleaching gold from non-biooxidized ESM In one step bioleaching, gold recovery from ESM by C.violaceum and P. fluorescens decreased with increasing pulp density over the range of 0.5-8%w/v, due to inhibition of ESM on the bacterial growth. Although C. violaceum grew better and produced more cyanide than P. fluorescens in fresh medium, it showed greater inhibition in the presence

122

Conclusions and Recommendations of ESM. It should be noted that in the absence of bacteria, a significant amount of copper was leached from ESM by LB medium in the shake flasks while only minor amount of copper was detected in a similar system with ESM in water. In two-step bioleaching, although C. violaceum has advantages in growth and production cyanide before the addition of ESM, it only showed better leaching gold at low pulp density (0.5 and 1%w/v). At higher pulp density (2, 4 and 8%w/v), gold concentration in culture of P. fluorescens was much higher of C. violacum. Two-step bioleaching improved gold recovery, and the highest gold concentration of 3.7 mg/l was attained with 2%w/v ESM by P. fluorescens. However, in both one-step and two-step bioleaching, while copper recovery was at high percentage (40% to more than 90%), gold recovery was generally no more than 10%, and the copper/gold ratio in the leachates was very high, thus inhibiting gold recovery. Bio-oxidation in comparison with chemical leaching Bio-oxidation by At. ferrooxidans showed better metal removal than chemical leaching (by ferric and sulphuric acid). It removed more than 80% of copper and nearly 60% of aluminum seven days after inoculation and it reduced copper/gold ratio in ESM from 5.8 to 3.1. Other acid chemical leaching also showed good performance in removal copper and other metals and should be considered in the pretreatment of ESM. Bioleaching gold from bio-oxidized ESM Gold recovery of C. violaceum improved significantly after bio-oxidation at all of pulp densities applied, while gold recovery of P. fluorescens was only improved at high pulp

123

Conclusions and Recommendations densities (2, 4, and 8%). The highest gold concentration of 3 mg/l was attained with 4% bio-oxidized ESM by C. violaceum. In contrast with improvement in leaching gold, leaching copper was reduced with both bacteria, leading to much lower copper/gold ratio in the leachate. Bioleaching in bioreactor The growth profile, cyanide production, as well as metal leaching of P. fluorescens were studied in a bioreactor. The growth and cyanide production of this bacterium in fresh medium in the bioreactor was similar to the growth profile in flasks. Bioleaching 2%w/v ESM by P. fluorescens in bioreactor showed higher leaching gold and lower copper leaching, in both cases with or without aeration. When no air was supplied, gold leaching was reduced marginally but copper concentration in culture was much lower.

5.2 Recommendations The potential of bioleaching gold from ESM by P. fluorescens and C. violaceum has been clearly demonstrated. Some recommendations for a better understanding and the optimization of gold recovery are given as follows: 1. Metal leaching may be improved if the non-metallic components are separated from metallic components in the ESM. 2. The lixiviant(s) responsible for the leaching copper and other base metals in LB medium should be investigated and determined. Since these metals interfere (and

124

Conclusions and Recommendations compete) with gold during bioleaching, pretreatment by removing these metals will enhance gold bioleaching. 3. Supplementation of glycine at the appropriate dosage and time should be considered since this may enhance cyanide production, and thereby lead to enhanced metal leaching. 4. The precipitation of iron on ESM by At. ferrooxidans during bio-oxidation may limit cyanide production. Thus bio-oxidation by the bacterium without the addition of iron (beyond the iron already present in ESM) may result in enhanced gold recovery. 5. Since organic acids from some fungi reportedly showed good copper removal, biooxidation of ESM by fungi may be considered. It is important, however, to recognize that the fine ESM particle may be accumulated within the fungal biomass, and thus lead to loss of non dissolved gold in the ESM. To avoid this, the use of spent medium from the fungal culture may be used as the pretreatment or bio-oxidation step. (The cost of the organic carbon sources would have to be considered). 6. Optimization of the gold bioleaching process could be investigated by examining the various factors affecting the process. This would include factors such as pulp density, particle size, glycine concentration and time of addition, aeration, as well as temperature.

125

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132

Appendices Appendix A: Sample Calculations A.1 Calculation of metal compositions in ESM If: c: concentration of a metal analyzed by ICP-OES, mg/l w: weight of ESM added in acid digestion, g Then weight of the metal dissolved in acid digestion is calculated as follow: m = c(mg / l ) Content of the metal in ESM is: content = 0.2 c mg g , or w g kg 200(ml ) = 0.2 c(mg ) 1000(ml / l )

A.2 Calculation of free cyanide concentration Compute slope and intercept of standard curve as follow:
m= n ca c a n a 2 ( a ) 2
2

a c a ac b= n a ( a )
2 2

Where: a: absorbance of standard solution c: concentration of CN- in standard, mg/l n: number of standard solutions m: slope of standard curve b: intercept on c axis

133

Appendices

Include the blank concentration, 0 ppm and blank absorbance in the calculations above. The cyanide concentration of a sample is calculated as follow:
CN = (ma1 + b) a1: absorbance of sample solution X: volume of sample, l 1500 , mg / l X

A.3 Calculation of metal recovery

Assumption: 100% metal recovery is reached when amount of metal leached out equals to amount of metals leached by acid digestion. If: c: metal concentration, mg/l c100: metal concentration at 100% metal recovery, mg/l p: pulp density of ESM, %w/v m: content of metal in ESM, g/kg (in non-biooxidized ESM, m=4.872 for gold and m=28.32 for copper in bio-oxidized ESM, m=0.931 for gold and m=2.835 form copper) 100% metal recovery is reached if leached metal concentration in liquor:
c100 = 10 m p, mg / l

Metal recovery is calculated as follow:

x =

c c100

100 =

10 c , % m p

A.4 Calculation of Standard Error

134

Appendices

Standard Error (S.E) used to show Y error bars on Figures (M S.E.) was calculated as follow:

M =

y
i =1

S .E . =

(y
i =1

M )2

(n 1)n

Where: M: arthimetic mean i: point number in series n: number of point in series yi: data value at the ith point

A.5 Calculation of growth rates and doubling times

The maximum growth rates and the doubling times were calculated following Equations (Shuler and Kargi, 1992):

= d =
Where: : maximum growth rate, hour-1 d: doubling time, hour

ln X 2 ln X 1 t 2 t1 ln 2

1, 2: two points in the exponential growth phase t1, t2: time after inoculation at points 1 and 2, hour

135

Appendices

X1, X2: cell count at two point t1, and t2, number of viable cells/ml The maximum growth rates of C. violaceum and P. fluorescens were calculated as follow:

C .violaceum = P. fluorescens

ln(3.98 E + 07) ln(1.2 E + 06) = 2.69 (hour 1 ) 5.3 4.0 ln(2.55 E + 08) ln 45583333 = = 1.23 (hour 1 ) 3.0 1.6

The doubling times of C. violaceum and P. fluorescens were calculated as follow:

d (C .violaceum ) = d ( P. fluorescens )

ln 2 = 0.26 (hour ) 2.69 ln 2 = = 0.56 (hour ) 1.23

136

Appendices Appendix B: Experimental Data B.1 Characterization of ESM B.1.1 Size range of as-received ESM Table B.1 Size range of as-received ESM

Size range (m) Percentage (wt%) Normalized (wt%) > 150 100-150 75-100 <75 Total 51.22 5.42 2.63 35.26 94.53* 54.18 5.73 2.79 37.30 100.00

*: Due to the light density of the ESM, 5.47% of the material lost during the sieving

