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0

SYNTHESIS AND CHARACTERIZATION OF CHOLINE BASED IONIC LIQUIDS

AND THEIR UTILIZATION IN THE RECOVERY OF BASE METALS FROM BCL

SLAG ASSISTED BY GOLD 1 MINE BACTERIAL ISOLATES A Dissertation submitted to the Faculty of Science, University of Johannesburg In

partial fulfilment of the requirement for the award of a Master’s Degree in

Technology: Biotechnology

January 2017

By

LETLHABILE THAPELO MOYAHA

STUDENT NUMBER: 200803857

Supervisor : Dr. V. Mavumengwana

Co-supervisor : Dr. S. Sekar

Co-Supervisor : Prof. A. Mulaba-Bafubiandi

I

ABSTRACT

Due to the depletion of rich ore bodies, the fact that conventional extractions become

obsolete and uneconomical and additional stringency on environment related regulations,

there is a call for cheaper and greener metal extraction methods. Through research and

innovation, new developments and improvements of processes and products are required in

both metallurgical and biological areas. As a result, research interests in the use of microbes

in the optimization of metal recovery from low grade sulphide ores have grown increasingly

popular. The involvement of microbes in the recovery process is thus dependent on the

growth of the microbes, which is influenced by their physicochemical parameters. The

availability of nutrient salts is thus essential for maintaining optimal growth, furthermore is

the dependence of metal dissolution with nutrient quantities on substrate accessibility.

Considering the “green chemistry” of some ionic liquids the important “technological”,

“toxicological ” and “eco-toxicological” assessment of the risks related with ionic liquid

design; choline based ionic liquids were selected by virtue of their low toxicity, low

environmental persistence and readily biodegradable nature. As a result of the selective

solubility strength of metal oxides in ionic liquids, choline based ionic liquids were

considered novel media for obtaining target metals and improving bioleaching kinetics. The

study thus sought to develop an economic and eco-friendly alternative technology, employing

choline based ionic liquids as biocompatible substrates for the optimal recovery of base

metals, improving bioleaching kinetics.

Biological and molecular characterization of ultra-deep mine isolates (Gold 1 Mine East

Rand, Springs Johannesburg, South Africa) revealed a halophilic community of gram positive

and negative bacteria namely Raoultella ornithinolytica, Bacillus sp., Bacillus thuringiensis

and Pseudomonas moraviensis. The halophilic bacterial strains indicated high

biocompatibility in choline lactate, choline chlorite, choline dihydrogen phosphate, choline

citrate and choline levulinate with poor biocompatibility noted in choline citrate and choline

tartarate. According to literature, the biocompatibility of the leading choline based ionic

liquids (ILs) was accredited to the quaternary- ammonium cation integrating a polar hydroxyl

and the limited branching of the side chain. Prominent logarithmic growth in choline lactate

was indicative of the metabolic advantage of choline lactate over conventional glucose

sources. Furthermore, the bioleaching of BCL slag by Bacillus species and Bacillus

II

thuringiensis employing choline lactate as a substrate was found to effect 97 % iron (Fe)

recoveries from the major iron fayalite phase in BCL slag. Producing moderate growth rates,

choline dihydrogen phosphate employing Bacillus species and Bacillus thuringiensis was

found to effect 31 % recovery of the zinc (Zn) element corroborated by the liberation of a

new zinc silicate phase ( Zn2SiO4) in the slag residue. The studies were thus suggestive of the

selective nature of choline lactate and choline dihydrogen phosphate ILs for the recovery of

iron (Fe) and zinc (Zn) base metals.

In contrast to the chemical leaching control, the use of halophilic bacteria in choline lactate

was found to accelerate bacterial growth, producing competitive iron recoveries; thus

alluding to the potential of these ionic liquid based reactions as an alternative to conventional

hydrometallurgy methods.

Keywords: Bioleaching kinetics, choline based ILs, biocompatibility, halophilic, recoveries

III

DECLARATION

I, Letlhabile Thapelo Moyaha hereby declare that this study entitled “Synthesis and

characterization of choline based ionic liquids and their utilization in the recovery of base

metals from BCL slag assisted by Gold 1 Mine bacterial isolates” is my work, conducted

under the supervision of Dr. Vuyo Mavumengwana, Dr. Sekar Sudharshan and Prof Antoine

Mulaba-Bafubiandi. This study represents my original work and has not been submitted for

any degree or examination in any other university. All other sources used, have been duly

cited in text and acknowledged by complete references.

XLetlhabile Thapelo Moyaha

IV

DEDICATION

I dedicate this work to my Heavenly Farther and maker for He’s abundant favour upon my

life and to my guardian angels Mrs E.B Moyaha and Mr N.N Moyaha for their continual

support. To God almighty be all the glory.

V

ACKNOWLEDGEMENTS

Trust in the Lord with all your heart and lean not on your own understanding; in all your

ways acknowledge him, and he will make your paths straight (Proverbs 3:5-6). Blessed is the

man who finds wisdom, the man who gains understanding, for she is more profitable than

silver and yields better returns than gold (Proverbs 3:13-14) .Glorifying God in all that he has

done and been for me during this remarkable journey.

My sincere gratitude to my supervisors Dr. Vuyo Mavumengwana, Dr. Sekar Sudharshan and

Prof Antoine Mulaba-Bafubiandi for their generous contribution to the success of this project.

I thank you for your mentorship, asking me insightful questions and offering invaluable

guidance towards improving and enhancing the quality of this work. Above all I thank you

for allowing me the creative space and freedom to learn.

A heartfelt appreciation goes to my family, my mother Mrs E.B Moyaha and father Mr N.N

Moyaha, my loving siblings Karabo Moyaha, Tlou Moyaha, Kibi Sebotja, Nomfundo

Morutse, and Nonhlanhla Maphanga, my grandmother Mrs P. Huma and late grandmother

Mrs A. Moyaha. I thank you for supporting me in my goals, always helping me to put my

best foot forward and spurring me on with my favourite guilty pleasures when fatigue has set

in. I thank you for you for your invaluable love, support and always dreaming with me. You

are one in a million.

My sincere gratitude to my laboratory colleagues, my partners in crime Bongeka Mbambo

and Evonia Kanyane Nchabeleng for the stimulating conversations and for all the laughs we

had that brightened up the late nights in the lab. I would also like to acknowledge my mentor

Ms Daphney Mogano and my friends Mapula Pale, Matshidiso Tlhapane, Ayanda Timothy

and Nondumiso Shongwe for their unwavering support.

My infinite gratitude to Mr. E Malenga Ntumba and Ms. N. P Baloyi for their insight,

expertise and mentorship in the field of Extraction Metallurgy, you always went beyond the

call of duty and for that I am eternally grateful. I also sincerely thank Professor Taddese

Wondimu Godetto, Nombuzo Mabuza, Harold Hussein Shiri and Bienvenue Gael Mbanga-

Fouda from the Analytical Chemistry Department. A special thank you goes to Mr. E.Van

Zyl, Dr. N. Niemann, Dr. Derek Dinteh, for their generous assistance and support.

For the financial endorsement of this study, I sincerely thank the University of Johannesburg,

through the National Research Foundation (NRF).

VI

PRESENTATIONS AND ARTICLES PUBLISHED OR WRITTEN FOR

PUBLICATION

Presentations:

Moyaha, L., Mavumengwana, V., Sidu, S., Mulaba-Bafubiandi, A., (2015).The recovery of

base metals from slag using bacterial isolates in the presence of biocompatible ionic liquids

as substrate(s).Poster presentation. DST: Howard University-IT woman in stem conference.

Johannesburg, Hyatt Regency hotel in Rosebank.

Publications:

Sekar, S., Moyaha, L., Mavumengwana, V., Sidu, S., Mulaba-Bafubiandi, A., (2016)

Metagenomics: DNA sequencing of environmental samples from Gold 1 Mine East Rand,

Springs Johannesburg, South Africa. New Biotechnology, 33, 179.

Moyaha, L., Sekar, S., Mavumengwana, V., Sidu, S., Mulaba-Bafubiandi, A. The

valorisation of slag from BCL using biocompatible choline based ionic liquids as a support.

http://www.sciencedirect.com/science/article/pii/S1871678416321264

http://www.sciencedirect.com/science/article/pii/S1871678416321264

VII

TABLE OF CONTENTS

ABSTRACT................................................................................................................................I

DECLARATION ..................................................................................................................... III

DEDICATION ......................................................................................................................... IV

ACKNOWLEDGEMENTS ...................................................................................................... V

PRESENTATIONS AND ARTICLES PUBLISHED OR WRITTEN FOR PUBLICATION

.................................................................................................................................................. VI

TABLE OF CONTENTS........................................................................................................VII

LIST OF FIGURES .............................................................................................................. XIII

LIST OF TABLES ............................................................................................................... XVII

LIST OF ABBREVIATIONS ............................................................................................... XIX

LIST OF UNITS .................................................................................................................. XXII

DISSERTATION OUTLINE............................................................................................. XXIV

CHAPTER ONE ........................................................................................................................ 1

1.0 GENERAL INTRODUCTION ................................................................................... 1

1.1 Background ................................................................................................................. 1

1.2 Problem statement ....................................................................................................... 2

1.3 Hypothesis ................................................................................................................... 3

1.4 Aim and Objectives of study ....................................................................................... 3

1.4.1 Aim of the study................................................................................................... 3

1.4.2 Objectives of the study......................................................................................... 3

CHAPTER TWO ....................................................................................................................... 5

2.0 LITTERATURE REVIEW ......................................................................................... 5

2.1 Introduction ................................................................................................................. 5

2.2 Historical perspective .................................................................................................. 5

2.3 Biomining motivation ................................................................................................. 8

2.4 Bioleaching microorganisms ....................................................................................... 8

VIII

2.4.1 Mesophiles ......................................................................................................... 10

2.4.2 Moderate thermophiles ...................................................................................... 11

2.4.3 Extreme thermophiles ........................................................................................ 11

2.5 Halophilic bacteria .................................................................................................... 15

2.6 Principle of microbial leaching ................................................................................. 15

2.6.1 Metal sulphide dissolution mechanism .............................................................. 16

2.7 Leaching technique ................................................................................................... 18

2.7.1 In situ leaching ................................................................................................... 18

2.7.2 Dump leaching ................................................................................................... 19

2.7.3 Heap leaching..................................................................................................... 19

2.7.4 Vat leaching ....................................................................................................... 20

2.8 Factors affecting bioleaching .................................................................................... 22

2.8.1 Physiochemical parameters................................................................................ 23

2.8.2 Microbiological parameters ............................................................................... 27

2.9 Recovery of base metals from slag ........................................................................... 30

2.10 Ionic liquids as an alternative medium...................................................................... 31

2.10.1 Properties of Ionic Liquids (ILs)........................................................................ 31

2.10.2 Synthesis of Ionic Liquids (ILs) ........................................................................ 33

2.10.3 Types of Ionic Liquids (ILs) .............................................................................. 34

2.10.4 Biodegradable designer solvents........................................................................ 35

2.10.5 Biological Activity of Ionic Liquids (ILs) ......................................................... 36

2.11 Metal recovery potential of Ionic Liquids (ILs) ........................................................ 37

2.12 Choline/cholinium derived Ionic Liquids (ILs) ........................................................ 38

2.13 Concluding remarks .................................................................................................. 39

REFERENCES..................................................................................................................... 40

CHAPTER THREE ................................................................................................................. 51

IX

CHARACTERIZATION OF HALOPHILIC BACTERIA FROM GOLD1 MINE EAST

RAND, SPRINGS JOHANNESBURG, SOUTH AFRICA FOR MICROBIAL MINING

APPLICATIONS ..................................................................................................................... 51

Abstract ................................................................................................................................ 51

3.1 Introduction ............................................................................................................... 52

3.2 Methodology ............................................................................................................. 53

3.2.1 Study area of interest for sampling .................................................................... 53

3.2.2 Collection of samples......................................................................................... 54

3.2.3 Isolation and enrichment of moderately halophilic bacteria .............................. 54

3.2.4 Phenotypic classification of bacteria: Microbiological identification of bacteria .

............................................................................................................................ 54

3.2.5 Biochemical characterization of bacteria API 20E® strips ................................ 54

3.2.6 Genotypic classification of bacteria: 16S rRNA sequencing ............................. 55

3.2.7 Phylogenetic tree................................................................................................ 55

3.3 Results and Discussion.............................................................................................. 55

3.3.1 Microbiological characterization of bacterial isolates ....................................... 55

3.3.2 Biochemical characterization of bacterial isolates............................................. 57

3.3.3 Genotypic characterization of bacterial isolates ................................................ 58

3.3.4 Phylogenetic study of bacterial strains .............................................................. 60

3.4 Conclusion................................................................................................................. 62

Acknowledgement................................................................................................................ 62

References ............................................................................................................................ 63

CHAPTER FOUR.................................................................................................................... 67

SYNTHESIS AND CHARACTERIZATION OF BIOCOMPATIBLE CHOLINE DERIVED

IONIC LIQUIDS FOR BIOTECHNOLOGY APPLICATIONS ............................................ 67

Abstract ................................................................................................................................ 67

4.1 Introduction ............................................................................................................... 68

4.2 Methodology ............................................................................................................. 69

X

4.2.1 Choline hydroxide properties............................................................................. 69

4.2.2 Acids .................................................................................................................. 70

4.2.3 Ionic liquid synthesis ......................................................................................... 71

