Gold bioleaching of electronic scrap material by cyanogenic bacteria and its enhancement with biooxidation

ACKNOWLEDGEMENTS iSUMMARY viNOMENCLATURE viii 2.1 Electronic Scrap Material ESM as a secondary gold ore 4 2.3 Cyanogenic microorganisms and cyanide producing mechanism 13 2.3.2 Chromoba

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

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

ACKNOWLEDGEMENTS i

SUMMARY viNOMENCLATURE viii

2.1 Electronic Scrap Material (ESM) as a secondary gold ore 4

2.3 Cyanogenic microorganisms and cyanide producing mechanism 13

2.3.2 Chromobacterium violaceum 14 2.3.3 Pseudomonas fluorescens 15

2.4 Applying cyanogenic microorganisms in gold bioleaching 16 2.4.1 Potentials in bioleaching precious metals 16

2.4.2 Gold bioleaching by C violaceum and P fluorescens 17

2.5.2 ESM 21

2.5.2.2 Effects of ESM on microbial growth 22

4.1 Characterization of ESM 42

4.1.4 Toxicity Characteristic Leaching Procedure tests 45

4.4.3 Comparison of Chromobacterium violaceum and Pseudomonas

fluorescens, one-step and two-step in bioleaching non-biooxidized ESM

94

by Chromobacterium violaceum and Pseudomonas fluorescens

4.6 Bioleaching ESM by Pseudomonas fluorescens in bioreactor 115

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 oxidation

bio-The ESM used in this project were fine particles, of size <75μm 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 step bioleaching, the ESM was introduced into the culture one day after bacterial inoculation

two-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

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

ATCC American Type Culture Collection

EDX Energy Dispersive X-Ray

ESM Electronic Scrap Material

ICP-OES Inductively Coupled Plasma Optical Emission Spectrometer

SEM Scanning Electron Microscopy

TCLP Toxicity Characteristic Leaching Procedure

US EPA United States Environmental Protection Agency

vvm Volume air per volume medium per minute

Table 2.1 Improvement of leaching gold from gold ore by biooxidation 24

Table 3.2 The wavelengths (nm) used for metal analysis by ICP-OES 31Table 4.1 Metal composition of the liquor after acid digestion 44Table 4.2 Metal concentration of the TCLP extract compared with Regulatory

Table 4.6 Increase in gold/copper ratio in leachate by bio-oxidation 113

Table B.2 Particle size distribution (%volume) of original <75µm ESM 139Table B.3 Particle size distribution (%volume) of acid digested <75µm ESM 141Table B.4 Particle size distribution (%volume) of bio-oxiddized <75µm ESM 143Table B.5 Particle size distribution (%volume) of original, acid digestion and

bio-oxidized <75µm ESM

145

Table B 6 Elemental content of <75µm (SEM/EDX) 155Table B.7 Elemental content of acid digested <75µm (SEM/EDX) 157

Table B.9 Metal composition of ESM in literature 160Table B.10 Metal composition of the liquor after acid digestion 161Table B.11 pH profile of one-step bioleaching non-biooxidized ESM in shake

flasks

162

Table B.12 pH profile of two-step bioleaching non-biooxidized ESM in shake 163

Table B.13 pH profile of bioleaching bio-oxidized ESM in shake flasks 164Table B.14 Free cyanide concentration (mg/l) in one-step bioleaching non-

biooxidized ESM in shake flasks

165

Table B.15 Free cyanide concentration (mg/l) in two-step bioleaching

non-biooxidized ESM in shake flasks

166

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

in shake flasks

167

Table B.17 Gold concentration in cultures (mg/l) in one-step bioleaching non-

biooxidized ESM in shake flasks

168

Table B.18 Gold concentration in cultures (mg/l) in two-step bioleaching non-

biooxidized ESM in shake flasks

169

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

ESM in shake flasks

Table B.22 Copper concentration in cultures (mg/l) in two-step bioleaching non-

biooxidized ESM in shake flasks

173

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

ESM in shake flasks

174

Table B.24 Copper recovery (%) in bioleaching ESM in shake flasks 175Table B.25 Growth of C violaceum in fresh medium in shake flasks 176Table B.26 Growth of P fluorescen in fresh medium in shake flasks 177Table B.27 Growth of P fluorescens in fresh medium in bioreactor 178Table B.28 Metal removal (%) of bio-oxidation ESM by At ferrooxidation 179

Table B.29 Metal removal (%) of bio-oxidation ESM in control 179Table B.30 Growth and bioleaching ESM of P fluorescens in bioreactor (with

aeration)

180

Table B.32 Metal removal of acid leaching 181

Figure 2.1 Market price of gold over the last decade 6Figure 3.1 Experimental set up of bioreactor system 39Figure 4.1 Particle size distribution of < 75μm ESM 42Figure 4.2 Images of < 75μm ESM under Scanning Electron Microscope

(SEM)

43

Figure 4.3 (a) Removal of metals from < 75μm ESM by nitric acid 48Figure 4.3 (b) Removal of metals from < 75μm ESM by sulfuric acid 48Figure 4.3 (c) Removal of metals from < 75μm ESM by citric acid 49Figure 4.3 (d) Removal of metals from < 75μm ESM by oxalic acid 49Figure 4.3 (e) Removal of metals from < 75μm ESM by gluconic acid 50Figure 4.4 (a) Bio-oxidation of ESM by A ferrooxidans 52