137

Appendices B.1.2 Particle size distribution B.1.2.1 Summary of particle size distribution of original <75m ESM

138

Appendices Table B.2 Particle size distribution (%volume) of original <75m ESM

139

Appendices B.1.2.2 Summary of particle size distribution of acid digested <75m ESM

140

Appendices Table B.3 Particle size distribution (%volume) of acid digested <75m ESM

141

Appendices B.1.2.3 Summary of particle size distribution of bio-oxidized <75m ESM

142

Appendices Table B.4 Particle size distribution (%volume) of bio-oxiddized <75m ESM

143

Appendices B.1.2.4 Changes in particle size distribution of <75m ESM after bio-oxidation and acid digestion

144

Appendices Table B.5 Particle size distribution (%volume) of original, acid digestion and bio-

oxidized <75m ESM

145

Appendices

146

Appendices

147

Appendices B.1.3 SEM images B.1.3.1 SEM images of as-received ESM

Figure B.1 SEM image of as-received ESM at magnification of 50x

Figure B.2 (a) SEM image (a) of as-received ESM at magnification of 250x

148

Appendices

Figure B.2 (b) SEM image (b) of as-received ESM at magnification of 250x

B.1.3.2 SEM images of <75m ESM

Figure B.3 SEM image of <75m ESM at magnification of 50x

149

Appendices

Figure B.4 SEM image of <75m ESM at magnification of 250x

Figure B.5 SEM image of <75m ESM at magnification of 500x

150

Appendices

Figure B.6 SEM image of <75m ESM at magnification of 2000x

B.1.3.3 SEM images of acid digested <75m ESM

Figure B.7 SEM image of acid digested <75m ESM at magnification of 50x

151

Appendices

Figure B.8 SEM image of acid digested <75m ESM at magnification of 500x

Figure B.9 SEM image of acid digested <75m ESM at magnification of 1000x

152

Appendices

Figure B.10 SEM image of acid digested <75m ESM at magnification of 2000x

153

Appendices B.1.4 SEM/EDX B.1.4.1 SEM/EDX of <75m ESM

Figure B.11 (a) Selected area in elemental analysis by EDX/SEM of <75m ESM

Figure B.11 (b) SEM/EDX spectra of <75m ESM

154

Appendices Table B.6 Elemental content of <75m (SEM/EDX)

Spectrum processing : Peak possibly omitted : 9.441 keV Processing option : All elements analyzed (Normalised) Number of iterations = 6 Element CK OK Al K Si K SK Fe K Ni K Cu L Sn L Total Weight% 34.29 39.19 0.40 19.51 0.75 2.47 1.24 1.33 0.83 100.00 Atomic% 46.57 39.96 0.24 11.33 0.38 0.72 0.34 0.34 0.11

155

Appendices B.1.4.2 SEM/EDX report of acid digested ESM

Figure B.12 (a) Selected area in elemental analysis by EDX/SEM

of acid digested <75m ESM

Figure B.12 (b) SEM/EDX spectra of acid digested <75m ESM

156

Appendices Table B.7 Elemental content of acid digested <75m (SEM/EDX)

Spectrum processing : No peaks omitted Processing option : All elements analyzed (Normalised) Number of iterations = 6 Element CK OK Si K Total Weight% 39.10 40.45 20.45 100.00 Atomic% 49.99 38.83 11.18

B.1.4.3 SEM/EDX report of carbon tape

Figure B.13 (a) Selected area in elemental analysis by EDX/SEM of carbon tape

157

Appendices

Figure B.13 (b) SEM/EDX spectra of carbon tape

Table B.8 Elemental content of carbon tape

Spectrum processing : No peaks omitted Processing option : All elements analyzed (Normalised) Number of iterations = 5 Element CK OK Cu L Zn L Pt M Total Weight% 55.74 19.65 -0.33 -0.09 25.04 100.00 Atomic% 77.47 20.50 -0.09 -0.02 2.14

158

Appendices B.1.5 BET analysis for Specific Surface Area

Figure B.18 Analysis of specific surface area

159

Appendices

B.1.6 Metal composition of ESM Table B.9 Metal composition of ESM in literature

Electronic waste Fe 28 7 5 23 62 4 4.5 12 Cu 10 20 13 21 5 3 14.3 10

Weight (%) Al 10 5 1 1 2 5 2.8 7 Pb 1.0 1.5 0.3 0.14 0.3 0.1 2.2 1.2 Ni 0.3 1 0.1 0.03 0.05 0.5 1.1 0.85

TV board scrap Hagelulen, 2002 PC board scrap Hagelulen, 2002 Mobile phone scrap Hagelulen, 2002 Portable audio scrap Hagelulen, 2002 DVD player scrap Hagelulen, 2002 Calculator scrap Hagelulen, 2002 PC main board scap Legarth et al., 2002 PCB scrap Zhang and Forssberg, 1997 TV scrap (CRTs removed) 3.4 1.2 0.2 0.038 20 <10 <10 Cui and Forssberg, 2007 Electronic scrap 8.3 8.5 0.71 3.15 2.0 29 12 Ilyas et al., 2007 PC scrap 20 7 14 6 0.85 189 16 3 (*) Typical electronic scrap 8 20 2 2 2 2000 1000 50 Sum, 1991 E-scrap 37.4 18.2 19 1.6 6 12 Busselle, 1999 E-scrap 27.3 16.4 11.0 1.4 210 150 20 Busselle, 1999 Printed circuit boards 5.3 26.8 1.9 0.47 3300 80 Theo, 2002 E- scrap 26.2 18.6 1800 220 30 Hoffmann, 1992 E- waste mixture 36 4.1 4.9 0.29 1 Morf et al., 2007 Dust from shredding process of ESM 8 23.7 2 1.5 sa sa Brandl et al., 2001 PCB scrap (70% non- metallic) 3 16 2 500 300 100 Goosey and Kellner, 2002 Electronic scrap 8.3 8.5 0.71 3.15 2.0 29 12.4 Ilyas et al., 2007 Electronic scrap 3.6 4.96 2.7 2.26 0.075 476 876 Ten and Ting, 2003 Electronic scrap 1.77 25.4 0.28 0.23 0.79 3436 2432 This work PCB: Printed Circuit Boards; sa: small amount; (*): http://www.cpcb.nic.in/Electronic%20Waste/chapter4.html 160

Weight (ppm/ g/ton) Ag Au Pd 280 20 10 1000 250 110 1380 350 210 150 10 4 115 15 4 260 50 5 639 566 124 280 110

References

Appendices Table B.10 Metal composition of the liquor after acid digestion

Content (g/kg ESM) Metal Au Ag Al Cu Fe Ni Pb As-received ESM 2.432 3.436 2.825 253.9 17.73 7.951 2.341 <75m ESM 4.872 nt 2.880 28.32 20.59 5.806 1.005 Bio-oxidized ESM 0.931 nt 0.471 2.835 119.4 0.96 1.471

161

Appendices B. 2 Bioleaching in flasks B.2.1 pH profiles Table B.11 pH profile of one-step bioleaching non-biooxidized ESM in shake flasks