4.2.4 Evaporation process ........................................................................................... 71

4.2.5 Analytical Spectral study/Characterization........................................................ 71

4.2.5.1 1H NMR Spectra............................................................................................. 72

4.2.5.2 13C NMR Spectra ........................................................................................... 72

4.2.5.3 FT-IR .............................................................................................................. 72

4.2.6 Preliminary growth studies of halophilic bacteria in the presence of choline

based ILs ........................................................................................................................... 72

4.2.6.1 Inoculum preparation for halotolerant bacteria strains .................................. 72

4.2.6.2 Media preparation for growth plate studies (solid media) ............................. 73

4.2.6.3 Direct colony count ........................................................................................ 73

4.2.6.4 Media preparation for shaker growth studies (liquid media) ......................... 73

4.2.6.5 Indirect colony count...................................................................................... 73

4.3 Results and Discussion.............................................................................................. 74

4.3.1 NMR................................................................................................................... 74

4.3.1.1 Characterization of Choline Lactate 1H NMR ............................................... 74

4.3.1.2 Characterization of Choline Lactate 13C NMR .............................................. 75

4.3.1.3 Characterization of Choline Dihydrogen Phosphate 1H NMR....................... 76

4.3.1.4 Characterization of Choline Dihydrogen Phosphate 13C NMR ..................... 77

4.3.1.5 Characterization of Choline Citrate 1H NMR ................................................ 78

4.3.1.6 Characterization of Choline Citrate 13C NMR ............................................... 79

4.3.1.7 Characterization of Choline Tartarate 1H NMR............................................. 80

4.3.1.8 Characterization of Choline Tartarate 13C NMR............................................ 81

4.3.1.9 Characterization of Choline Chloride 1H NMR ............................................. 82

4.3.1.10 Characterization of Choline Chloride 13C NMR ........................................ 83

4.3.1.11 Characterization of Choline Levulinate 1H NMR ...................................... 84

4.3.1.12 Characterization of Choline Levulinate 13C NMR ..................................... 85

4.3.2 FTIR ................................................................................................................... 85

4.3.2.1 FTIR: 2-Hydroxy-N, N, N-trimethylethanaminium 2-hydroxypropanoate ... 86

XI

4.3.2.2 FTIR: 2-Hydroxy-N, N, N-trimethylethanaminium dihydrogen phosphate .. 87

4.3.2.3 FTIR:2-Hydroxy-N,N,N-trimethylethanaminium 3-carboxy-2-

(carboxymethyl)-2-hydroxypropanoate ........................................................................ 87

4.3.2.4 FTIR:2-Hydroxy-N,N,N-trimethylethanaminium3-carboxy-2,

3dihydroxypropanoate. ................................................................................................. 88

4.3.2.5 FTIR: 2-Hydroxy-N, N, N-trimethylethanaminium chloride......................... 89

4.3.2.6 FTIR: 2-Hydroxy-N, N, N-trimethylethanaminium 4-oxopentanoate ........... 89

4.3.3 Preliminary growth studies: ............................................................................... 90

4.3.3.1 Growth Plate studies....................................................................................... 90

4.3.4 Shaker growth studies ........................................................................................ 96

4.4 Conclusion............................................................................................................... 101

Acknowledgement.............................................................................................................. 102

References .......................................................................................................................... 103

CHAPTER FIVE ................................................................................................................... 105

THE VALORISATION OF SLAG FROM BCL USING BIOCOMPATIBLE CHOLINE

BASED IONIC LIQUIDS AS A SUPPORT ......................................................................... 105

Abstract .............................................................................................................................. 105

5.1 Introduction ............................................................................................................. 106

5.2 Methodology ........................................................................................................... 108

5.2.1 Slag processing ................................................................................................ 108

5.2.2 Particle size distribution................................................................................... 108

5.2.3 Scanning electron microscopy (SEM) ............................................................. 108

5.2.4 Slag prep -Microwave digestion ...................................................................... 109

5.2.5 Inductively coupled plasma optical emission spectroscopy (ICP-OES).......... 110

5.2.6 X-ray fluorescence (XRF) analysis.................................................................. 111

5.2.7 X-ray Powder Diffraction (XRD) analysis ...................................................... 112

5.2.8 Inductively coupled plasma mass spectrometry (ICP-MS) ............................. 112

5.2.9 Standard solution preparation: ......................................................................... 114

5.2.9.1 Internal standard ........................................................................................... 114

XII

5.2.9.2 Calibration standards .................................................................................... 114

5.2.10 Atomic absorption spectroscopy (AAS) .......................................................... 114

5.2.11 Bioleaching experiment of mineral sample (BCL slag) .................................. 115

5.3 Results and Discussion............................................................................................ 116

5.3.1 Particle size distribution................................................................................... 116

5.3.2 Slag morphology .............................................................................................. 116

5.3.3 XRF of BCL slag before and after leaching .................................................... 119

5.3.4 ICP-OES slag elemental analysis..................................................................... 122

5.3.5 XRD analysis of BCL slag before and after leaching ...................................... 123

5.3.6 Bacterial growth studies................................................................................... 127

5.3.7 pH studies......................................................................................................... 132

5.3.8 Bioleaching Studies.......................................................................................... 135

5.4 Conclusion............................................................................................................... 141

Acknowledgement.............................................................................................................. 141

References .......................................................................................................................... 142

CHAPTER SIX ...................................................................................................................... 145

6.0 GENERAL DISCUSSION AND CONCLUSION ................................................. 145

6.1 GENERAL DISCUSSION...................................................................................... 145

6.2 CONCLUSION ....................................................................................................... 147

REFERENCES ...................................................................................................................... 148

APPENDICES ....................................................................................................................... 150

XIII

LIST OF FIGURES

Figure 2-1: Extreme thermophile isolates located in hot springs A) Yellow State National

Park United States B) Hot coal dumps in Witbank South Africa (Sourced from Neale, 2006).

.................................................................................................................................................. 14

Figure 2-2: Bacteria classified according to optimal growth temperatures namely Extreme

thermophiles (left), spherical shaped with a dimension of 1-2µm. Moderate thermophiles

(centre) with comparable phenotypic characteristics to Mesophiles. Mesophiles (right) known

to be rod shaped microorganisms with an estimated dimension of 0.5X2.0µm (Sourced from

Neale, 2006). ............................................................................................................................ 14

Figure 2-3: Summery diagram of a) thiosulfate mechanisms and b) polysulfide mechanism

(Sourced from Mishra et al., 2005).......................................................................................... 18

Figure 2-4: Operating flow for In situ Leaching (Sourced from Thosar et al., 2014) ............. 19

Figure 2-5: Operating flow for Heap Leaching (Sourced from Bauer, n.d.) ........................... 20

Figure 2-6: Operating process for Vat Leaching (Sourced from Thosar et al., 2014)............. 21

Figure 2-7: Annual growth rates of ionic liquid published articles (Sourced from Park et al.,

2014) ........................................................................................................................................ 32

Figure 2-8: The application of ILs (Sourced from Pham et al., 2010) .................................... 32

Figure 2-9: Synthesis procedure of an ionic liquid (Sourced from Park et al, 2014) .............. 34

Figure 2-10: Metabolic activity of choline lactate by Stapylococcus lentus (Sourced from

Sekar et al., 2013). ................................................................................................................... 39

Figure 3-1: Gold 1 mine East Rand mine sites under study NRW Ramp water, W2E19,

N2W2, UK9 and E2 Reef drive south ..................................................................................... 53

Figure 3-2:Streak plates of Springs Gold1 mine bacterial isolates after 24hrs incubation at

37°C to attain pure cultures for further characterization studies: A) Strain 2KL, B) Strain

9KL, C) Strain 12KL, D) Strain 16KL and E)Strain 19KL. ................................................... 55

Figure 3-3: API (Analytical Profile index) Colour change: A) 9KL before incubation at 37 °C

for 24 hrs and B) 9KL after incubation (addition of TDA, James, VP1, VP2 Reagents) C)

19KL before incubation at 37 °C for 24hrs and B) 19KL after incubation (addition of TDA,

James, VP1, VP2 Reagents)..................................................................................................... 57

Figure 3-4: Phylogenetic tree of consensus sequence 2KL ..................................................... 60

Figure 3-5: Phylogenetic tree of consensus sequence 9KL ..................................................... 61

Figure 3-6: Phylogenetic tree of consensus sequence 12KL ................................................... 61


XIV


Figure 4-1: Structure of choline hydroxide.............................................................................. 69

Figure 4-2: Synthesised ionic liquids namely choline levulinate, choline chloride, choline

citrate, choline lactate, choline tartarate and choline dihydrogen phosphate ........................... 71

Figure 4-3: 1H NMR of choline lactate .................................................................................... 74

Figure 4-4: 13C NMR of choline lactate................................................................................... 75

Figure 4-5: 1H NMR of choline dihydrogen phosphate ........................................................... 76

Figure 4-6: 13C NMR of choline dihydrogen phosphate.......................................................... 77

Figure 4-7: 1H NMR of choline citrate .................................................................................... 78

Figure 4-8: 13C NMR of choline citrate ................................................................................... 79

Figure 4-9: 1H NMR of choline tartarate ................................................................................. 80

Figure 4-10: 13C NMR of choline tartarate .............................................................................. 81

Figure 4-11: 1H NMR of choline chloride ............................................................................... 82

Figure 4-12: 13C NMR of choline chloride .............................................................................. 83

Figure 4-13: 1H NMR of choline levulinate ............................................................................ 84

Figure 4-14: 13C NMR of choline levulinate ........................................................................... 85

Figure 4-15: FTIR of choline lactate........................................................................................ 86

Figure 4-16: FTIR of Choline dihydrogen phosphate.............................................................. 87

Figure 4-17: FTIR of choline citrate ........................................................................................ 88

Figure 4-18: FTIR of choline tartarate ..................................................................................... 88

Figure 4-19: FTIR of choline chloride ..................................................................................... 89

Figure 4-20: FTIR of choline levulinate .................................................................................. 90

Figure 4-21: Shaker growth studies of Bacillus aryabhattai in choline based ionic ILs vs.

glucose as an alternative carbon source at a speed of 120 rmp and temperature of 37 °C

(5days)...................................................................................................................................... 96

Figure 4-22: Growth curve of Raoultella ornithinolytica in choline ILs vs. glucose as an

alternative carbon source at speed of 120 rpm and temperature of 37 °C ............................... 97

Figure 4-23: Growth curve of Bacillus sp. in choline based ILs vs. glucose as an alternative

carbon source at speed of 120 rpm and temperature of 37 °C. ................................................ 98

Figure 4-24: Growth curve of Bacillus thuringiensis in choline based ILs vs. glucose as an

alternative carbon source at speed of 120 rpm and temperature of 37 °C. .............................. 99

Figure 4-25: Growth curve of Pseudomonas moraviensis in choline based ionic liquids vs.

glucose as an alternative carbon source at speed of 120 rpm and temperature of 37 °C. ...... 100

XV

Figure 4-26: Growth curve in the absence of bacteria in choline based ionic liquids vs.

glucose as an alternative carbon source at speed of 120 rpm and temperature of 37 °C ....... 101

Figure 5-1: Schematic of BCL slag (Adapted from Malema & Legg, 2006) ........................ 108

Figure 5-2: Particle size distribution of BCL slag before bioleaching process ..................... 116

Figure 5-3: SEM of raw BCL slag (Before bioleaching process) indicative of an amorphous

structure.................................................................................................................................. 116

Figure 5-4: EDS spectrum of BCL slag as directed in Figure 5.3 ......................................... 117

Figure 5-5: Scanning electron photomicrograph of BCL slag after bioleaching in mixed

culture..................................................................................................................................... 118

Figure 5-6: Scanning electron photomicrograph of BCL slag after bioleaching in mixed

culture..................................................................................................................................... 118

Figure 5-7: Raw BCL slag sample before bioleaching process (feed) .................................. 123

Figure 5-8: BCL slag subsequent to bioleaching with Bacillus aryabhattai (2) in choline

based ILs vs. glucose ............................................................................................................. 124

Figure 5-9: BCL slag subsequent to bioleaching with Raoultella ornithinolytica (9) in choline


Figure 5-10: BCL slag subsequent to bioleaching with Bacillus sp. (12) in choline based ILs

vs. glucose .............................................................................................................................. 125

Figure 5-11: BCL slag subsequent to bioleaching with Bacillus thuringiensis (16) in ILs vs.

glucose ................................................................................................................................... 125

Figure 5-12: BCL slag subsequent to bioleaching with Pseudomonas moraviensis (19) in

choline based ILs vs. glucose................................................................................................. 126

Figure 5-13: BCL slag subsequent to bioleaching with a mixed culture (All) of bacteria in


Figure 5-14: BCL slag subsequent to leaching in the absence (0) of bacteria in choline based

ILs vs. glucose........................................................................................................................ 127

Figure 5-15: Growth curve of Bacillus aryabhattai in choline based ILs vs. glucose for the

recovery of base metals at a speed of 120 rmp and temperature of 37 °C (3 weeks) ............ 128

Figure 5-16: Growth curve of Raoultella ornithinolytica in choline based ILs vs. glucose for

the recovery of base metals at a speed of 120 rmp and temperature of 37 ⁰C (3 weeks) ...... 128

Figure 5-17: Growth curve of Bacillus sp. in choline based ILs vs. glucose for the recovery of

base metals at a speed of 120 rmp and temperature of 37 °C (3 weeks) ............................... 129

Figure 5-18: Growth curve of Bacillus thuringiensis in choline based ILs vs. glucose for the