Figure 4.5 (a) Growth of C violaceum in shake flask in the absence of ESM 57Figure 4.5 (b) pH profile and cyanide production of C violaceum in shake flask

treated ESM

63

Figure 4.9 (b) Cyanide profile in one-step bioleaching with 1% pulp density 66Figure 4.9 (c) Cyanide profile in one-step bioleaching with 2% pulp density 67Figure 4.9 (d) Cyanide profile in one-step bioleaching with 4% pulp density 67Figure 4.9 (e) Cyanide profile in one-step bioleaching with 8% pulp density 68Figure 4.10 (a) Gold leaching in one-step bioleaching with 0.5% pulp density 70Figure 4.10 (b) Gold leaching in one-step bioleaching with 1% pulp density 71Figure 4.10 (c) Gold leaching in one-step bioleaching with 2% pulp density 71Figure 4.10 (d) Gold leaching in one-step bioleaching with 4% pulp density 72Figure 4.10 (e) Gold leaching in one-step bioleaching with 8% pulp density 72Figure 4.11 Influence of pulp density on one-step bioleaching gold from non-

treated ESM

73

Figure 4.12 (a) Copper leaching in one-step bioleaching with 0.5% pulp density 75Figure 4.12 (b) Copper leaching in one-step bioleaching with 1% pulp density 76Figure 4.12 (c) Copper leaching in one-step bioleaching with 2% pulp density 76Figure 4.12 (d) Copper leaching in one-step bioleaching with 4% pulp density 77Figure 4.12 (e) Copper leaching in one-step bioleaching with 8% pulp density 77Figure 4.13 Influence of pulp density on leaching copper in one-step

bioleaching

78

Figure 4.14 (a) pH profile in two-step bioleaching with 0.5% pulp density 79Figure 4.14 (b) pH profile in two-step bioleaching with 1% pulp density 80Figure 4.14 (c) pH profile in two-step bioleaching with 2% pulp density 80Figure 4.14 (d) pH profile in two-step bioleaching with 4% pulp density 81Figure 4.14 (e) pH profile in two-step bioleaching with 8% pulp density 81Figure 4.15 (a) Cyanide production in two-step bioleaching with 0.5% pulp

density

84

Figure 4.15 (c) Cyanide production in two-step bioleaching with 2% pulp density 85Figure 4.15 (d) Cyanide production in two-step bioleaching with 4% pulp density 85Figure 4.15 (e) Cyanide production in two-step bioleaching with 8% pulp density 86Figure 4.16 (a) Gold leaching in two-step bioleaching with 0.5% pulp density 88Figure 4.16 (b) Gold leaching in two-step bioleaching with 1% pulp density 88Figure 4.16 (c) Gold leaching in two-step bioleaching with 2% pulp density 89Figure 4.16 (d) Gold leaching in two-step bioleaching with 4% pulp density 89Figure 4.16 (e) Gold leaching ESM in two-step bioleaching with 8% pulp density 90Figure 4.17 Influence of pulp density on gold leaching in two-step bioleaching 90Figure 4.18 (a) Copper leaching in two-step bioleaching with 0.5% pulp density 92Figure 4.18 (b) Copper leaching in two-step bioleaching with 1% pulp density 92Figure 4.18 (c) Copper leaching in two-step bioleaching with 2% pulp density 93Figure 4.18 (d) Copper leaching in two-step bioleaching with 4% pulp density 93Figure 4.18 (e) Copper leaching in two-step bioleaching with 8% pulp density 94Figure 4.19 Comparison of gold recovery in one-step and two-step bioleaching 95Figure 4.20 Comparison of copper recovery in one-step and two-step

pulp density

103

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

Figure 4.31 Bioleaching gold and copper from 2% ESM by P fluorescens in

bioreactor, at 300C, 200 rpm, with no aeration

120

Figure 4.32 Comparison of bioleaching in shake flasks and bioreactor 121Figure B.1 SEM image of as-received ESM at magnification of 50x 148Figure B.2 (a) SEM image (a) of as-received ESM at magnification of 250x 148Figure B.2 (b) SEM image (b) of as-received ESM at magnification of 250x 149Figure B.3 SEM image of <75µm ESM at magnification of 50x 149Figure B.4 SEM image of <75µm ESM at magnification of 250x 150Figure B.5 SEM image of <75µm ESM at magnification of 500x 150Figure B.6 SEM image of <75µm ESM at magnification of 2000x 151Figure B.7 SEM image of acid digested <75µm ESM at magnification of 50x 151Figure B.8 SEM image of acid digested <75µm ESM at magnification of 500x 152Figure B.9 SEM image of acid digested <75µm ESM at magnification of

Figure B.11 (a) Selected area in elemental analysis by EDX/SEM of <75µm ESM 154

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

<75µm ESM

156

Figure B.12 (b) SEM/EDX spectra of acid digested <75µm ESM 156Figure B.13 (a) Selected area in elemental analysis by EDX/SEM of carbon tape 157

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

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:

- 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

CHAPTER 2: LITERATURE REVIEW

2.1 Electronic Scrap Material (ESM) as a secondary gold ore

2.1.1 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 Earth’s 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)

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

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)

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)

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

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

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

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

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 sulfur-oxidizing 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)

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

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

can grow in the temperature range from 10-40 0C, and optimum temperature is at

30-350C (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 β-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

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 å 2Au(CN)

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

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

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)

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

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

25-300C 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)

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 μM) 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

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,

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)