Sample 0.5A 0.5B 0.5A CV 0.5B CV 0.5A PF 0.5B PF 1A 1B 1A CV 1B CV 1A PF 1B PF 2A 2B 2A CV 2B CV 2A PF 2B PF 4A 4B 4A CV 4B CV 4A PF 4B PF 8A 8B 8A CV 8B CV 8A PF 8B PF 0 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 1 7.24 7.17 7.77 7.73 7.84 7.85 7.20 7.20 8.00 8.00 7.99 7.98 7.19 7.23 8.00 7.95 7.96 7.94 7.19 7.22 8.07 8.04 7.91 7.84 7.26 7.32 7.33 7.27 8.02 7.86 2 7.18 7.19 8.18 8.10 8.31 8.27 7.22 7.21 8.56 8.52 8.32 8.25 7.24 7.24 8.43 8.12 8.75 8.26 7.32 7.32 8.48 8.31 8.73 8.63 7.40 7.40 7.99 7.43 8.83 8.73

Time(days) 3 4 7.19 7.20 7.19 7.19 8.73 8.56 8.42 8.63 8.88 8.78 8.58 8.23 7.19 7.19 7.26 7.16 8.64 8.40 8.53 8.35 8.39 8.70 8.39 8.62 7.26 7.27 7.24 7.29 8.37 8.31 8.14 8.10 8.88 8.80 8.84 8.90 7.29 7.29 7.32 7.28 8.45 8.37 8.30 8.20 8.87 8.76 8.89 8.83 7.43 7.36 7.40 7.34 7.95 7.89 7.41 7.37 8.89 8.85 8.87 8.77

5 7.19 7.20 8.74 8.78 9.00 8.96 7.20 7.21 8.56 8.46 9.08 9.05 7.24 7.26 8.30 8.05 8.83 8.92 7.30 7.34 8.37 8.17 8.80 8.94 7.44 7.43 7.92 7.44 8.83 8.82

6 7.19 7.17 8.79 8.79 9.01 9.14 7.22 7.21 8.50 8.31 9.01 9.14 7.20 7.22 8.29 8.07 8.84 8.90 7.28 7.34 8.39 8.18 8.79 8.97 7.42 7.41 7.98 7.44 8.85 8.84

7 7.21 7.24 8.75 8.75 8.99 9.10 7.20 7.24 8.46 8.40 9.18 9.20 7.20 7.20 8.20 8.00 8.78 8.80 7.28 7.32 8.19 8.00 8.76 8.90 7.42 7.43 7.90 7.40 8.81 8.80

162

Appendices Table B.12 pH profile of two-step bioleaching non-biooxidized ESM in shake flasks

Sample 0.5A 0.5B 0.5A CV 0.5B CV 0.5A PF 0.5B PF 1A 1B 1A CV 1B CV 1A PF 1B PF 2A 2B 2A CV 2B CV 2A PF 2B PF 4A 4B 4A CV 4B CV 4A PF 4B PF 8A 8B 8A CV 8B CV 8A PF 8B PF

0 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20

1 7.19 7.17 8.05 8.14 8.20 8.32 7.20 7.17 8.20 8.17 8.37 8.20 7.16 7.20 8.46 8.33 8.37 8.34 7.28 7.28 8.48 8.43 8.43 8.50 7.36 7.39 8.44 8.47 8.76 8.66

2 7.20 7.18 8.17 8.28 8.63 8.65 7.20 7.23 8.83 8.76 8.57 8.67 7.15 7.24 8.82 8.50 9.10 9.10 7.34 7.32 8.56 8.55 9.07 9.00 7.40 7.50 8.56 8.60 9.01 8.87

Time (days) 3 4 5 7.20 7.20 7.21 7.20 7.19 7.20 8.70 9.25 9.55 8.80 9.31 9.58 9.00 9.40 9.65 9.00 9.37 9.64 7.20 7.20 7.21 7.22 7.20 7.20 9.00 9.19 9.21 8.95 9.14 9.27 9.01 9.44 9.54 9.02 9.35 9.55 7.20 7.20 7.30 7.25 7.27 7.35 8.85 8.78 8.81 8.55 8.60 8.63 9.18 9.26 9.35 9.18 9.24 9.41 7.35 7.38 7.48 7.34 7.36 7.46 8.55 8.54 8.60 8.53 8.50 8.48 9.14 9.20 9.29 9.03 9.06 9.18 7.47 7.50 7.60 7.55 7.57 7.64 8.55 8.53 8.57 8.50 8.50 8.57 9.00 8.92 8.97 8.86 8.86 8.95

6 7.23 7.20 9.55 9.52 9.65 9.75 7.25 7.20 9.24 9.26 9.55 9.59 7.40 7.41 8.83 8.62 9.36 9.42 7.50 7.47 8.56 8.59 9.27 9.03 7.60 7.67 8.52 8.56 8.90 8.83

7 7.21 7.21 9.55 9.49 9.65 9.70 7.23 7.20 9.15 9.20 9.48 9.50 7.40 7.40 8.70 8.60 8.30 9.35 7.50 7.50 9.00 8.55 9.20 9.00 7.60 7.60 8.54 8.55 8.92 8.82

8 7.19 7.20 9.55 9.48 9.69 9.66 7.20 7.17 9.08 9.14 9.40 9.48 7.40 7.39 8.62 8.62 9.26 9.28 7.55 7.53 9.55 8.54 9.20 8.99 7.60 7.66 8.56 8.56 8.93 8.83

163

Appendices Table B.13 pH profile of bioleaching bio-oxidized ESM in shake flasks

Sample 0.5A 0.5B 0.5A CV 0.5B CV 0.5A PF 0.5B PF 1A 1B 1A CV 1B CV 1A PF 1B PF 2A 2B 2A CV 2B CV 2A PF 2B PF 4A 4B 4A CV 4B CV 4A PF 4B PF 8A 8B 8A CV 8B CV 8A PF 8B PF

0 6.97 6.94 6.95 6.95 6.95 6.96 6.90 6.91 6.90 6.90 6.90 6.91 6.84 6.82 6.83 6.83 6.83 6.83 6.63 6.66 6.62 6.66 6.65 6.62 6.32 6.32 6.34 6.32 6.34 6.31

1 6.96 6.92 8.14 8.14 8.23 8.24 6.88 6.90 8.15 8.15 8.17 8.22 6.82 6.80 8.13 8.13 8.22 8.22 6.61 6.64 8.13 8.16 8.22 8.20 6.30 6.30 8.07 8.03 8.22 8.14

2 6.87 6.86 8.27 8.28 8.64 8.64 6.84 6.84 8.28 8.29 8.59 8.61 6.75 6.75 8.30 8.29 8.60 8.60 6.58 6.58 8.37 8.40 8.59 8.59 6.27 6.27 8.38 8.32 8.57 8.57

Time (days) 3 4 6.89 6.90 6.88 6.89 8.76 9.25 8.76 9.24 8.89 9.14 8.91 9.16 6.85 6.86 6.85 6.85 8.74 9.22 8.76 9.23 8.84 9.07 8.86 9.11 6.74 6.74 6.74 6.74 8.77 9.25 8.76 9.24 8.86 9.12 8.87 9.13 6.55 6.52 6.56 6.55 8.79 9.19 8.80 9.22 8.85 9.12 8.86 9.13 6.21 6.15 6.21 6.16 8.72 9.11 8.72 9.11 8.84 9.11 8.84 9.11

5 6.86 6.85 9.03 9.04 9.19 9.17 6.82 6.84 9.02 9.03 9.15 9.15 6.70 6.72 8.95 8.94 8.97 9.17 6.49 6.52 8.97 8.98 9.15 9.19 6.14 6.14 8.91 8.93 8.90 8.91

6 6.90 6.86 9.00 8.98 9.03 9.00 6.82 6.67 8.94 8.97 8.99 9.02 6.75 6.73 8.89 8.88 8.99 9.00 6.48 6.50 8.88 8.89 8.97 8.98 6.12 6.12 8.82 8.82 8.83 8.86