XVI

Figure 5-19: Growth curve of Pseudomonas moraviensis in choline based ILs vs. glucose for

the recovery of base metals at a speed of 120 rmp and temperature of 37 °C (3 weeks) ...... 130

Figure 5-20: Growth curve of a mixed culture of bacteria in choline based ILs vs. glucose for

the recovery of base metals at a speed of 120 rmp and temperature of 37 °C (3 weeks) ...... 130

Figure 5-21: Growth curve in the absence of bacteria in choline based ILs vs. glucose for the


Figure 5-22: The pH profile of BCL slag bioleaching by Bacillus aryabhattai in choline


Figure 5-23: The pH profile of BCL slag bioleaching by Raoultella ornithinolytica in choline


Figure 5-24: The pH profile of BCL slag bioleaching by Bacillus sp. in choline based ILs vs.

glucose ................................................................................................................................... 133

Figure 5-25: The pH profile of BCL slag bioleaching by Bacillus thuringiensis in choline


Figure 5-26: The pH profile of BCL slag bioleaching by Pseudomonas moraviensis in choline


Figure 5-27: The pH profile of BCL slag bioleaching by a mixed culture in choline based ILs

vs. glucose .............................................................................................................................. 134

Figure 5-28: The pH profile of BCL slag bioleaching without bacteria in choline ILs vs.

glucose ................................................................................................................................... 135

Figure 5-29: The recovery rates of base metals from BCL slag using Bacillus aryabhattai in

choline ILs vs. glucose........................................................................................................... 136

Figure 5-30: The recovery rates of base metals from BCL slag using Raoultella

ornithinolytica in choline ILs vs. glucose .............................................................................. 136

Figure 5-31: The recovery rates of base metals from BCL slag using Bacillus sp. in choline


Figure 5-32: The recovery rates of base metals from BCL slag using Bacillus thuringiensis in


Figure 5-33: The recovery rates of base metals from BCL slag using Pseudomonas

moraviensis in choline based ILs vs. glucose ........................................................................ 138

Figure 5-34: The recovery rates of base metals from BCL slag using a mixed culture in


Figure 5-35: The recovery rates of base metals from BCL slag in choline based ILs vs.

glucose (absence of bacteria) ................................................................................................. 139

XVII

LIST OF TABLES

Table 2-1: Historic timeline of bio mining; Nearly 2000 years of documentation to the

identification of Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans)

(Sourced from Watling et al., 2016) .......................................................................................... 6

Table 2-2: Industrial copper bio heap leaching plants (Sourced from Mishra, 2005) ............... 7

Table 2-3: Development of Bioxidation Processes-Commercial plants for pre-treatment of

gold concentrates (Sourced from Mishra, 2005)........................................................................ 8

Table 2-4: Categories of Bioleaching microorganisms based on optimal growth temperatures

(Sourced from Gentina & Acevedo, 2013) .............................................................................. 10

Table 2-5: Microbial inoculation used in a ‘top down’ approach to evaluate mesophilic and

moderately thermophilic mineral leaching Consortia (Sourced from Rawlings & Johnson,

2007) ........................................................................................................................................ 13

Table 2-6: Bio-mining technologies (Sourced from Johnson, 2013)....................................... 21

Table 2-7: Factors affecting the bioleaching kinetics (Sourced from Celik, 2008 & Riekkola-

Vanhanen, 2010) ...................................................................................................................... 22

Table 2-8: Acidithiobacillus pH Profile (Sourced from Hamidian et al., 2009)...................... 23

Table 2-9: pH effects on metal recovery from bauxite ore by T. ferrooxidans (Sourced from

Shaikh et al., 2010) .................................................................................................................. 24

Table 2-10: Medium formulations ........................................................................................... 26

Table 2-11: Formulation sheet of nutrient medium used in bioleaching and bioxidation

research (Sourced from Deveci et al, 2015) ............................................................................ 26

Table 2-12: Medium formulation studies for bioleaching of heavy metals from ores

(Sourced from Mulligan et al., 2004) ...................................................................................... 26

Table 2-13 : Defined growth media for Thiobacillus ferrooxidans (Sourced from Acevedo et

al., 1989) .................................................................................................................................. 27

Table 2-14: Bioleaching potential of microorganism for metal liberation (Sourced from

Thosar et al., 2014) .................................................................................................................. 29

Table 2-15: Bioleaching investigations using an array of minerals......................................... 31

Table 2-16: Unique properties of ionic liquids (Sourced from Park et al., 2014) ................... 33

Table 2-17: Metal studies employing ionic liquids.................................................................. 38

Table 3-1: Characteristics of five bacterial isolates obtained from mine water samples ......... 56

Table 3-2: Biochemical characterisation of bacterial isolates ................................................. 57

Table 3-3: Identification of bacterial sequence........................................................................ 58

Table 4-1: Properties of Choline Hydroxide Solution ............................................................. 69

XVIII

Table 4-2: Ionic Liquids Reagents ........................................................................................... 70

Table 4-3: Nicolet iS10 FT-IR instrument specifications ........................................................ 72

Table 4-4: Growth plate studies of Bacillus aryabhattai (CFU) in Ionic liquids .................... 90

Table 4-5: Plate growth studies of Raoultella ornithinolytica (CFU) in Ionic Liquids ........... 91

Table 4-6: Growth plate studies of Bacillus sp. (CFU) in Ionic Liquids ................................. 92

Table 4-7: Growth plate studies of Bacillus thuringiensis (CFU) in ionic liquids ................ 93

Table 4-8: Plate growth studies of Pseudomonas moraviensis................................................ 94

Table 4-9: Plate growth studies -Control ................................................................................. 95

Table 5-1: SEM Tescan EDX measurement specification..................................................... 109

Table 5-2: Microwave digestion specification....................................................................... 109

Table 5-3: ICP-OES measurement specifications for slag analysis ....................................... 110

Table 5-4: ZXS Primus II measurement specification........................................................... 111

Table 5-5: XRD RigakuUltimalV measurement specification .............................................. 112

Table 5-6:Perkin- Elmer SCIEX-ELAN 6100 ICP-MS measurement specifications ........... 113

Table 5-7: Elements of interest for ICP-MS analysis ............................................................ 113

Table 5-8: Calibration standard preparation specification for ICP-MS ................................. 114

Table 5-9: Formulation of mineral salt medium .................................................................... 115

Table 5-10: XRF of slag before and after leaching with Bacillus aryabhattai in ILs vs.

Glucose................................................................................................................................... 119

Table 5-11: XRF of slag before and after leaching with Raoultella ornithinolytica in ILs vs.

Glucose................................................................................................................................... 120

Table 5-12: XRF of BCL slag after leaching with Bacillus sp. in ILs vs. Glucose ............... 120

Table 5-13: XRF of BCL slag before and after leaching with Bacillus thuringiensis in ILs vs.

Glucose................................................................................................................................... 120

Table 5-14: XRF of BCL slag before and after leaching with Pseudomonas moraviensis in

ILs vs. Glucose....................................................................................................................... 121

Table 5-15: XRF of BCL slag before and after leaching with a mixed culture in ILs vs.

Glucose................................................................................................................................... 121

Table 5-16: XRF of BCL slag before and after leaching with no bacteria in ILs vs. Glucose

................................................................................................................................................ 122

Table 5-17: ICP-OES Characterization of BCL slag ............................................................. 122

XIX

LIST OF ABBREVIATIONS

ABC Adapted bacteria culture

Al Aluminium

API Analytical profile index

AD Anno Domini

As Arsenic

AAS Atomic absorption spectrophotometer

BCL Bangwato Concessions Ltd Mine

BC Before Christ

Bio Biological

Bi Bismuth

Ca Calcium

C Carbon

C Chalcopyrite

Cl Chlorine

CCh Choline chloride

CC Choline citrate

CDP Choline dihydrogen phosphate

CLa Choline lactate

Cle Choline levulinate

CT Choline tartarate

Cr Chromium

Co Cobalt

CFU Colony forming units

Cu Copper

DNA Deoxyribonucleic acid

D2O Deuterated water

DF Dilution factor

EDS Energy Dispersive X-ray Spectroscopy

F Fayalite

FTIR Fourier Transform Infrared Spectroscopy

Ga Gallium

XX

G Glucose

Au Gold

H Hydrogen

OH Hydroxide

IND Indole

ICP-MS Inductively coupled mass spectrometry

ICP-OES Inductively coupled plasma optical emission spectrometry

IL/s Ionic Liquid/s

Fe Iron

Pb Lead

Lu Luthinium

Mg Magnesium

Mn Manganese

CH3 Methyl

Mo Molybdenum

NCBI National centre for biotechnology

Ni Nickel

NC Not countable

NMR Nuclear magnetic resonance

No Number

O Olivine

O Oxygen

PBS Phosphate buffered saline

PCR Polymerase chain reaction

K Potassium

P Pyrite

P Pyrrhotite

Q Quartz

r RNA Ribosomal ribonucleic acid

Rb Rubidium

SEM Scanning electron microscopy

Si Silicone

Ag Silver

XXI

sp. Specie

S Sulphur

TMS Tetramethylsilan

Ti Titanium

NC Too much to count

UBC Unadapted bacteria culture

U Uranium

V Vaesite

V Vanadium

VP Voges-Proskauer

XRD X-ray diffraction

XRF X-ray fluorescence

Zn Zinc

Z Zinc silicate

XXII

LIST OF UNITS

cP Centipoises

Cps Counts per second

cm3/dm3 Cubic centimetre per cubic decimetre

°C Degree

G Gram

g/l Gram per litre

g/ml Gram per millilitre

Hrs Hours

kA Kiloamps

Kg/m3 Kilograms per cubic metre

kV Kilovolts

kW Kilowatt-hour

L Litres

L/min Litre per minute

MHz Megahertz

µA Microamps

µm Micrometre

mA Milliamp

Mg Milligram

Ml Millilitre

Mm Millimetre

mM Millimolar

Ms Millisecond

mS/cm Milli siemen per centimetre

Nm Nanometre

Ns Nanosecond

N Newton

OD Optical density

Ppb Parts per billion

Ppm Parts per million

Pa Pascal

XXIII

% Percentage

pH Potential of hydrogen

N Refractive index

Rpm Revolutions per minute

Scm2/mol Siemens square centimetre per mol

V Volts

V Volume

W Watt

D Wavelength

Wt Weight

w/v weight per volume

XXIV

DISSERTATION OUTLINE

A synopsis of the chapters presented in this dissertation is as follows:

Chapter One: Introduction

This chapter presents the research topic, providing pertinent information on the context of the

study and a brief explanation of the research problem. The chapter ends with the hypothesis,

aim and objectives.

Chapter Two: Literature review

This chapter presents a comprehensive literature review on the focus of this research,

describing bioleaching and its implications. The use of novel microorganism and alternative

media as technologies for the development of bioleaching kinetics is also thoroughly

reviewed.

Chapter Three: Characterization of halophilic bacteria from Gold 1 Mine, East Rand,

Springs Johannesburg, South Africa for microbial mining applications

This chapter describes the microbial diversity of Gold 1 Mine aquifers, East Rand, Springs

Johannesburg, South Africa employing microbial and molecular characterization techniques.

Chapter Four: Synthesis and characterization of biocompatible choline derived ILs for

biotechnology applications

This chapter describes the synthesis of choline derived ILs relative to glucose as a carbon

source and the biocompatibility studies of these ILs.

Chapter Five: The valorisation of slag from BCL using biocompatible choline based

ILs as a support

This chapter describes the bioleaching application of these ILs for base metal recovery from

Bamangwato Concessions Ltd Mine (BCL) slag.

Chapter Six: General discussion and conclusion

This is the concluding chapter of this study and presents the general discussions and

conclusions made from the studies undertaken from Chapters three to Chapter five. The

chapter also presents closing remarks and recommendations for future work.

1

CHAPTER ONE

1.0 GENERAL INTRODUCTION

1.1 Background

Biomining describes the use of micro-organisms in facilitating the recovery of the

metallurgical value from sulphide/iron bearing ores (Çelik, 2008). Biomining includes two

similar processes, namely bioleaching and biooxidation. Bioleaching is a solubilizing process

occurring in natural environments where optimal conditions exist, allowing the development

of ubiquitous bioleaching microbes. Then there is biooxidation, an oxidation process initiated

by microorganisms which maintains the precious metals or the related minerals in the solid

state (Celik, 2008; Schippers et al., 2013). Due to the exhaustion of high grade ores and

stringent antipollution legislations, bioleaching is growing increasingly popular as an

alternative technology to conventional hydrometallurgy methods.

While bioleaching is being researched for its ability to recover precious metals from unique

ore bodies, thus far industrial applications is restricted mainly to lower grade ores. The main

problem facing the industrialization of this process is the result of the slow bioleaching

kinetics affecting the dissolution of metals from sulphide ores (Das et al., 1999). As a result

improvement is required in both technical and biological areas affecting bioleaching

(Bosecher Klaus, 1997; Mulligan et al., 2004). Investigations have demonstrated the selective

solubility strength of metal oxides in Ionic liquids, thus providing a novel technique for

obtaining target metals in ionic liquids advancing recovery and separation processes. Crucial

factors that had to be observed in enhancing the green properties of biological processes were

namely the mineral salt employed to increase microbial populations, which had to be non-

toxic to the atmosphere; and biomass production, which could not be a pollutant.

Considering these factors and the biodegradable nature of choline based ionic liquid

(Vijayaraghavan et al., 2010) one could conclude that choline based Ionic liquids offer a

green alternative to traditional organic solvents and can potentially be used as media in

leaching of low grade ores and refractory oxide ores ( Guo-cai et al.,2010).