7 6.90 6.88 8.98 8.90 9.02 9.00 6.82 6.82 8.95 8.95 9.00 9.02 6.73 6.73 8.87 8.87 8.95 8.95 6.49 6.50 8.85 8.90 8.90 8.95 6.10 6.10 8.80 8.80 8.80 8.80

8 6.89 6.88 9.00 8.90 9.03 8.92 6.83 6.80 8.96 8.97 9.02 9.02 6.75 6.70 8.85 8.84 8.90 8.85 6.50 6.48 8.82 8.84 8.90 8.92 6.10 6.10 8.78 8.75 8.75 8.78

164

Appendices B.2.2 Free cyanide concentration Table B.14 Free cyanide concentration (mg/l) in one-step bioleaching non-biooxidized ESM in shake flasks

Sample 0.5A 0.5B 0.5A CV 0.5B CV 0.5A PF 0.5B PF 1A 1B 1A CV 1B CV 1A PF 1B PF 2A 2B 2A CV 2B CV 2A PF 2B PF 4A 4B 4A CV 4B CV 4A PF 4B PF 8A 8B 8A CV 8B CV 8A PF 8B PF

1 0.050 0.070 14.210 17.995 14.030 14.670 0.020 0.030 2.670 1.840 1.050 1.410 0.020 0.030 0.830 0.641 0.720 0.348 0.017 0.023 0.072 0.034 0.100 0.146 0.090 0.097 0.090 0.095 0.163 0.144

2 0.060 0.080 13.250 17.393 12.950 13.470 0.030 0.040 6.320 4.900 4.850 5.790 0.030 0.040 1.540 1.172 1.250 0.750 0.030 0.037 0.200 0.093 0.253 0.301 0.090 0.094 0.090 0.097 0.193 0.160

3 0.069 0.105 11.661 15.639 11.123 11.411 0.043 0.054 5.056 3.674 3.435 3.608 0.041 0.041 2.684 1.829 2.339 1.451 0.072 0.079 0.302 0.198 0.290 0.343 0.100 0.120 0.089 0.100 0.218 0.154

Time (days) 4 5 0.046 0.066 0.073 0.059 10.031 8.758 12.287 10.966 9.458 8.548 8.665 7.648 0.031 0.069 0.018 0.073 3.773 4.640 2.094 3.226 2.145 3.088 2.612 3.253 0.030 0.064 0.031 0.057 1.133 2.145 0.920 1.644 1.343 2.350 1.039 1.959 0.163 0.071 0.130 0.104 0.375 1.211 0.252 0.850 0.334 0.812 0.486 1.017 0.105 0.117 0.108 0.123 0.095 0.358 0.118 0.281 0.224 0.545 0.157 0.539

6 0.065 0.056 8.310 9.968 8.981 8.584 0.073 0.061 6.075 3.387 3.669 3.878 0.087 0.062 3.974 3.107 3.723 2.323 0.074 0.084 2.341 1.820 1.549 1.834 0.118 0.139 0.822 0.467 1.311 0.854

7 0.061 0.064 7.217 7.592 7.933 9.077 0.088 0.070 3.586 3.817 6.406 4.160 0.073 0.080 3.901 3.013 3.468 4.042 0.083 0.110 1.826 2.173 2.545 1.983 0.129 0.123 1.181 0.175 1.048 0.997

165

Appendices Table B.15 Free cyanide concentration (mg/l) in two-step bioleaching non-biooxidized ESM in shake flasks

Sample 0.5A 0.5B 0.5A CV 0.5B CV 0.5A PF 0.5B PF 1A 1B 1A CV 1B CV 1A PF 1B PF 2A 2B 2A CV 2B CV 2A PF 2B PF 4A 4B 4A CV 4B CV 4A PF 4B PF 8A 8B 8A CV 8B CV 8A PF 8B PF

1 0.023 0.026 62.322 56.350 18.200 19.980 0.023 0.027 61.200 55.620 19.350 18.260 0.027 0.026 54.260 58.350 17.890 18.950 0.022 0.020 59.620 59.680 19.360 19.480 0.036 0.029 54.260 56.690 18.280 17.380

2 0.045 0.040 18.230 15.280 8.826 9.167 0.032 0.028 10.350 10.050 6.355 5.935 0.015 0.029 4.429 4.600 2.753 2.962 0.017 0.023 2.523 2.586 2.426 2.585 0.090 0.097 2.023 2.152 1.685 1.728

3 0.056 0.060 3.240 2.056 4.253 5.462 0.035 0.043 2.423 2.532 1.399 1.248 0.038 0.036 1.035 1.257 3.455 3.587 0.030 0.037 1.329 1.574 2.635 2.217 0.090 0.094 0.125 0.148 0.138 0.147

Time (days) 4 5 0.069 0.056 0.105 0.062 2.560 2.965 2.017 2.735 1.532 0.482 1.598 0.358 0.062 0.058 0.058 0.035 2.045 1.686 2.356 1.923 1.035 0.862 0.992 0.800 0.040 0.030 0.040 0.035 0.296 0.148 0.313 0.185 3.896 3.214 4.232 3.023 0.072 0.163 0.079 0.130 2.053 2.354 2.248 2.348 2.748 2.356 2.513 2.241 0.100 0.105 0.120 0.108 0.129 0.119 0.127 0.115 0.135 0.135 0.147 0.136

6 0.065 0.045 4.185 3.520 0.355 0.324 0.068 0.068 0.532 0.685 0.623 0.452 0.062 0.065 0.072 0.079 1.257 1.341 0.071 0.104 1.354 1.480 0.489 0.443 0.117 0.123 0.129 0.135 0.428 0.438

7 0.062 0.059 3.441 4.040 0.235 0.423 0.075 0.052 0.233 0.435 0.185 0.234 0.067 0.057 0.069 0.084 0.102 0.120 0.074 0.084 0.532 0.623 0.128 0.147 0.118 0.139 0.132 0.142 0.147 0.152

8 0.050 0.067 0.536 0.726 0.201 0.113 0.076 0.060 0.075 0.157 0.125 0.148 0.070 0.082 0.085 0.105 0.086 0.095 0.083 0.110 0.236 0.321 0.089 0.139 0.129 0.123 0.138 0.142 0.144 0.162

166

Appendices Table B.16 Free cyanide concentration (mg/l) in bioleaching bio-oxidized ESM in shake flasks

Sample 0.5A 0.5B 0.5A CV 0.5B CV 0.5A PF 0.5B PF 1A 1B 1A CV 1B CV 1A PF 1B PF 2A 2B 2A CV 2B CV 2A PF 2B PF 4A 4B 4A CV 4B CV 4A PF 4B PF 8A 8B 8A CV 8B CV 8A PF 8B PF

1 0.010 0.010 36.327 42.256 14.362 14.538 0.015 0.018 13.256 15.684 6.850 8.673 0.020 0.023 6.695 5.328 3.526 4.319 0.021 0.024 3.202 3.185 2.305 2.248 0.023 0.027 2.548 2.475 1.125 1.218

2 0.010 0.017 32.640 36.762 12.358 12.475 0.020 0.020 11.157 12.832 7.326 7.655 0.022 0.019 4.528 3.348 2.754 3.355 0.024 0.023 2.655 2.662 1.526 1.328 0.025 0.030 1.725 1.624 0.524 0.637

3 0.015 0.018 20.566 25.374 9.864 10.478 0.025 0.028 9.653 10.092 5.834 6.756 0.031 0.029 3.095 2.254 1.535 2.207 0.030 0.031 1.942 2.000 0.826 0.592 0.031 0.035 0.785 0.700 0.323 0.408

Time (days) 4 5 0.020 0.021 0.020 0.023 16.255 13.635 17.356 14.858 7.895 6.834 8.137 7.362 0.026 0.030 0.028 0.031 6.935 6.035 8.524 6.872 5.234 4.351 5.830 4.694 0.035 0.037 0.035 0.038 1.863 1.653 1.522 1.208 1.035 0.535 1.635 0.786 0.036 0.041 0.037 0.040 1.235 0.521 1.344 0.548 0.520 0.214 0.475 0.200 0.035 0.041 0.040 0.045 0.500 0.284 0.460 0.235 0.235 0.155 0.254 0.174

6 0.034 0.035 9.320 10.522 6.308 6.820 0.032 0.035 3.619 4.852 3.682 3.932 0.041 0.042 1.227 0.864 0.200 0.248 0.043 0.045 0.258 0.215 0.187 0.170 0.050 0.052 0.305 0.282 0.205 0.217

7 0.040 0.045 7.358 8.950 4.863 4.900 0.045 0.047 3.262 3.489 1.845 2.318 0.045 0.047 0.855 0.634 0.231 0.240 0.051 0.055 0.230 0.200 0.170 0.175 0.056 0.055 0.326 0.297 0.215 0.254

8 0.042 0.043 6.820 7.053 3.510 3.726 0.050 0.049 3.866 4.285 1.632 1.864 0.050 0.050 0.554 0.428 0.251 0.287 0.053 0.058 0.180 0.164 0.187 0.170 0.060 0.062 0.284 0.235 0.187 0.206

167

Appendices B.2.3 Gold leaching Table B.17 Gold concentration in cultures (mg/l) in one-step bioleaching nonbiooxidized ESM in shake flasks

Sample 0.5A 0.5B 0.5A CV 0.5B CV 0.5A PF 0.5B PF 1A 1B 1A CV 1B CV 1A PF 1B PF 2A 2B 2A CV 2B CV 2A PF 2B PF 4A 4B 4A CV 4B CV 4A PF 4B PF 8A 8B 8A CV 8B CV 8A PF 8B PF

1 0.085 0.000 0.168 0.035 0.074 0.072 0.000 0.000 0.041 0.045 0.130 0.122 0.000 0.000 0.077 0.076 0.336 0.213 0.000 0.000 0.300 0.242 0.380 0.296 0.000 0.000 0.000 0.000 0.140 0.027

2 0.156 0.048 0.371 0.163 0.269 0.352 0.008 0.007 0.409 0.435 0.757 0.638 0.001 0.001 0.296 0.127 1.047 0.796 0.000 0.000 0.227 0.185 0.326 0.245 0.001 0.000 0.002 0.002 0.111 0.034

3 0.110 0.037 0.621 0.382 0.540 0.406 0.004 0.004 0.612 0.598 0.889 0.854 0.001 0.001 0.253 0.084 1.082 0.928 0.000 0.000 0.153 0.128 0.271 0.194 0.001 0.000 0.004 0.003 0.082 0.041

Time (days) 4 0.063 0.026 0.870 0.600 0.810 0.460 0.000 0.000 0.814 0.760 1.020 1.070 0.000 0.000 0.209 0.041 1.117 1.059 0.009 0.008 0.108 0.090 0.195 0.179 0.011 0.000 0.005 0.004 0.072 0.052

5 0.000 0.000 2.140 2.180 1.380 1.240 0.004 0.002 1.710 1.349 1.870 2.030 0.004 0.000 0.227 0.066 1.430 1.267 0.004 0.002 0.108 0.091 0.237 0.202 0.010 0.007 0.009 0.017 0.118 0.096

6 0.001 0.000 1.798 1.948 1.568 1.448 0.002 0.000 1.038 0.979 1.038 1.198 0.000 0.000 0.138 0.057 1.290 1.079 0.000 0.000 0.084 0.080 0.217 0.212 0.000 0.001 0.014 0.003 0.113 0.082

7 0.078 0.032 2.228 2.408 2.628 2.348 0.008 0.010 1.458 1.219 1.628 1.728 0.005 0.000 0.084 0.150 1.008 0.908 0.000 0.003 0.062 0.054 0.165 0.194 0.000 0.009 0.009 0.000 0.068 0.091

168

Appendices Table B.18 Gold concentration in cultures (mg/l) in two-step bioleaching nonbiooxidized ESM in shake flasks

Sample 0.5A 0.5B 0.5A CV 0.5B CV 0.5A PF 0.5B PF 1A 1B 1A CV 1B CV 1A PF 1B PF 2A 2B 2A CV 2B CV 2A PF 2B PF 4A 4B 4A CV 4B CV 4A PF 4B PF 8A 8B 8A CV 8B CV 8A PF 8B PF 2 0.000 0.005 0.037 0.038 0.067 0.054 0.001 0.005 0.346 0.304 0.291 0.288 0.010 0.000 0.010 0.020 1.582 1.838 0.001 0.000 0.181 0.185 1.020 0.515 0.000 0.000 0.108 0.077 0.047 0.033 3 0.000 0.000 0.100 0.110 0.200 0.160 0.004 0.006 0.817 0.623 1.200 0.816 0.012 0.002 0.040 0.160 2.493 2.747 0.010 0.000 0.150 0.150 1.500 0.818 0.000 0.000 0.072 0.064 0.047 0.033 4 0.010 0.024 0.289 0.302 0.304 0.343 0.012 0.013 1.180 1.000 2.150 1.450 0.017 0.006 0.342 0.575 3.270 3.530 0.026 0.005 0.108 0.108 1.902 1.118 0.025 0.000 0.043 0.052 0.048 0.030

Time (days) 5 0.000 0.000 0.563 0.683 0.473 0.354 0.006 0.010 1.220 0.919 1.200 1.130 0.000 0.000 0.200 0.300 3.012 3.408 0.000 0.000 0.052 0.050 1.500 0.860 0.000 0.000 0.028 0.032 0.020 0.020

6 0.010 0.015 0.978 1.000 0.519 0.553 0.019 0.000 1.600 1.270 1.810 1.680 0.000 0.000 0.081 0.223 2.790 3.210 0.011 0.000 0.024 0.021 1.000 0.595 0.035 0.000 0.006 0.010 0.008 0.007

7 0.000 0.000 1.480 1.520 0.700 0.760 0.010 0.002 1.620 1.350 1.850 2.164 0.000 0.000 0.080 0.120 2.990 3.472 0.020 0.000 0.018 0.018 1.000 0.580 0.000 0.000 0.006 0.000 0.008 0.004

8 0.000 0.040 2.190 2.250 0.877 1.120 0.004 0.000 1.650 1.420 1.890 2.660 0.014 0.000 0.079 0.194 3.300 4.120 0.029 0.000 0.016 0.017 1.000 0.569 0.038 0.000 0.002 0.000 0.010 0.012

169

Appendices Table B.19 Gold concentration in cultures (mg/l) in bioleaching bio-oxidized ESM in shake flasks

Sample 0.5A 0.5B 0.5ACV 0.5BCV 0.5APF 0.5BPF 1A 1B 1ACV 1BCV 1APF 1BPF 2A 2B 2ACV 2BCV 2APF 2BPF 4A 4B 4ACV 4BCV 4APF 4BPF 8A 8B 8ACV 8BCV 8APF 8BPF

1 0.019 0.006 0.055 0.050 0.042 0.039 0.004 0.003 0.128 0.126 0.054 0.045 0.000 0.002 0.219 0.211 0.104 0.100 0.001 0.001 0.408 0.280 0.188 0.201 0.005 0.001 0.535 0.525 0.359 0.339