2

1.2 Problem statement

Owing to the exhaustion of the earth’s reserves of high grade ores, the recovery of base metal

values from low grade sulphide ores has grown significantly over the last decade. The poor

solubilisation of these ores in traditional leaching solvents, their complex mineralogical

structure reducing the concentrations of accessible metal values and the demand of ores to be

processed, has restricted the use of conventional hydrometallurgy processes (Olubambi,

Ndlovu & Potgieter, 2008). Furthermore are the heavy implications of the toxic, volatile

solvents employed in traditional hydrometallurgy methods that instigate ozone destruction.

Notwithstanding the slow bioleaching kinetics that obstruct further commercialization,

biohydrometallurgy presents an alternative method for processing ores and recovering metal

values, proving to be an eco-friendly technology (Bosecher, 1997; Rawlings et al., 1995). As

a result further research is required in improving bioleaching kinetics, thus increasing the

economic viability of this process.

Ionic liquids (ILs) are regarded as potential alternatives to traditional organic solvents due to

their minimal toxicity and by virtue of being environmentally benign (Guo-cai et al., 2010).

As traditional organic solvents are toxic, volatile and highly flammable resulting in oxidant

smog and ozone destruction, ionic liquids (ILs) are thus becoming increasingly popular as

replacements of conventional solvents in chemical and biological processes (Lee et al., 2005;

Silva et al., 2014).

Despite the awareness of improved leaching rates of “mixed cultures” to “pure cultures” the

effects of ore “mineralogy” which are a key element in the leaching kinetics of Mesophiles,

have not been thoroughly investigated (Olubambi, Ndlovu & Potgieter, 2008). In addition,

minimal quantitative studies have been executed on the solvability of metal salts in the

presence of ILs (Guo-cai et al., 2010). The selective design of non-toxic, biodegradable and

biocompatible ionic liquid that promote microbial growth (Hou et al., 2013); and the

continued selection of microbes that catalyse mineral destruction (Rawlings et al., 2007) have

also been nominal or entirely absent in bioleaching processes. However, due to the

characteristic nature of ILs as “designer solvents” cation and anion combinations can be

manipulated to synthesis unique ionic liquids with characteristics suitable for green leaching

(Docherty et al., 2007). Considering the factors impeding the further commercialization of

bioleaching processes, the choice of an eco-friendly and affordable energy source was a

crucial aspect for developing this technology and ensuring a process that produces optimal

3

profitable yields. Mineralogical studies were also crucial in determining the leaching

response and behavioural pattern of complex ores/slags in choline derived media, due to the

unique effects of microbial-mineral interactions of individual minerals and microorganisms in

different media.

1.3 Hypothesis

It was hypothesized in the study that:

Biocompatible choline derived ionic liquids are a reliable alternative of nutrients for the

optimal growth of bacteria for base metal recovery as they are a cheaper, easily synthesized,

and an environmentally friendly alternative.

1.4 Aim and Objectives of study

1.4.1 Aim of the study

The aim of the study was to uncover the understanding of significant pathways in

microorganisms activated by base metal extraction utilizing choline ILs relative to glucose

as carbon sources for optimizing bioleaching processes. Their synthesis, characterization

and base metal adsorption mechanisms onto such IL systems would be elucidated.

1.4.2 Objectives of the study

To achieve the aim specified in section 1.4, the subsequent objectives were met:

o Isolation and identification (sequencing) of bacterial isolates from Gold 1 Mine East

Rand, Springs Johannesburg

o Synthesis of choline based ILs: (1) Choline lactate, (2) Choline dihydrogen phosphate,

(3) Choline citrate, (4) Choline levulinate , (5) Choline tartarate and (6) Choline

chloride

o Characterization of choline based ILs using NMR and FTIR

o Biocompatibility determination:

Preliminary growth studies of bacteria in the presence of choline based ILs by

means of direct plate counts and spectrophotometry

Finally establishing growth factors and physiochemical parameters pH, temperature,

incubation time, carbon and nitrogen source for optimizing growth conditions

o Characterization of Bamangwato Concessions Ltd (BCL) mine slag using SEM, XRD ,

XRF and ICP-OES

4

o Bioleaching shaker studies in the presence of a) Bacteria and BCL slag in the absence

of choline based ILs and b) Bacteria, BCL slag and choline based ILs, investigating

bioleaching factors and the effects of choline based ILs on optimizing bioleaching

processes etc.

5

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Introduction

The leading drawbacks in bioleaching applications are the slow paced reaction kinetics

affecting the extraction of metals from sulphide minerals. Hence significant improvement is

required in manipulating operating parameters granting the process commercially feasible

and efficient. These challenges thus call for a pragmatic approach towards optimizing

bioleaching processes using green technology

2.2 Historical perspective

The ability of microorganisms to facilitate the dissolution of minerals was not established up

until the late 1900s. As such, the only historical information available on biological leaching

dates back to 100-200BC, a prehistoric Chines practice involving natural copper (Cu)

recovery from ores. The 2nd century also revealed Europe and Asia Minor practicing similar

processes (Mishra et al., 2005). Gaius Plinius Secundus, a roman author (23-19 A.D) was one

of the first to report possible mobilization of metal species due to leaching; from 1494 to

1555 Georgius Agricola a German physician and mineralogist referred to his findings

involving copper leaching from ores and metal rich leachates from mines. Though the

commercialisation of biological mining was initiated in Tharsis Spain ten years before,

including Sweden, Germany and China (Brandl et al., 2008; Mishra et al., 2005), the Rio

Tinto mines in Spain are regarded as the “cradle of bio-hydrometallurgy.” Dating back to the

pre- Roman period the Rio Tinto mines yielded precious metals values such as copper (Cu),

gold (Au) and silver (Ag). In the 1890s attempts were made at the Rio Tinto mines in

establishing biological leaching involving heaps of low grade ores (Mishra et al., 2005).The

run-of-mine low grade copper ores were arranged in waste dumps to heights of 100m and

recovered using iron (III) acidic fluids for economic copper extraction at the Bingham mines

of Kennecott. Heap and in situ mining by means of native microorganisms was later

established.

In 1947 Acidithiobacillus ferrooxidans were established as one of the microbial communities

discovered in acid mine drainages (Brandl et al., 2008). However it was only in 1961 upon

the discovery of Acidithiobacillus ferrooxidans (Thiobacillus ferrooxidans) in the leachate

that bacteria were recognised for their role in solubilizing metals. Parameters contributing to

6

biological leaching such as heap heights, particle dimension, acid pre-treatment, temperature,

water volumes etc. were later reported. The 1880s revealed the oxidation of reduced sulphur

and sulphur minerals leading to the production of sulphuric acids. However it was only in

1992 upon the analysis of the dissolution of zinc from zinc sulphide metal values, that the

redox reaction of metal sulphides was scientifically elucidated. The reaction was also

revealed to be facilitated by natural microbial processes. The 1980s saw the

commercialization of bio-hydrometallurgy processes involving copper recovery from heaps.

Since then multiple heap bioleaching projects have been commissioned (Watling et al.,

2016), as indicated in Table 2.1 to 2.3:

Table 2-1: Historic timeline of bio mining; Nearly 2000 years of documentation to the identification of Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) (Sourced from Watling et al., 2016)

Timeline Records

25–220

A.D.

The Roman Gaius Plinius Secundus (Pliny the Elder, 23–79 A.D.) documented

that chrysocolla is also synthetically formed water gently flowing through the mine over the winter season up until June; there after the water evaporates

throughout the months of June and July

Records in ancient Chinese manuscripts based on rock leaching including the

production of gall (CuSO4) springs / copper (Cu+) extraction was by cementation on iron

The researcher Galen explained in-situ leaching at a mining site around Cyprus

1086 A.D. The Gall (CuSO4):Copper development is defined: Cu+ leaching and extraction by

cementation on iron as a commercialized operation; the Cu+ was employed for coinage

1500’s In-situ Cu+ leaching of specific ores (in situ) and mine sites (Spain, Hungary, Germany) and Cu+ recovery by using cementation on iron. A woodcut image in

Agricola’s De re metallica symbolizes the labour-intensive gathering of Cu+

solutions in woody vats and transferral to evaporation pools for concentrating the solutions

1600’s Copper recovery from mining water samples (Peru) stated by Alvaro Alonso

Barba de Garfias (priest and metallurgist); His patent was approved for Cu+

recovery from mine water

1800’s Upscale Cu+ heap leaching (Spain): Stacks of low grade ores are deserted for 1to

3 years to decompose naturally

1900–1920 Commercialized Cu+ processing using leaching techniques, including Cu+

cementation (Butte, MT, USA); in-place leaching (Cananea, Sonora, Mexico); extraction of Cu+ from below ground mine waters (Bisbee, AZ, USA)

1921– 1940 The oxidizing principal of sulphur by ground microbes defined; Acidithiobacillus ferrooxidans (formerly Thiobacillus) isolated and characterised

The theory relating to the biosphere wherein live organisms formed the

environment (biogeochemistry) developed by Vladimir Vernadskii (1863–1945).

Reports on the biotic manufacturing of organic acids

First report on the bio-oxidation of pyrite and recommendations for the commercialization of bio-hydrometallurgical processing of zinc-sulphide

Large-scale dump leaching of open mine excess rock by recirculating the leaching solution (Bingham Canyon, UT, USA)

7

The role of microbes in rock weathering process is documented

1941–1951 The isolation of Acidithiobacillus ferrooxidans from coal mine acid effluent and

its part in the oxidation of ferrous and reduced in-organic sulphur complexes (RISC) in acid mine effluents (AMD) is explained

1960’s Dump and heap leaching for copper, uranium lean ores – in-situ is leaching.

1965 Iron (Fe) – and sulphur (S) oxidizing archaea

1977 First international biohydrometallurgy conference in Braunschweig, Germany

1980 Lo Aguirre, first heap bioleaching plant

1985 In-situ uranium (U) heap leaching using intermittent flooding and forced aeration

1986 Fairview S.Africa: first commercial refractory gold biooxidation plant (many gold

bioreactors to follow)

1987 Paques anaerobic systems for effluent treatment

1993 Forced aeration on heap bioleach systems

1995 Bioleaching of chalcopyrite concentrate developed and evaluated on commercial

scale

1997 BioNic ® and BioZinc ® for nickel and zinc bioleaching

1999 Cobalt bioreactor plant, Uganda

2000 Commercial scale applications of archaea

2002 Penoles, Mintek, BacTech Chalcopyrite concentrate bioleach pilot plant, Mexico

2002 BHP Billiton / Alliance Copper commercial demonstration plant for copper- enargite concentrate

2002 GEOCOAT Thermophilic Bioleachng of Chalcopyrite Concentrates (Field Trials

2003 Exhibition. BioCopTM, BHP. Billiton(chalcopyrite)

2006 High temperature heap bioleaching, transitional primary / secondary copper ore (Mintek)

Where AD indicates Anno Domini. Information dating between 1960s-2006 was sourced from Van Staden

et al. (2009)

Table 2-2: Industrial copper bio heap leaching plants (Sourced from Mishra, 2005)

Plant and Location Processed quantities

(tonnes/day)

Years’ operating

Lo Aguirre, Chile 16000 1980-1996

Gunpowder’s Mammoth Mine, Australia

In situ- 1.2 million tonnes 1991-Current

Mt.Leyshon,Australia 1370 1992-1997

Cerro Colorado ,Chile 16000 1993-Current

Girilambone, Australia 2000 1993-Current

Ivan-Zar,Chile 1500 1994-Current

Quebrada Blanca,Chile 17300 1994-Current

Andacollo, Chile 10000 1996-Current

Dos Amigos, hile 3000 1996-urrent

Cerro Verde, Peru 15000 1996-Current

Zaldivar, Chile 20000 1998-Current

S&K Copper Project ,Myanmar 15000 1998-Current

8

Table 2-3: Development of Biooxidation Processes-Commercial plants for pre-treatment of gold

concentrates (Sourced from Mishra, 2005)

Plant and Location Processed quantities Technology Years’

operating

Fairview, South Africa Originally 10, Increased to 40

BIOX 1986-Current

Sao Berto, Brazil Originally 150,

Increased

BIOX Eldorado 1990-Current

Harbour Lights, Australia 40 BIOX 1992-1994

Wiluna, Australia Originally 115,

Increased to 158

BIOX 1993-Current

Sansu, Ghana Originally 720, Increased to 960

BIOX 1994-Current

Youanmii, Australia 120 Bac Tech 1994-1998

Tamboraque, Peru 60 BIOX 1990-Current

Beaconsfield, Australia 70 Bac Tech 200-Current

Laizhou, China 100 Bac Tech 2001-Current

2.3 Biomining motivation

According to Brierly (2008), in the past century factors impacting the processing of metal

values globally have namely been: (a) The call for an array of metal values due to

industrialization and urbanization in China, (b) Discovery of ore minerals that are challenging

to mine, (c) Stringent environmental laws, (d) Increasing capital cost resulting from the rising

price of steel, (e) Increasing operation cost resulting from hiking energy prices, (f) A shortage

of skills, (g) Technology challenges. As such, these needs necessitated for innovative

hydrometallurgy and bio-mining applications that could: reduce expenses related to smelting

and refining, including transport costs and pollution fines; recover metals from lower-grade

ores that cannot be processed by conventional smelting methods; and recover metals from

polymetallic minerals which have sophisticated mineralogy and are not easily treated by

metallurgy techniques such as smelting.