2 0.001 0.001 0.050 0.054 0.061 0.061 0.002 0.005 0.103 0.110 0.106 0.100 0.000 0.000 0.206 0.195 0.216 0.221 0.000 0.000 0.395 0.394 0.417 0.382 0.000 0.003 0.711 0.713 0.758 0.774

3 0.002 0.003 0.100 0.103 0.070 0.070 0.001 0.002 0.130 0.136 0.126 0.120 0.000 0.001 0.500 0.489 0.260 0.286 0.000 0.000 1.352 1.263 0.620 0.600 0.000 0.000 1.760 1.830 0.762 0.783

Time (days) 4 5 0.008 0.008 0.007 0.008 0.128 0.261 0.133 0.200 0.088 0.120 0.095 0.141 0.000 0.000 0.000 0.002 0.211 0.450 0.247 0.502 0.133 0.242 0.130 0.212 0.002 0.000 0.000 0.000 0.765 1.023 0.755 1.100 0.315 0.452 0.344 0.482 0.000 0.000 0.001 0.000 1.515 2.200 1.425 2.162 0.765 0.866 0.675 0.850 0.002 0.001 0.001 0.002 2.605 2.655 2.775 2.702 0.785 0.550 0.705 0.485

6 0.010 0.011 0.388 0.281 0.149 0.204 0.004 0.001 0.686 0.765 0.332 0.312 0.001 0.001 1.305 1.425 0.571 0.617 0.001 0.000 2.895 2.885 0.975 1.025 0.000 0.000 2.685 2.745 0.306 0.259

7 0.008 0.010 0.500 0.450 0.195 0.170 0.002 0.003 0.685 0.793 0.340 0.310 0.002 0.002 1.502 1.502 0.600 0.603 0.000 0.000 3.005 2.910 0.800 0.810 0.002 0.001 2.146 2.200 0.212 0.230

8 0.010 0.007 0.627 0.428 0.175 0.161 0.003 0.006 0.689 0.815 0.350 0.302 0.004 0.003 1.585 1.515 0.615 0.582 0.004 0.002 2.995 3.025 0.609 0.563 0.002 0.002 1.105 1.155 0.175 0.121

170

Appendices Table B.20 Gold recovery (%) in bioleaching ESM in shake flasks

Bacteria 0.5 C. violaceum (a) (b) (c) P. fluorescens (a) (b) (c) 9.516 9.113 11.34 10.21 4.099 3.922 1 3.139 3.151 8.080 4.002 4.670 3.503

Pulp density (%) 2 0.217 0.471 8.327 1.384 3.489 3.215 4 0.139 0.094 8.085 0.173 0.775 2.305 8 0.003 0.024 3.646 0.027 0.010 1.037

(a): one-step bioleaching non-biooxidized ESM (b): two-step bioleaching non-biooxidized ESM (c): bioleaching bio-oxidized ESM

171

Appendices B.2.4 Copper Table B.21 Copper concentration in cultures (mg/l) in one-step bioleaching nonbiooxidized ESM in shake flasks

Sample 0.5A 0.5B 0.5A CV 0.5B CV 0.5A PF 0.5B PF 1A 1B 1A CV 1B CV 1A PF 1B PF 2A 2B 2A CV 2B CV 2A PF 2B PF 4A 4B 4A CV 4B CV 4A PF 4B PF 8A 8B 8A CV 8B CV 8A PF 8B PF 0 15.5 14.4 15.4 14.5 14.3 15.9 25.1 27.4 26.1 26.4 26.2 26.3 39 33.5 37.5 35.6 36.7 36 74.4 47.2 64.3 53.8 61.2 57.9 107 95.6 98.5 101 105 99.3 1 79.5 75.6 75.8 65.7 79.7 96 135 155 164 167 180 180 222 204 299 262 339 330 349 407 417 482 513 480 633 696 564 603 795 717 2 90.3 77.4 85.1 73.7 78.5 97.1 147 164 174 188 174 174 247 210 318 270 356 325 350 423 400 469 490 431 630 686 560 617 820 836

Time (days) 3 4 94.8 99.3 87.05 96.7 95.05 109 84.85 92 78.5 78.6 81.2 65.3 150 154 167 169 178 182 188 189 179 184 180 187 264 280 205 200 320 327 280 283 355 352 311 296 350 352 532 438 395 393 458 447 467 447 420 402 632 633 678 669 555 551 625 636 873 925 855 874

5 111 105 138 108 61.6 56.8 181 205 210 219 208 219 300 214 356 311 393 254 462 572 481 558 500 450 829 844 710 833 1020 1010

6 115 106 127 97.1 56.3 58.9 133 158 166 144 137 154 319 220 367 202 365 309 471 589 508 573 563 520 869 896 676 835 986 996

7 100 104 59.7 96.9 134 99.2 181 205 224 223 204 211 335 200 370 245 298 281 424 525 457 376 456 506 1136 1224 896 1064 1480 1480

172

Appendices Table B.22 Copper concentration in cultures (mg/l) in two-step bioleaching nonbiooxidized ESM in shake flasks

Sample 0.5A 0.5B 0.5A CV 0.5B CV 0.5A PF 0.5B PF 1A 1B 1A CV 1B CV 1A PF 1B PF 2A 2B 2A CV 2B CV 2A PF 2B PF 4A 4B 4A CV 4B CV 4A PF 4B PF 8A 8B 8A CV 8B CV 8A PF 8B PF 2 46.1 47.3 100.2 97.1 72.7 75.1 143.2 132.8 195.3 185 163.2 160.8 184.2 179.8 329 271 281.3 297.8 249.3 259.2 450.2 454.8 483 509 543 561 670 686 851 825 3 53.2 54.92 133 131.7 100.3 106.9 150.2 130.4 214.5 208.2 190 188.5 205.3 192.8 348.6 291.4 295.4 304.6 276 300 468 469 522 520.6 666.2 678.4 713.2 710.8 949 913 4 58 62.1 135 123.4 120.5 125.5 155 135.5 244.8 239.8 237.5 232.5 248.5 237.5 360.3 304.7 322 338 387.5 393.6 501.3 503.7 576 561 765 795 792.5 822.5 907 1061

Time (days) 5 59.68 65.78 133.8 122.2 122 123.6 151 136.3 230 229.1 198.5 193.5 246.5 235.5 367.8 316.2 340.4 356.6 440.3 454 548.3 551.7 613 587 826 874.8 799 1013 1050 1028

6 63.5 72.4 129.4 123.6 121.5 118.5 150 136.5 213 203 195 189 246 235.2 376.1 328.4 373.1 388.9 434 467 525 536.4 652 632 867 880 894.2 949.8 1200 1078

7 60.2 69.8 129 125 120 114 162 138 206 202.7 197.3 193.6 229 215.6 315 262.8 326.6 337.4 426 438 520 534 651.4 617.6 870.3 905.7 927 935 1246 1232

8 60 68 129.2 126 120 118 172 138 205 193 206.3 199.5 220 211.5 298 204 243 279 490.7 306.6 516.3 523.7 645 615.4 883 917 1160 1210 1560 1560

173

Appendices Table B.23 Copper concentration in cultures (mg/l) in bioleaching bio-oxidized ESM in shake flasks

Sample 0.5A 0.5B 0.5ACV 0.5BCV 0.5APF 0.5BPF 1A 1B 1ACV 1BCV 1APF 1BPF 2A 2B 2ACV 2BCV 2APF 2BPF 4A 4B 4ACV 4BCV 4APF 4BPF 8A 8B 8ACV 8BCV 8APF 8BPF