2.4 Bioleaching microorganisms

Since ancient times, for over 2000 years the Greeks and Romans recovered copper from mine

streams possibly using biotechnology methods. However, it’s only been 50yrs since it came

to light that microbes play a crucial role in the enrichment of metals occurring in ore

9

precipitates and mine effluents (Bosecher, 1997). Since then bio-mining also known as bio-

hydrometallurgy has become a popular biotechnology process for the treatment of ores in

the mining sector (Schippers et al., 2013).With the global depletion of high grades ore

deposits increasing metal demands, conventional methods such as pyro metallurgy, chemical

processes etc. are becoming unfeasible. Microorganisms are more advantageous due to their

economic feasibility and their green technology properties (Das et al., 1999; Siddiqui et al.,

2009). Tailings from biological mining processes present minimal chemical activity and

sustain nominal biological life due to the degree of biological leaching, resulting in the

mitigation of environmental threats. In contrast, physiochemical methods exposed to the

atmosphere can undergo microbial leaching; releasing toxins into the environment (Celik,

2008). Microorganisms like bacteria and fungi transform metals into their soluble form

functioning as biological catalysts in leaching reactions. In addition microbial leaching can be

implemented in the recovery of valuable metals from industry waste. This process is gaining

increasing popularity in recovering valuable minerals including copper, gold, iron and

uranium (Siddiqui et al., 2009).

The microorganisms generally employed in biological leaching applications are known as

chemolithotrophs (Nakade, 2013). Chemolithotrophs are mainly acidophilic autotrophs that

play a role in the release of precious minerals from sulphide ores. These microbes develop in

inorganic media of low pH. They are able to withstand environments of elevated metal-ion

content. These microbes are mostly CO2 fixing chemolithoautotrophs and play a critical role

in oxidising the reaction of iron (II) to iron (III) and sulphur to sulphuric acid, which are

responsible for controlling the reaction kinetics (Das et al., 1999; Schippers et al., 2013).

Comprehension of the reaction kinetics is dependent on bacterial characterization and optimal

oxidation environments (Das et al., 1999).

The microbes responsible for the oxidation of iron (II) to iron (III) and sulphur to sulphuric

acid are namely Acidithiobacillus (Thiobacillus), Leptospirillium, Acidianus and Sulfolobus.

Acidithiobacillus (Thiobacillus) are a class of gram negative, non-sporing rods growing in

aerobic environments, which are mesophiles with the exception of Acidithiobacilli

(Thiobacilli) categorized as thermophiles which are capable of growing at greater

temperatures. Leptospirillium are gram negative, non-sporing, spiral structured bacteria.

Acidianus bacteria are sphere-shaped having saucer structured lobes. Sulfobacillus are gram

positive, rod structured bacteria having curved or narrowing ends and are capable of growing

at great temperatures (Bosecher, 1997; Das et al., 1999; Schippers et al., 2013). Even though

10

heap and tank bioleaching processes mostly operate at temperatures less than 40 °C, reactions

occurring at elevated temperatures are favoured promising greater bioleaching rates

(Schippers et al., 2013). Based on their optimal temperature ranges as shown in Table 2.4,

acidophilic microbes can be classified into three groups (Celik et al., 2008).

Table 2-4: Categories of Bioleaching microorganisms based on optimal growth temperatures (Sourced

from Gentina & Acevedo, 2013)

Mesophiles Moderate Thermophiles Extreme Thermophiles

Acidithiobacillus albertensis

Acidimicrobium ferrooxidans Acidianus brierleyi

Acidithiobacillus

ferrooxidansT

Acidithiobacillus caldus Metallosphaera sedula

Acidithiobacillus thiooxidans

Sulfobacillus acidophilus Sulfolobus acidocaldarius

Leptospirillum

ferrooxidansT

Sulfobacillus

thermosulfidooxidans

Sulfolobus acidophilus

Leptospirillum ferriphilum Sulfobacillus thermotolerans Sulfolobus metallicus

Thiobacillus prosperus Ferroplasma acidiphilum Sulfolobus thermosulfidooxidans

2.4.1 Mesophiles

Mesophilic bacteria are known to grow optimally at room temperatures, from 28 °C to 37 °C

in biological oxidation reactions occurring at 40 °C and lower (Celik et al., 2008; Das et al.,

1999).The most prevalent and commonly applied mesophilic bacteria in bioleaching

processes are Acidithiobacillus ferrooxidans formerly known as Thiobacillus ferrooxidans,

initially discovered in 1947 by Colmer and Hickle at an acid coal mine drain (Bosecher,

1997; Das et al., 1999). Acidithiobacillus ferrooxidans (Thiobacillus ferrooxidans) strains

from diverse environments predominately display optimal growth rates at pH’s 1.5 to 2.5 and

at temperatures of 28 °C to 37 °C. They are lithotrophic microorganisms that derive energy

for their growth by the oxidation of iron (II) to iron (III) and unique oxoanions of sulphur to

sulphate. Carbon fixation then occurs by means of the Calvin Benson cycle and the nitrogen

(N) demand is supplied by the ammonium composition of the media. Acidithiobacillus

ferrooxidans (Thiobacillus ferrooxidans) strains have proven to be dissimilar in their genetic

homology. Leptospirillium ferrooxidans for instance is only capable of iron (II) oxidation and

Acidithiobacillus thiooxidans formerly known as Thiobacillus thiooxidans though resembling

similar morphology to Acidithiobacillus ferrooxidans (Thiobacillus ferrooxidans) are only

capable of Sulphur oxidation (Das et al., 1999). Rawlings, Tributsch & Hansford (1999)

11

stated that in stirred-tanks the fixed Fe (III) concentrations are predominantly elevated, in

which case Acidithiobacillus ferrooxidans (Thiobacillus ferrooxidans) are regarded of

secondary importance to combinations of Leptospirillium ferrooxidans and Acidithiobacillus

thiooxidans (Thiobacillus thiooxidan ) or Acidithiobacillus caldus .These combinations have

also been noted to be dominant in copper heap leaching occurring at acidic pH ranges of pH

0.7 (Celik et al., 2008).

2.4.2 Moderate thermophiles

Moderate thermophiles are known to grow optimally at temperatures close to 50 °C,

oxidising Fe (II) and sulphur minerals at temperatures 45 °C to 65 °C (Celik et al., 2008; Das

et al., 1999). Compared to the study of mesophiles, minimal research has been conducted on

moderate thermophiles and as a result, the characterization and nomenclature of moderate

thermophiles has yet to be well established. Moderate thermophiles Sulfobacillus

thermosulfidooxidans are vital in bioleaching processes as sulphur and Fe (II) oxidizing

bacteria (Das et al., 1999). Other moderate thermophiles include Sulfobacillus acidophilus,

Acidophilus ferrooxidans, and Thiobacillus caldus (Celik et al., 2008). Moderate

thermophiles function at elevated temperatures exhibiting greater reaction rates in biological

leaching processes than mesophiles (Das et al., 1999). In cases where the temperature

exceeds a maximum of 45 °C, mesophiles are compromised and their protein is denatured

leading to cell death whiles moderate thermophiles thrive. Okibe et al. (2003) noted

Thiobacillus caldus as the most prevalent sulphur oxidizing bacteria in biological reactors

processing arsenopyrite/copper (Celik et al., 2008).

2.4.3 Extreme thermophiles

Extreme thermophiles are found growing optimally at extraordinary temperatures ranging

from 60 °C to 80 °C (Das et al., 1999).They fall under the domain Arcahae rather than

Bacteria. The genus Sulfolobus are among the most popular extreme thermophiles namely

Sulfolobus acidocaldarius, Sulfolobus sofataricus, Sulfolobus brierley, and Sulfolobus

ambioalous. They are capable of growing anaerobically whiles reducing sulphur elements,

growing aerobically whiles oxidizing sulphur and are capable of both Fe (II) and sulphur

oxidation. The application of extreme thermophiles in bioleaching processes is still under

development in contrast to popular processes that involve bacteria growing at optimum

temperatures below or at 50 °C (Celik, 2008; Das et al., 1999). In comparison to mesophiles

and moderate thermophiles, extreme thermophiles exhibit greater biological leaching

12

kinetics. As a result, heat exchangers in bioleaching reactions involving these extremophilic

thermophiles (Figure 2.1) might not be necessary in controlling bioleaching temperatures

(Das et al., 1999).

13

Table 2-5: Microbial inoculation used in a ‘top down’ approach to evaluate mesophilic and moderately thermophilic mineral leaching Consortia (Sourced from

Rawlings & Johnson, 2007)

(i) Mesophilic (30 °C) consortium

Acidithiobacillus ferrooxidansT

At. ferrooxidans-like (strain NO37) Leptospirillum ferrooxidans (strain CF12)

ß-Proteobacterium isolate PSTR ‘Ferrimicrobium acidiphilum’ (proposed strain)

Gram-positive iron-oxidizing isolate SLC66

‘Sulfobacillus montserratensis’ Thiomonas intermedia (strain WJ68)

Acidithiobacillus thiooxidansT

Acidiphilium cryptum-like (strain SJH) ‘Acidocella aromatica’

(ii) Moderately thermophilic (45 °C) consortium Leptospirillum ferriphilum (strain MT6)

Acidimicrobium ferrooxidans (strain TH3) Ferroplasma acidiphilum (strain MT17)

Actinobacterium isolate Y005

Sulfobacillus thermosulfidooxidansT Sulfobacillus acidophilus

Sulfobacillus isolate BRGM2

Autotrophic Fe2+/S-oxidizer, Fe3+-reducer

Autotrophic Fe2+/S-oxidizer, Fe3+-reducer Autotrophic Fe2+ -oxidizer

Autotrophic Fe2+-oxidizer Heterotrophic Fe2+-oxidizer, Fe3+-reducer

Heterotrophic Fe2+-oxidizer

Mixotrophic Fe2+/S-oxidizer, Fe3+-reducer Mixotrophic Fe2+/S

Autotrophic S-oxidizer

Heterotrophic Fe3+-reducer Heterotrophic Fe3+-reducer

Autotrophic Fe2+-oxidizer

Mixotrophic Fe2+-oxidizer, Fe3+-reducer Heterotrophic Fe2+-oxidizer, Fe3+-reducer

Heterotrophic Fe2+-oxidizer, Fe3+-reducer

Mixotrophic Fe2+/S-oxidizer, Fe3+-reducer Mixotrophic Fe2+/S-oxidizer, Fe3+-reducer

(Mixotrophic) Fe2+/S-oxidizer

Temple & Colmer (1951)

Johnson et al. (2001a) Coram & Rawlings (2002)

Hallberg et al. (2006) Johnson et al. (2001b)

Johnson et al. (2001b)

Yahya & Johnson (2002) Battaglia-Brunet et al. (2006)

Waksman & Joffe (1921)

Hallberg & Johnson (2001) Hallberg et al. (1999)

Okibe et al. (2003)

Clark & Norris (1996) Okibe et al. (2003)

Johnson et al. (2003)

Tourova et al. (1994) Norris et al. (1996)

D. B. Johnson and others,

Unpublished

14

Figure 2-1: Extreme thermophile isolates located in hot springs A) Yellow State National Park United

States B) Hot coal dumps in Witbank South Africa (Sourced from Neale, 2006).

Even though the three categories of microorganisms thrive at unique temperatures, they have

some shared characteristics (Figure 2.2). They all attain their energy from ferrous iron and/or

chemically reduced sulphur complexes functioning as electron donors and oxygen sources

functioning as electron acceptors. The microbes are acidophiles, growing in acidic conditions

commonly pH 1.4-1.6 for bioleaching processes. Important dissimilarities are to be noted

among mesophiles and thermophiles. In contrast to mesophiles, thermophiles are extremely

sensitive due to the absence of a cell wall in their structure. As a result shear rate is a

challenge for thermophiles and lower agitation rates are essential with low concentrations of

slurries to insure their survival. Having a short life cycle thermophiles have to be monitored

and not over agitated without a continuous supply of concentrate (Celik et al., 2008).

Figure 2-2: Bacteria classified according to optimal growth temperatures namely Extreme thermophiles

(left), spherical shaped with a dimension of 1-2µm. Moderate thermophiles (centre) with comparable

phenotypic characteristics to Mesophiles. Mesophiles (right) known to be rod shaped microorganisms

with an estimated dimension of 0.5X2.0µm (Sourced from Neale, 2006).

15

Microbial heterotrophs such as bacteria and fungi also form a significant portion of the

bioleaching community. These microorganisms make use of extracellular metabolic products

and cellular lysates from autotrophic microbes as carbon sources thereof removing any

hindering effects of excessive carbon, and encouraging the development and iron-oxidation

of heterotrophs. Additionally heterotrophic microorganism also participate in solubilizing

metals by extracting organic acids known as citrates, gluconates, oxalates and succinates

(Brandl et al., 2008).

2.5 Halophilic bacteria

Biological leaching employing autotrophs though highly economical, can be ineffective for

specific elements (Zn, As). To overcome this shortfall, bioleaching using heterotrophs has

been established using organic matter for instance, yeast extracts or glucose which functions

as a nutrient source (Wang et al., 2015). Aerobic spore forming halophiles are a taxonomy of

gram positive rods that have been recovered from salt environments namely saltern

environments, estuarine-water, salt-lakes, salt foods, sea-ice, and deep sea hydro-thermal

vents. Halophiles are quickly growing popularity, due to their biotechnological prospective as

producers of harmonious solutes and hydrolysing enzymes. It has been indicated from 16s

rRNA gene sequencing that Bacillus species contain six distinctive phylogenetic groups with

numerous alkaliphilic or halophilic bacillus belonging to the rRNA Bacillus group (Lim et

al., 2006). Acidithiobacillus ferrooxidans halophiles isolated from saline-soil were discovered

to recover 72 % of Cu from ores (Nakade, 2013). Halophilic Bacillus species isolated by

Nithya & Pandian (2010) from the oceans depths were able to tolerate high metal volumes of

As.

2.6 Principle of microbial leaching

The principals that govern the mineralytic effect of bacteria / fungi on minerals include

“acidolysis”, “complexolysis” and “redoxolysis”. The mobilization of metals is facilitated by

the microbes through a) the development of organic / inorganic acids (protons), b) oxidation-

reduction reactions and c) elimination of coordination compounds. Sulphuric acids (H2SO4)

are common inorganic acids in leaching reactions and are the product of sulphur oxidising

microbes, Thiobacillus. The metabolic activity of theses bacteria /fungi produces chains of

organic acids causing organic acidolysis, complexolysis and chelating (Brandl, 2008).

16

2.6.1 Metal sulphide dissolution mechanism

Direct vs. Indirect mechanisms of bioleaching for describing microbial metal solubilisation of

sulphide minerals, have been one of the main discussions of past literature. Direct leaching

describes the ability of the microorganism to oxidize metal sulphides by the direct transferal

of electrons from reduced metal sulphides to the microbial cells bound to the minerals surface

(Shippers et al., 2013). Indirect leaching occurs via reduced metals facilitated by ferric (III)

ion which is produced through the microbial oxidation of ferrous (II) ions found in the

minerals. Ferric (III) ion thus functions as oxidizing agents, oxidizing metal sulphides

resulting in its reduction to ferrous (II) iron, which can then undergo microbial oxidation.

Nonetheless, the binding of microbes to the metal surface is not a clear suggestion of the

presence of a direct mechanism (Shippers et al., 2013). More especially, because of the

absence of direct electron transferal by enzymes and the lack of information around the

attraction of metal sulphides to the bound cell, the direct mechanism remains a theory.

Contrary, the bound cells demonstrate an effective “EPS-filled reaction area” supportive of

indirect leaching facilitated by ferric (III). Data has also been presented establishing indirect

leaching with none to support direct enzymatic processes (Brandl, 2008; Shippers et al.,

2013). As a result the labels “ direct-leaching” and “ indirect-leaching” have been replaced

by “contact-leaching” and “non-contact leaching” ,indicating the pertinence of microbial

attachment to the surface of the mineral (Mishra et al., 2005). Nonetheless the “direct-

leaching” and “indirect-leaching” models are still under review. Instead, an alternative model

has been proposed which is independent of the distinction between direct and indirect

leaching mechanisms. The model reviews all former knowledge formulating a comprehensive

leaching mechanism, characteristic of the following (Brandl, 2008; Mishra et al., 2005):

a) Cells have to be attached to the minerals and in physical contact with the surface;

b) Cells form and excrete exopolymers;

c) These exopolymeric cell envelopes contain ferric iron compounds that are complexed to

glucouronic residues. These are regarded as part of primary attack;

d) Thiosulfate is formed as intermediate during the oxidation of sulphur compounds;

e) Sulphur particles accumulate in the periplasmic-space alternatively occurring in the cell-

envelope;

Metal sulphide dissolution occurs through two unique reaction mechanism namely the

thiosulfate pathway and the polysulfate pathway. Dissolution generally couples proton attack

17

and oxidation reactions. The reaction mechanism depends on the mineral species (Brandl,

2008). According to Mishra et al. (2005) observations, this is because different metal

sulphides produce different intermediates in their reactions. As a result metal sulphides are

characterised according to their ability to solubilize in acids. This includes the (Mishra et al.,

2005; Siddiqui et al., 2009):

a) Thiosulfate pathway proposed for acid-insoluble metal sulphides such as pyrite(FeS2),

molybdenite (MoS2) and tungstenite(WS2);

b) Polysulfate mechanisms for acid-soluble metal sulphides spalerite (ZnS), chalcopyrite

(CuFeS2), galena (PbS), arsenopyrite (FeAsS) and hauerite (MnS2).

Microbes in bioleaching reactions attain energy by facilitating the breakdown of minerals into

their principal /basic elements. In the Thiosulfate mechanism, dissolution occurs through the

oxidation of acid-insoluble metal sulphide facilitated by electron extraction by ferric (III)

ions. The reaction outputs include a thiosulfate intermediate and a sulphate end product

(Siddiqui et al., 2009). The name Thiosulfate thus originates from the first liberated sulphur

product (Mishra et al., 2005).

The equation below illustrates the Thiosulfate mechanisms using pyrite (Siddiqui et al., 2009):

FeS2 + 6Fe3+ +3H2O S2O3

2- + 7Fe2+ + 6H+ (1)

S2O32- + 8 Fe3 + 5 H2O 2SO4

2- + 8Fe2+ + 10H+ (2)

In the Polysulfide mechanisms, dissolution occurs through a combination of electron

extraction facilitated by ferric (III) ions and proton attack (Siddiqui et al., 2009). The metal

sulphide chemical bond is broken by the binding of two protons to the sulphide moiety of the

molecule producing hydrogen sulphide (H2S). The sulphide cation then undergoes

spontaneous dimerization forming disulphide (H2S2) which then undergoes additional

oxidation by polysulfide/s producing intermediate product elemental sulphur, hence the name

“polysulfide mechanism” (Mishra et al., 2005). Though elemental sulphur is fairly stable,

sulphur oxidizing microorganisms can be employed to further oxidize elemental sulphur to

sulphate (Siddiqui et al., 2009).

18

The equation below illustrates the polysulfide mechanisms (Siddiqui et al., 2009):

MS + Fe3++ H+ M2+ + 0:5H2Sn + Fe 2+ (n ≥2) (3)

0:5 H 2Sn + Fe3+ 0:125S8+ Fe2+ + H+ (4)

0:125 S8 + 1.5O2+ H2O SO42- + 2H+ (5)

Figure 2-3: Summery diagram of a) thiosulfate mechanisms and b) polysulfide mechanism (Sourced from

Mishra et al., 2005).

2.7 Leaching technique

Biological mining is based on two principal methods know as percolation and agitation. The

percolation process consists of a “lixiviant” liquid medium percolating through a stationery

bed, while the agitation process consists of fine sized particles agitated in a “lixiviant” media.

Commercial bio-mining processes occur at a large scale and thus favour percolation methods.

The primary techniques applied commercially are in situ, dump, heap and vat leaching

(Siddiqui et al., 2009).

2.7.1 In situ leaching

This leaching process is applicable for mineral and metal recovery of ores found below the

earth’s surface. It’s characterised as solution-mining involving the extraction of ground

minerals through leaching and fluid-recovery. A pump action and gravity-flow facilitate the

movement of the solution (Figure 2.4). The extraction process produces a leachate labelled a

“pregnant solution” and the fluid returning to the extraction process is labelled a “barren

19

solution.” This process is dependent on the ore-permeability, which can be enhanced by

“rubblizing” which involves the fragmentation of ores-in-place. In situ leaching is commonly

used for the recovery of U (uranium), Cu (copper) and Ag (gold) (Thosar et al., 2014).

Figure 2-4: Operating flow for In situ Leaching (Sourced from Thosar et al., 2014)

2.7.2 Dump leaching

Dump leaching requires the piling up of uncrushed waste rock (Siddiqui et al., 2009). It is

commonly used for recovering low grade ores. Large rocks are ruptured by blasting, forming

bulky rock fragments for dump leaching. An acidic liquid solution is discharged on the

uppermost layer and filters through the dump creating a favourable environment for microbial

growth. The microbes initiate mineral oxidation for the dissolution of metal values. Dump

leaching is commonly employed for Cu2S (copper sulphide) ores (Thosar et al., 2014).

2.7.3 Heap leaching

Heap leaching differs from dump leaching techniques as the large rock material is reduced to

fragments increasing mineral and lixiviant interactions and piled in rotating drums containing

acidic liquid media. This process functions to condition the ores for the microorganisms. The

treated ores are dispersed on specially designed padding consisting of a porous synthetic

drain system (Figure 2.5) to encourage the drainage of the mineral infused liquid from the

bottom of the ores. Air is also fed into the system for the optimal growth of the

microorganisms. The copious amount of sulphide and iron complemented by an acidic

environment organically encourage the optimal development of microbes, catalysing

metal/copper recovery. Heap leaching is commonly employed for Cu2S (copper sulphide)

leaching and for the pre-treatment of Au (gold) (Brierley, 2008).

20

Both dump and heap leaching techniques are similar in their use of a lixiviant solution on the

ore surface and the extraction of metal values in solution seeping to bottom of the dump/heap

under gravity (Siddiqui et al., 2009)

Figure 2-5: Operating flow for Heap Leaching (Sourced from Bauer, n.d.)

2.7.4 Vat leaching

Vat leaching is operated for mineral extraction from oxide-ores. The technique essentially

involves the retention of ore-slurry and solvent for numerous hours in tanks while applying

continuous agitation (Figure 2.6). It is employed for the cyanidation of ores highly

concentrated with gold and to recover other precious metals from ores (Siddiqui et al., 2009).

21

Figure 2-6: Operating process for Vat Leaching (Sourced from Thosar et al., 2014)

Table 2-6: Biomining technologies (Sourced from Johnson, 2013)

Period Location Technique

Middle ages China, Spain, UK Leaching of copper ores using precipitation ponds

1960s Utah, USA Copper dump leaching implemented

1960s-1980s Canada In situ mining of uranium

1980s-current

Chile Heap leaching of copper

1986-current South Africa Bio-oxidation of refractory gold ores in aerated stirred tanks

1995 Nevada, USA Bio heap leaching of gold ores

1999-current Kasese, Uganda Bioleaching of cobalt ,ferrous pyrite in aerated stirred tanks

20004-2005 Chuquicamata, Chile

Thermophilic bioleaching of chalcopyrite concentrate

2008-current Talvivaara, Finland Bio heap leaching of polymetallic black schist

Regardless of the employment of tank or heap leaching methods as shown in Table 2.6 the

microbes that facilitate biological mining reactions need to be cultivated in inorganic, oxygen

prevalent, low pH conditions. The microbes of supreme importance are thus autotrophs and

even though the energy source among different types of minerals can change, the microbes

grow through the oxidation of reduced sulphur / iron (II) or both. The pH in tank and heap

leaching environments can differ but are commonly high in acidity ranging from pH 1.5- 2.0.

As a result of these extreme conditions in stirred-tank and heap leaching systems, the sum of

microorganisms expected to perform a pivotal part in the biological mining system are

reduced (Rawlings et al., 2007).

22

2.8 Factors affecting bioleaching

The biological leaching of mineral deposits and contaminated soil saturated with metal,

remains an easy and efficient technique for treating sulphide ore minerals and heavy metal

sources, commonly practised for the extraction of copper and uranium minerals. The

efficiency and feasibility of bioleaching operations are mostly dependent on bacteria action,

including the chemistry and mineralogy of the ores. Prior to applications of such

biotechnologies, optimal leaching environments need to be established for individual mineral

types (Simona et al., 2011). Optimal conditions for bioleaching are dependent on a string of

factors, as stated in Table 2.7.

Table 2-7: Factors affecting the bioleaching kinetics (Sourced from Celik, 2008 & Riekkola-Vanhanen,

2010)

Factor Parameters Outcome

Physicochemical parameters

of a bioleaching environment

Temperature

pH

Redox potential Water potential

Oxygen content and

availability Carbon dioxide content

Mass transfer Nutrient availability

Iron(III) concentration

Light Pressure

Surface tension

Presence of inhibitors

-Temperature has an impact on the

leaching rates , the microbial

organization and productivity -Low pH is required to promote rapid

leaching and to maintain Fe3+ irons and

metals in solution -In biological/chemical oxidation

electron accepting chemical species are required

Microbiological parameters

of a bioleaching environment

Microbial diversity

Population density Microbial activities

Spatial distribution of microorganisms

Metal tolerance

Adaptation abilities

-Bacterial consortiums apt to be

resilient and effective than pure cultures

-Increased population densities apt to elevate the rates of leaching

-Increased metal volumes can be of

extreme toxicity to microbes

Properties of the minerals to be leached

Mineral composition Mineral dissemination

Grain size

Surface area Porosity

Hydrophobicity Galvanic interactions

Formation of secondary

minerals

-Supplies electron donors and trace-elements

-Grain size determines mineral

availability and contact areas -Bioleaching is equivalent to the

increase in mineral surface area -Porosity in mineral particle is the

results of the internal area

23

Factor Parameters Outcome

Processing Leaching mode (in situ, heap, dump, or tank

leaching)

Pulp density Stirring rate (in case of

tank leaching

operations) Heap geometry (in case

of heap leaching)

2.8.1 Physicochemical parameters

Temperatures: Optimal temperatures for Acidithiobacillus ferrooxidans (Thiobacillus

ferrooxidans) range from 28-35 °C. At a reduced temperature a reduction in the recovery of

metals is recorded, nevertheless at 4 °C bacteria solubilisation of Cu+, Co, Ni and Zn is still

noted. At high temperature ranges 50 °C to 80 °C thermophiles can be employed for leaching

processes (Simona et al., 2011).

pH and Fe(III) Concentrations: The modification of pH to the right values is essential for

growing leaching microorganisms and is a determining factor for metal solubilisation. The

pH ranges 2.0 to 2.5 offer optimal conditions for microbes to oxidise iron (II) and sulphide.