1 0.492 0.138 2.26 2.54 0.572 0.722 1.11 0.475 4.88 5.16 1.57 0.929 1.3 1.63 10.5 9.16 2.93 2.5 3.39 3.5 13.6 12.5 3.63 4.4 6.84 6.86 16.7 14.7 3.62 3.68

2 0.433 0.014 3.51 4.09 2.17 2.38 2.16 0.73 6.36 7.9 3.94 3.21 1.87 2.61 14.6 12.7 6.96 6.59 4.05 4.37 17.8 17.7 11.4 11 8.73 9.26 22.8 17.5 22.3 22

3 2 0.85 5.16 5.63 3.35 3.6 4.62 1.16 7.9 9.4 5.52 4.72 2.89 4.18 20.5 18.9 9.82 11.3 5.82 6.43 28 28 19.5 18.2 13.4 12.7 46.8 40.2 30.1 30.2

Time (days) 4 5 3.53 3.8 1.69 1.86 6.8 7.1 7.15 7.32 4.57 5 4.81 5.48 7.03 9 1.55 2.89 9.5 13.6 10.7 14.5 6.96 10.1 6.22 8.4 3.91 4.82 5.8 6.85 26.8 30 24.5 28.6 13.1 15.6 16.1 19 7.76 10.6 8.32 10.6 38.9 50.3 38.6 50 27.1 29.1 25.6 29 18.2 19 16.2 19 68.8 75.6 60.5 66.3 36.2 33.5 38.1 38.1

6 4.15 1.8 7.34 7.45 5.23 6.15 12.3 3.13 17.6 18.3 13 10.6 5.74 7.78 35.9 30.6 17.9 22 13.3 12.9 60.6 58.3 30.8 33.7 21.4 22.2 81.4 71.9 30.1 38.3

7 4.93 1.65 6.92 6.6 4.8 4.79 13 3.67 15.6 16.3 11.5 9.52 5.52 8.07 35.7 27.8 16.7 19 13.2 12.1 54 54.2 27.3 27.6 19 20.2 67.5 62.3 30 34.2

8 5.6 1.42 5.76 5.71 4.3 3.63 15 2.45 13.1 14.1 10.3 8.22 5.15 8.15 35 24.7 15.4 16.8 12.5 11.5 48.4 49.3 22.9 22.1 16 18.4 56 49.1 28.9 29.4

174

Appendices Table B.24 Copper recovery (%) in bioleaching ESM in shake flasks

Bacteria 0.5 C. violaceum (a) (b) (c) P. fluorescens (a) (b) (c) 86.86 93.47 52.15 82.34 86.86 40.13 1 78.92 85.56 63.30 75.39 82.98 41.61

Pulp density (%) 2 58.88 62.19 58.62 60.12 67.27 35.17 4 47.71 48.55 52.41 47.80 56.67 28.43 8 43.26 52.30 33.79 65.32 68.86 16.37

(a): one-step bioleaching non-biooxidized ESM (b): two-step bioleaching non-biooxidized ESM (c): bioleaching bio-oxidized ESM

175

Appendices B.3 Growth of C. violaceum and P. fluorescens in fresh medium Table B.25 Growth of C. violaceum in fresh medium in shake flasks

Time (hours) 0 0.5 17 2.8 4.0 4.3 4.7 5.3 5.8 6.3 6.9 7.4 8.0 8.6 9.0 9.6 10.2 10.7 11.2 11.9 12.7 13.7 14.6 15.5 19.4 20.3 21.0 22.5

pH 7.14 7.12 7.11 7.17 7.12 7.12 7.16 7.13 7.27 7.24 7.25 7.27 7.35 7.40 7.45 7.52 7.60 7.67 7.64 7.73 7.80 7.90 7.89 7.91 8.12 8.15 8.17 8.20

OD600nm 0.0579 0.0629 0.0623 0.0876 0.2886 0.3568 0.4969 0.8359 1.0597 1.3527 1.5392 1.6772 1.7935 1.8927 1.9434 2.2180 2.6700 3.0000 3.2132 3.2984 3.3136 3.5640 3.7655 4.0528 4.2100 4.3480 4.3680 4.4852

No of Viable Cell/ml Cyanide (mg/l) 1.60E+05 0.0179 1.62E+05 0.132 1.85E+05 0.109 4.42E+05 0.122 1.20E+06 0.113 3.27E+06 0.132 8.89E+06 0.158 3.98E+07 0.272 6.00E+07 1.096 1.08E+08 2.849 2.94E+08 4.819 4.85E+08 7.195 1.32E+09 10.03 1.39E+09 13.80 1.46E+09 15.05 1.9E+09 18.20 2.57E+09 26.91 2.83E+09 50.93 3.23E+09 38.96 3.13E+09 25.40 2.78E+09 20.57 3.75E+09 19.67 3.28E+09 18.83 5.68E+09 16.25 1.9E+09 12.05 1.61E+09 10.35 4.85E+08 10.14 4.5E+08 8.26

176

Appendices Table B.26 Growth of P. fluorescen in fresh medium in shake flasks

Time (hours) 0 0.8 1.6 2.0 3.0 3.5 4.5 6.0 6.8 8.0 10.0 11.0 12.3 14.0 15.3 16.3 17.0 17.7 19.0 20.0

pH 7.26 7.31 7.38 7.42 7.49 7.53 7.60 7.65 7.70 7.76 7.78 7.82 8.03 8.13 8.16 8.17 8.25 8.30 8.32 8.40

OD450nm 0.215 0.2971 0.3441 0.4037 0.9172 1.0057 1.3644 1.5825 1.6606 1.7677 1.7526 1.7994 1.8268 2.0356 2.2458 2.5036 2.745 3.1242 3.1854 3.2220

No of Viable Cell/ml Cyanide (mg/l) 6.00E+06 2.25E+07 4.56E+07 8.50E+07 2.55E+08 5.6E+08 5.18E+08 6E+08 7.5E+08 8.13E+08 8E+08 8.5E+08 6.63E+08 6E+08 6.65E+08 5.02E+08 3.37E+08 3.13E+08 6.30E+07 4.70E+07 0.104 0.050 0.029 0.040 0.065 0.200 0.544 1.456 3.786 8.453 18.02 13.27 12.21 10.36 8.235 6.325 5.233 5.023 2.032 1.805

177

Appendices Table B.27 Growth of P. fluorescens in fresh medium in bioreactor

Time Agitation Temperature (min) (hour) (rpm) (0C) 0.0 0.00 200 30.0 33.0 0.55 200 30.0 96.0 1.60 200 28.6 276.0 4.60 200 29.9 345.5 5.76 200 30.6 427.0 7.12 200 29.2 510.5 8.51 200 29.2 569.0 9.48 200 30.1 673.5 11.23 200 30.3 756.5 12.61 200 30.2 790.0 13.17 200 29.7 846.0 14.10 200 30.2 871.0 14.52 200 28.9 929.5 15.49 200 28.9 1006.0 16.77 200 30.5 1034.0 17.23 200 28.9 1070.0 17.83 200 30.3 1128.5 18.81 200 29.0 1195.0 19.92 200 28.3 1251.0 20.85 200 28.8 1284.5 21.41 200 29.0 1338.0 22.30 200 29.4 1397.5 23.29 200 29.0 1459.0 24.32 200 29.7 1515.5 25.26 200 30.8 1582.5 26.38 200 30.4 1623.5 27.06 200 30.3 1645.0 27.42 200 30.4 1682.5 28.04 200 29.2 1767.5 29.46 200 29.8 2118.0 35.30 200 30.6 DO: dissolved oxygen; OD: optical density

pH 7.31 7.33 7.38 7.35 7.36 7.41 7.42 7.44 7.46 7.49 7.60 7.62 7.79 7.88 7.89 8.06 7.94 8.03 8.19 8.25 8.10 8.05 8.07 8.01 7.94 7.96 8.18 8.23 8.36 8.36 8.34