Microbes that biologically oxidize sulphide mineral deposits at reduced pH levels have been

reported to demonstrate resilience to acid prone environments and heavy metal resistance

(Hamidian et al., 2009; Simona et al., 2011). In pH environments less than 2.0 bacteria

Acidithiobacillus ferrooxidans (Thiobacillus ferrooxidans) can be inhibited, however they

can also adapt to reduced pH when sulphuric acid is added (Simona et al., 2011).

Characteristically, microorganisms are initially grown and acclimatized to specific ore feed in

the lab. It is crucial for biological mining microbes to have the means to develop in low pH

conditions and be tolerable to elevated acidic volumes (Table 2.8), so as to be able to

facilitate iron-cycling and permit reverse electron transporting (Hamidian et al., 2009). In

addition, acclimatization reduces the lag phase, therefore improving the general bioleaching

rate (Das et al., 1999).

Table 2-8: Acidithiobacillus pH Profile (Sourced from Hamidian et al., 2009)

Microbes Optimal pH pH Range

Acidithiobacillus albertensis 3.5 to 4.0 2.0 to 4.5

Acidithiobacillus ferrooxidansT 2.0 to 2.5 1.3 to 4.5

Acidithiobacillus thiooxidans 2.0 to 3.0 0.5 to 5.5

Acidithiobacillus caldus 2.0 to 2.5 1.0 to 3.5

24

Upon the oxidation of iron (II) to iron (III) Acidithiobacillus ferrooxidans (Thiobacillus

ferrooxidans) absorb hydrogen ion (H+) from their surroundings. Hence hydrogen irons are

regarded as vital nutrients for bacterial cultures. On a broad spectrum Acidithiobacillus

ferrooxidans are not able to grow using iron (III) at pH levels above 3. It has been reported

that reduced iron (III) levels enhance the absorption of oxygen by Acidithiobacillus

ferrooxidans (Thiobacillus ferrooxidans). However high concentrations of iron (III) are

known to inhibit Fe (II) oxidation initiated by Acidithiobacillus ferrooxidans (Thiobacillus

ferrooxidans) (Hamidian et al., 2009). In a study by Shaikh et al. (2010), the best bioleaching

rate was accomplished in oxidized environments at reduced pH range (Table 2.9).

Table 2-9: pH effects on metal recovery from bauxite ore by T. ferrooxidans (Sourced from Shaikh et al.,

2010)

Where Wt indicates weight of the ores

Nutrients: Microbes utilised for the recovery of metals from sulphide minerals are namely

chemolithoautotrophs which are bacteria that require inorganic components for maximum

development. The nutrients are generally sourced from the surrounding ecosystem and from

the minerals undergoing leaching processes (Bosecher, 1997; Waites et al., 2001), supplied

by oxidation of iron and/or sulphur containing minerals suspended in an aqueous otherwise

in a heap-irrigation system (Rawlings et al., 2005). Carbon, hydrogen, and oxygen are

obtained from the environment and soil waters. The outstanding vital elements namely

nitrogen, phosphorus, potassium, calcium, magnesium, sulphur, chlorine and metals iron,

zinc, manganese, copper, molybdenum etc. are obtained from ground minerals / inorganic

fertilisers (Bosecher, 1997; Rawlings et al., 2005). The use of these nutrients for the

development of bacteria in dump bioleaching and heap bioleaching process has proven to be

unfeasible (Celik, 2008), hence the need for alternative nutrient sources.

pH Bauxite ore(g) Metal recovered (%) Bio extraction efficiency of

Al and Fe (%) Initial wt Final wt Al Fe

1.0 2 2.789 66.20 80.01 70.49 2.0 2 2.896 81.13 84.82 82.28

3.0 2 2.846 77.59 80.64 78.54

4.0 2 2.856 68.95 75.20 70.89

5.0 2 2.734 63.87 65.92 64.51 6.0 2 2.72 55.67 59.88 56.98

7.0 2 2.679 52.74 54.47 53.28

8.0 2 2.523 40.12 49.39 43.56

25

Various nutrient media (Table 2.10-2.12) have been suggested for bioleaching as derived

from the cited nutrients in variable quantities, such as the much quoted 9K media. High

recovery of zinc, copper and iron metal concentration were reported in 9K media than in

Norris media. The salt concentrations in the additional media formulations were

suggestively lower (Deveci et al., 2015). The main uncertainty surrounding the application

of 9K media is the possible sedimentation of phosphate, potassium and ammonium in the

form of jarosite compounds as a result of their great concentrations in the media (Deveci et

al., 2015).

Metal elements such as calcium and magnesium are found in adequate concentrations from

degrading rocks in acid leachates, whereas nitrogen and potassium can be scarce and need to

be added in the leaching reaction. In a study by Sarcheshmehpour et al. (2009)

characterization of low sulphide ores established that calcium ,magnesium and sulphur

quantities were abundant for supporting microbial growth ,whiles nitrogen, phosphorous and

potassium were lacking. Thus the addition of nitrogen, phosphate and potassium as an

affordable fertiliser revealed that, the addition of potassium has optimal results on the

biological leaching of copper ores whiles nitrate and chloride inhibit bacterial growth. On the

contrary, it was reported by Celik (2008) that the addition of phosphate and ammonium

should be avoided due to the expenses of such chemicals and the danger of the precipitation

of these chemicals in the form of jarosites and ferric phosphates. The addition of phosphate

and ammonium nutrients takes place in stirred-tank bioleaching processes and the occasional

addition of ammonium takes place in heap leaching processes. Potassium addition in tank

bioleaching process acts as hydroxides, sulphates and sometimes phosphates (Celik, 2008).

For optimization of metal recovery Bosecker (1997) and Simonan et al. (2011) reported that

iron and sulphur components can be augmented along with NH4, PO43 and Mg salt. While

Acevedo et al. (1989) reported that Thiobacillus ferrooxidans are able to grow in defined

media (Table 2.13) and obtain energy from oxidizing reduced sulphur, elemental sulphur and

ferrous (Acevedo et al., 1989).

26

Table 2-10: Medium formulations

Basal Salt medium

(Sugio et al., 1984) Concentration Mineral Medium

(Pronk et al.,1992) Concentration

Fe2(SO4)3 0.0 g Fe2(SO4)3 50 mM

(NH4)2SO4 3.0 g (NH4)2SO4 132 mg

KCl 0.1 g KCl 52 mg

K2HPO4 0.5 g K2HPO4 41 mg

MgSO4.7H2O 0.5 g MgSO4.7H2O 490 mg

Ca(NO3)2 0.01 g Ca(NO3)2 -

Distilled water 1000 ml Deionized water 1000 ml

H2SO4 2.5 ml (pH 2.5) H2SO4 (pH 1.9)

Table 2-11: Formulation sheet of nutrient medium used in bioleaching and bioxidation research (Sourced

from Deveci et al., 2015)

Medium (NH4)2SO4

(g/l)

MgSO4.7H2O

(g/l)

KH2PO4

(g/l)

KCl

(g/l)

Ca(NO3)2.H2O

(g/l)

References

9K 3.0 0.5 0.5 0.1 0.01 Silverman et al. (1959)

T&K 0.4 0.4 0.4 - - Tuovinen et al. ( 1973)

ES 0.2 0.4 0.1 0.1 - Norris et al .(1985)

Leathen 0.15 0.5 0.01 0.05 0.05 Leathen et al. (1956)

Norris 0.2 0.2 0.2 - - Gomez et al. (1999)

Table 2-12: Medium formulation studies for bioleaching of heavy metals from ores (Sourced from

Mulligan et al., 2004)

Medium

number

Substrates Preliminary treatment

1 100 g/L sucrose Autoclaving

2 100 mL/L molasses Autoclaving

3 40 g/L potato peels Autoclaving

4 40 g/L potato peels & 1 g/L

sucrose

Autoclaving ,no yeast extract

5 40 g/L potato peels & 1 g/L sucrose

Not autoclaved; no yeast extract

6 40 g/L sawdust Sulphuric acid (pH 2) not autoclaved; no yeast extract

7 40 g/L leaves Sulphuric acid (pH 2) not autoclaved; no yeast extract

27

Medium

number

Substrates Preliminary treatment

8 1 L/L potato chips waste Sulphuric acid (pH 2) not autoclaved; no yeast extract

9 40 g/L potato peels Sulphuric acid (pH 2) not autoclaved; no

yeast extract

10 20 g/L corn kernel & 20 g/L corn husk

Sulphuric acid (pH 2) not autoclaved; no yeast extract

Table 2-13 : Defined growth media for Thiobacillus ferrooxidans (Sourced from Acevedo et al., 1989)

Reagent Concentration kg/m3

FeSO4.7H2O 44.2 44.2 1.00 33.3

(NH4 )2SO4 3.0 3.0 0.15 0.4

KCl 0.1 0.1 0.05 -

K2HPO4 0.5 0.5 0.05 0.04

MgSO4.7H2O 0.5 0.5 0.50 0.4

Ca(NO3)2 0.01 0.01 0.01 -

H2SO4(10N) 1.0cm3/dm3 1.0cm3/dm3 (pH 3.5) (pH 1.5)

2.8.2 Microbiological parameters

Microbial Activity: The activity of Thiobacillus and acidophilic bacteria are calculated

according to iron and sulphur oxidizing rates. The iron and sulphur oxidizing rates differ

according to different strains. The oxidation rates differ based on aspects like, morphological

characteristics, lipopolysaccharide cell envelope chemistry, nutrient metabolic rates,

including DNA nucleobase ratios. High activity microorganisms can be acquired from unique

mining environments and microbial activity can be multiplied through ultraviolet radiation

treatment or genetic engineering processes (Das et al., 1999).

Oxygen and carbon dioxide: Sufficient excess to oxygen is required for optimal growth and

energy for leaching microbes. Lab techniques such as “aeration, stirring and shaking” can be

implemented. In technologies such as dump leaching and heap leaching, adequate aeration

supplies can result in problems. Carbon sources are only required in the form of carbon

dioxide, nonetheless carbon dioxide supply is not required (Simona et al., 2011).

Mineral substrate: The mineralogy of bioleaching substrates is crucial to the process. In the

presence of elevated carbonate composition of ore/gangue minerals, the pH of the

bioleaching media can escalate, inhibiting or completely suppressing microbial growth.

Reduced pH levels for optimal development of leaching microbes can be obtained through

the supply of sulphuric acid, which can also cause the iron to precipitate and impact the

28

costing of the operation. The bioleaching rates are dependent on substrate surface area as

well. A reduction in the particle magnitude implies increased surface area and thus increased

metal yield short of changes in total particle mass. Particle sizes with a range of 42 µm are

considered as optimal. An increase in the particle surface area is achieved by increasing pulp

density. The increasing of pulp density can elevate metal recovery (Simona et al., 2011). It

has been reported that in the mineralogy of low grade sulphide ores, decreasing particles sizes

expressed larger quantities of Pb, Fe, S and Si whiles Zn and Cu volumes reduced (Olubambi

et al., 2008). Olubami et al. (2007) reported significant recoveries acquired at particle sizes in

the range of 75 µm whiles 106 µm particles expressed minimal recovery.

Heavy metals: Bioleaching of metal sulphides is associated with increased metal

concentrations in the leaching solution. Generally bioleaching microbes more importantly

Acidithiobacillus /Thiobacillus (Table 2.14) are tolerant of heavy metals and other

microorganisms can have a tolerance for e.g. 50 g/L Ni, 55 g/L Cu or 112 g/L Zn. Unique

genus of the same species can display dissimilar heavy metal sensitivity. Single bacteria

species can also be adapted to high metal conditions and substrate through gradual increase of

metal/substrate concentration (Simona et al., 2011).

Water: Water makes up 80 % to 90 % of total cell mass and performs a pivotal function in

solubilizing nutrients, required for transport and hydrolysis reaction. Water activity

parameters quantify its accessibility. In nutrients water is associated with other compounds

e.g. salt, protein and is inaccessible for microbes requiring isolated water for growth (Simona

et al., 2011).

29

Table 2-14: Bioleaching potential of microorganism for metal liberation (Sourced from Thosar et al.,

2014)

Microorganism Bio-leaching proficiency Reference

A. ferrooxidans and

A. thiooxidans

Al: 29.3 %

Mo: 64.5 % Ni: 99.8 %

Aung et al.(2004)

Ni-adapted and Mo adapted

fungal strains

Ni: (45–50) %

Mo: (62–66) % Al: (30–33) %

Santhiya et al.(2006)

Al-adapted fungus Ni: 88.2 %,

Mo: 45.1 %, Al: 68.2 %


Ni:Mo:Al adapted fungus Ni: (45–50) %

Mo: 82.3 %

Al: 65.2 %


Chemolithotrophic sulphur

oxidizing bacteria

Ni: 89.5 %

V: 95.8 %

Mo: 21.5 %

Mishra et al.(2007)

A. ferrooxidansT Al: 63 % Co: 96 %

Mo: 84 %

Ni: 99 %

Gholami et al.(2010)

A. thiooxidans

Al: 2.4 %

Co: 83 %

Mo: 95 % Ni: 16 %

Gholami et al.(2010)

Adapted Bacteria Culture (ABC)

Ni: 95 %

V: 95 %

Kim et al.(2010)

Unadapted Bacteria Culture (UBC)

Ni: 85 % V: 85 %

Kim et al.(2010)

P. simplicissimum W: 100 %

Fe: 100 % Mo: 92.7 %

Ni: 66.43 %

Al: 25 %

Amiri et al.(2011)

30

2.9 Recovery of base metals from slag

The recovery of Cu, Ni and additional nonferrous minerals by means of smelting processes

is associated with the accumulation of large volumes of slag. With reference to copper ore, 1 t

of ore gives rise to 22 t of slag (Kalinkin et al., 2012). Slag recovered from nonferrous

smelting processes can often contain significant volumes of metal values such as cobalt,

copper and nickel which can be recovered by pyrometallurgy and hydrometallurgy processes

(Kalinkin et al., 2012; Maweja et al., 2010; Wang et al., 2015). Nonferrous slag or cleaned

nonferrous slag containing low quantities of metal values can also be applied in the

manufacturing of a tools, paving, concrete, tiles, glass and roofing (Yang et al., 2010). Slag is

often designed for optimizing the removal of gangue elements including the oxides produced

by ore smelters. The content of slag is thus determined by raw material (Table 2.15) and the

smelting process employed. The quantities of metal are automatically drawn into the slag in

the course of tapping, resulting in slag material containing base metals (Maweja et al., 2010).