DO (%) 74 73 82 58 54 49 36 33 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

OD450 0.1370 0.1370 0.1320 0.1624 0.1920 0.1940 0.2363 0.2603 0.3431 0.6195 0.7711 0.9661 1.0610 1.1329 1.5311 1.5867 1.7803 1.6606 1.8835 2.4104 2.7636 3.0560 3.3640 3.5296 3.6048 3.6760 3.7338 3.7506 3.8445 3.8943 4.2507

Cyanide (mg/l) 0.000 0.156 0.089 0.097 0.106 0.109 0.086 0.137 0.136 0.184 0.186 0.308 0.347 0.333 0.975 2.483 3.418 4.432 6.008 7.339 9.202 12.62 17.69 18.03 17.41 16.62 16.37 16.47 15.06 12.59 10.14

178

Appendices B4. Bio-oxidation ESM Table B.28 Metal removal (%) of bio-oxidation ESM by At. ferrooxidation

Metals Time (days) 0 1 2 3 4 5 6 7 Al 0 50.2 58.9 60.6 62.5 62.8 63.9 64.6 Cu 0 69.6 77.2 77.9 78.7 78.4 79.4 81.1 Ni 0 17.7 26.3 40.5 47.1 56.0 60.7 69.5

Table B.29 Metal removal (%) of bio-oxidation ESM in control

Day 1 2.1 3.2 4.8 5.7 6.7 7.6

Al 0 35.4 39.9 44.4 47.0 47.4 48.9

Cu 0 47.1 50.1 52.8 55.1 53.9 55.8

Ni 0 0 7.2 18.6 27.6 31.9 40.1

179

Appendices B.5 Bioleaching in bioreactor Table B.30 Growth and bioleaching ESM of P. fluorescens in bioreactor (with aeration)

Days 0 0.2 0.5 1.0 1.9 3.2 4.3 5.2 6.3 7.2 7.3

rpm 200 200 200 200 200 200 200 200 200 200 200

Temp 29.8 29.2 29.4 30 29.8 29 29.7 30.6 29.3 29.6 30

pH 7.25 7.34 7.75 8.25 8.72 8.79 8.65 8.52 8.53 8.78 8.79

D.O(%) 81 0 0 21 83 80 78 78 0 16 19

ln(CFU) 18.1 20.2 20.6 19.6 13.5 11.6 8.81 13.4 20.6 21.1 nt

Cu(mg/l) 0 152 169 196 226 293 282 299 301 306 314

Au(mg/l) 0 1.375 1.420 1.600 1.885 0.845 0.810 1.030 1.450 1.640 1.600

Table B.31 Growth and bioleaching ESM of P. fluorescens in bioreactor (without aeration)

day 0 0.3 0.9 2.0 3.1 4.1 5.2 6.2 8.9

rpm 200 200 200 200 200 200 200 200 200

Temp 30.6 30.6 24.6 29.1 29.4 30.3 30.8 30.8 30.1

pH 7.15 6.57 6.46 6.53 6.57 6.56 6.56 6.63 6.64

D.O (%) 47 0 0 0 0 0 0 0 0

ln(CFU) 18.00517 20.32279 20.39476 17.9899 18.62769 20.11446 19.99449 19.39524 20.67197

Cu(mg/l) Au(mg/l) 0 77.2 114 128 125 124 126 139 130 0 1.39 1.44 1.445 1.345 1.38 1.36 1.48 1.365

180

Appendices B.6 Chemical leaching Table B.32 Metal removal of acid leaching

Sample Control Control N10 N10' N50 N50' N100 N100' N200 N200' S10 S10' S50 S50' S100 S100' S200 S200' C10 C10' C50 C50' C100 C100' C200 C200' O10 O10' O50 O50' O100 O100' O200 O200'

Cu nd nd 209 235 295 177 226 202 250 244 233 212 237 224 234 240 276 274 229 202 250 315 231 242 362 288 60 76.5 64.5 69.5 70.9 75.4 75.7 71.3

Fe nd nd 61.4 37.9 143 142 147 150 154 156 110 104 127 131 126 134 124 133 95.8 91.6 98.8 97.8 100 94.5 81.8 87.6 135 122 155 164 177 178 163 174

Metal (mg/l) Ni nd nd 39.3 41.6 82 79.6 76.5 76.7 75.3 83.5 43 41.9 39.5 38.4 36.9 38.3 34.6 34.8 40 38.2 35.2 36.4 35.6 32.5 28.4 31.7 9.01 8.92 1.77 2.47 2.64 3.45 2.71 2.95

Al 0.11 0.12 6.56 6 8.81 8.91 9.65 10.1 10.7 11.9 9.41 9.65 12.6 12.6 15.5 15.6 15.4 16 8.19 7.85 8.36 8.47 8.8 8.41 7.25 8.09 15.6 13.9 17.4 18.1 18.8 18.6 17.6 17.4

Pb nd nd 8.39 7.64 12.2 11.6 12.4 12.2 11.6 12.8 5.65 5.33 3.3 2.71 2.81 2.7 3.18 3.03 12 11.2 11.5 12.6 11.1 11.5 12.1 10.3 1.68 1.67 1.66 4.14 4.24 5.14 1.39 5.97 181

Appendices

G10 152 38.9 31.9 5.39 G10' 186 34.6 29.1 5.04 G50 233 83.2 35.8 7.26 G50' 231 80.4 37.5 7.54 G100 202 84.8 33.6 7.56 G100' 204 85.8 35 7.77 G200 224 88.9 33.4 8.32 G200' 256 87 30.9 7.69 N: nitric acid, S: sulphuric acid, C: citric acid, O: oxalic acid, G: gluconic acid, nd: not detected
Table B.33 Metal removal of ferric leaching

8.99 11.8 11.8 11.6 10.2 10.4 9.85 9.4

Reagent(s)
H2SO4 water Fe3+

Metal concentration (mg/l)

No 1 2 3 4 5 6 7 8 13 14 15 16

Day 7 7 14 14

Cu

Co

Ni 0.6 0.4

Ag 0.00 0.00

Al 0.00 0.00 0.02 0.07 11.7

Pb 1.12 0.95 0.64

Zn

Mg

y y y y y y y y y y y y y y y y y y y y y y y y

0.03 0.06 0.00 0.12

0.027 2.71 0.00 2.46

0.01 0.11 0.48 0.00 0.02 0.00 0.92 0.00 148 155 152 161 150 153 156 155 3.75 50.8 1.35

0.004 2.74

0.272 0.047 3.04 12.95 10.6 4.25 4.45 4.30 4.55 4.35 4.35 4.55 4.40 5.60 5.25 6.35 6.45 6.60 6.00 6.85 6.40

7 7 14 14 7 7 14 14

2.50 50.8 1.45 11.45

2.75 56.8 1.55 13.05 13.35 3.00 56.3 1.75 14.25 3.15 54.3 1.55 12.6 14.2 13.75 13.8 13.3 18.45

2.50 52.8 1.65 12.75 2.70 59.3 0.9 14.25 13.6

2.15 56.3 1.15

182