Hydrometallurgy methods for recovering valuable metals have been extensively investigated,

such as the recovery of Zn, Co, Cu from copper ores and slag, with one study focusing on

atmospheric leaching using acid, base and salt as solvents; and high pressure oxidation acidic

leaching. The applications of roasting methods and additional processing for slag have also

been reported. Then again, this approach has proven to be costly and poorly selective .Slag

waste containing base metals (Co, Ni, Cu) has also been reported to pollute the atmosphere

(Kalinkin et al., 2012;Yang et al., 2010). Environmental contamination can result through

numerous systems e.g. the suspension of metals from slag dumps in acid rain water as well as

the air driven corrosion and transport of slag dust (Maweja et al., 2010). Taking into

consideration the environmental and financial implications of contamination, there is a need

for efficient and economic solutions for the recovery of these metal values and disposal of

slag waste material (Yang et al., 2010).

31

Table 2-15: Bioleaching investigations using an array of minerals

Solid material Reference

Fly ash Burgstaller et al. (1993); Bosshard et al. (1996); Brombacher et al. (1998)

Sewage sludge Jenkins et al. (1981); Tyagi et al. (1991); Chartier et al. ( (1997)

Spent batteries Cerruti et al. (1998)

Electronic scrap material

Brandl et al. (2001); Ilyas et al. (2007); Ilyas et al. (2010)

Ores Mousavi et al. (2005); Mousavi et al. (2006a); Mousavi et al. (2006b);

Mousavi et al. (2007); Mousavi et al. (2008); Soleimani, et al. (2009);

Soleimani et al. (2011)

Spent catalysts Mafi et al. (2010); Mafi et al. (2011); Amiri et al. (2011a); Amiri et al.

(2011b); Amiri et al. (2011c); Amiri et al. (2012)

2.10 Ionic liquids as an alternative medium

Reduced enzyme turnover rates have also been observed in organic media sparking an

interest in alternative, environmentally friendly solvents and reaction media (Silva et al.,

2014). Inorganic nutrients are necessary for the development of chemolithotrophs applied

in metal recovery from sulphide mineral deposits. Other bioleaching microbes make use of

critical nutrients such as C, H, O found in the environment and ground water. The

outstanding nutrients N, P, K, Ca, Mg, SO32

, Cl including heavy metal Fe, Zn, Mn, and Cu

etc. are sourced from ground mineral deposits/inorganic fertilizer. Such nutrients are

provided in the lab in the form of a media. The establishment of such media for the

development of bacteria for the bioleaching of heaps and dumps on an industrial scale is

costly. As a result of the cost and accessibility of these nutrients, optional compounds are

required. It’s imperative that individual elements initiate elevated microbial growth and not

hinder microbial activity (Sarcheshmehpour et al., 2009).

2.10.1 Properties of ionic liquids (ILs)

Ionic liquids gained significant popularity in 1914 when Paul Walden produced ethyl

ammonium nitrate (room temperature salts), since then from the year 2000 the quantity of

ionic liquid based articles published has mushroomed (Figure 2.7) more especially between

1998 and 2013.

32

Figure 2-7: Annual growth rates of ionic liquid published articles (Sourced from Park et al., 2014)

The use of ionic liquids has expanded to catalytic reactions, electrolytes for power storage

equipment, biological sensors, and extraction/recovery agents (Figure 2.8); and has gained

increasing popularity as replacements of conventional solvents in chemical and biological

processes (Davris et al., 2014; Lee et al., 2005; Park et al., 2014).

Figure 2-8: The application of ILs (Sourced from Pham et al., 2010)

33

According to Ferlin (2013) ionic liquids (ILs) are a combination of organic cations and

inorganic /organic anions. On the contrary Khupse & Kumar (2010) stated that though the

cation can be organic /inorganic the anion component should be inorganic. They are also

characterized as “room temperature ionic liquids (RTILs)” having a low melting point

below 100 °C (Ferlin et al., 2013; Khupse et al., 2010). The majority of ILs are

characterized as mass organic cations (e.g. imidazoliums, piperidinums, pyridiniums,

pyrrolidiniums, ammoniums, phosphoniums and lately choliniums also called cholines)

replaced with alkyl chains of varying lengths and functionalism (Silva et al., 2014). The

chemistry of ILs including liquefaction point, viscosity and solubility can be modified by

altering the anion or cation of the salt. Thus improving the effectiveness of engineering,

electrochemical and extraction processes (Pernak et al., 2007).

Owing to their capabilities to adapt their physicochemical conditions ILs are regarded as

designer solvents (Table 2.16). Hence hydrophobic/hydrophilic behaviour can be

surveillanced through the modification of the cation chain lengths, including their miscible

nature with liquid by employing unique anions (Davris et al., 2014).

Table 2-16: Unique properties of ionic liquids (Sourced from Park et al., 2014)

Properties Values Melting point

Liquidus range Thermal stability

Viscosity Dielectric constant

Polarity

Ionic conductivity Molar conductivity

Electrochemical window

Vapour pressure

Preferably below 100 °C

Often > 200 °C Usually high

Normally < 100 cP, workable Implied < 30

Moderate

Usually < 10 mS/cm <10 Scm2/mol

Often > 4 V

Usually negligible

2.10.2 Synthesis of ionic liquids (ILs)

To date, efforts to develop ILs as alternate medias have primarily been dedicated to the

design of ionic liquids with novel physical characteristics “first generation ILs”, designed

to be non-volatile or thermally stable; designing ionic liquids with specific chemistry

towards manipulating reactions “second generation ILs”; and the innovation of ionic liquids

with advantageous biological characteristics “third generation ILs” which have grown

popular and the blueprint for the design of task specific ionic liquids ( Samori et al.,2010).

34

Figure 2-9: Synthesis procedure of an ionic liquid (Sourced from Park et al, 2014)

Ionic liquids have been synthesised according to the following formulation (Figure 2.9): a)

Cations synthesised using quaternization reactions and, b) Anion exchange reactions.

Nuclear magnetic resonance is then employed for determining the ionic liquid

characteristics and purity. Karl –Fischer coulometer can be employed for quality,

establishing the presence of water from the purified and desiccated ionic liquids (Khupse et

al., 2010).

2.10.3 Types of ionic liquids (ILs)

Ionic liquids are classified under four categories according to their cation component,

namely alyklammonium-, dialkylimidazolium-, phosphonium and N-alkylpyridinium-

derived ionic liquids. Even though these ionic liquids have effectively been used for their

solvation and catalytic properties, they do pose certain restrictions. Hence the pros and cons

of all the classes will be discussed. Ethanolammonium nitrate was the first ionic liquid

produced in 1888 by Gabriel. Since then, ammonium based ionic liquids have extensively

been employed as electrolytes for electrochemical equipment due to their cathode

electrochemical stability, reduced melting point and reduced viscosity. Common

imidazolium based ionic liquids have been extensively analysed. The choice of the cation

imadozolium-rings is the result of its stable nature in oxidation and reduction reactions,

their reduced viscosity and effortless synthesis. There are numerous investigations

concerning the catalytic properties of imidazolium based ionic liquids for improving

reaction times, yields and chemo selective ability of carbon based reactions (Ghandi, 2014),

bioactivity (Samori et al., 2010).

Pyridinium based ionic liquids are fairly new compared to their imidazolium based

complements and studies on their reactive properties are still in their development stages.

35

Even though they have demonstrated low region-selectivity in palladium catalysed

telomerisation of butadiene and methanol; and proved to have adverse effects on the rates of

certain Diels Alder reactions, the application of pyridinium has been effective in the Friedel

Crafts and Grignard reactions. In their capacity as catalysts, pyridinum based ionic liquids

have proven extraordinary in synthesising pharmacological products namely 1, 4-

dihydropyridine (Ghandi, 2014).

Phosphonium based ionic liquids have more novelty than imidazolium and pyridinium based

ionic liquids; and exhibit greater thermal stability compared to ammonium and imidazolium.

Their extraordinary properties make them appropriate for processes executed at temperatures

above 100 °C. Phosphonium based ionic liquids are commonly employed as catalysts and

solvents in hydroformylation (Ghandi, 2014).

2.10.4 Biodegradable designer solvents

Ionic liquids were initially labelled as “green solvents”, sparking great interest in ILs as

alternatives to volatile solvents in the chemical manufacturing industry (Khupse et al.,

2010; Laus et al., 2005). This was due to their physicochemical properties such as

negligible vapour pressure (minimal risk as air pollutants), high thermal and

electrochemical stability; and high solvation power of an array of compounds. However,

the latest research has revealed that though ILs are alleged to be “green” due to their low

vapour pressure resulting in minimal atmospheric pollution, ILs are incorrectly branded as

green solvents. This is because of their varying solubility in water facilitating the flow of

ILs in aquatic environments. Several research studies have also established that some ionic

liquids used thus far are highly toxic and non-biodegradable (Laus et al., 2005; Lopes et al.,

2013; Ventura et al., 2014). Investigations on ecotoxicology and environmental safety of

ionic liquids have revealed that toxicity of ILs differs across organisms and trophic levels

(Ventura, 2014). The overall toxicology of ILs is dependent on both anion and cation, but

the length of the cation alkyl chain has the greatest impact (Lopes et al., 2013; Samori et

al., 2010).

The ionic liquid anion component has also been known to add to the IL toxicity, though its

role has been disregarded (Hou et al., 2013). Hence the selection of the cation and anion

component that constitute an ionic liquid has a great impact on the physical factors like

viscosity, conductivity and polarity. The generation of non-toxic and biodegradable ILs

makes use of cation and anion biological molecules derived from natural amino acids, lactic

36

acid, fructose and choline (Lopes et al., 2013). As a result, information regarding

developments in ILs toxicity, biodegradation potential and pathways are imperative for

designing green chemistry ILs (Docherty et al., 2007).

The features of designing a biodegradable ionic liquid as developed for biodegradables

surface-active agents were identified as; a) the existence of a probable site for enzyme

hydrolysis such as an ester or amide group, b) the institution of oxygen in the forms of

hydroxyls, aldehydes and carboxyl acids, including c) the existence of un-substituted

straight chain alkyls (particularly ±44 carbon groups) and phenyls, representing potential

target areas for oxgenase enzymatic activity (Gathergood, Garcia & Scammells, 2004).

2.10.5 Biological activity of ionic liquids (ILs)

Reduced enzyme turnover rates observed in organic media have ignited interest in ILs as

alternative media. However, there is not enough evidence to predict enzyme activity and

stability in ILs. However in one study bio-catalysis of microbial lipases did reveal catalytic

activity in ILs (1-alkyl-3lmethylimidazolium and 1-alkyl pyridinium) mixed with anion

tetrafluoroborate, hexafluorophosphate and bisimide. Drawbacks in the study were the

result of contaminations during IL preparations. As a result, purification procedures are

highly suggested (Rantwijk et al., 2003).

Denaturing gradient gel electrophoresis (DGGE) studies have also suggested that specific

microbes are enhanced by ILs utilized as a carbon supplies (Docherty et al., 2007).

Research based on baker’s yeast, Rhodococcus R312 and Escherichia coli (E. coli)

indicated that their activity was maintained in ILs with none/minimal water. Thus

indicating ionic liquids as having lower toxicity than traditional organic solvents to

microbial cell membranes. Various enzymes tolerating traditional organic solvents were

active in ILs. Enzyme performance was reported to be similar if not superior in ILs than in

traditional organic solvents (Rantwijk et al., 2003).

In another study Martins et al. (2013) attempted to comprehend the effect of ionic liquid

stress on the biochemistry and cells of fungi. The investigation revealed the growth of

Ascomycota in media containing elevated volumes of IL, leading to altered metabolism.

Mycelial proteins were monitored in Apergillusnidulans and Neurosporacrassain in ILs

cholinium chloride. Information obtained indicated the alteration of stress response proteins

and biological pathways as a result of the ionic liquids (ILs). Hulle cells responsible for the

production of osmolytes were identified, which have a critical part in stress response. The

37

ILs analysed produced high levels of proteins responsible for the biological synthesis of

novel metabolic compounds (Martins et al., 2013).

2.11 Metal recovery potential of ionic liquids (ILs)

Pilot research has revealed the possibility of ionic liquids as solvents and electrolytes for

recovering metals. Ionic liquids have shown to be promising solvents in extracting gold and

silver from mineral matrices, recovering uranium and plutonium from used nuclear power,

metal electrodeposition and electroextraction specifically lithium, aluminium, magnesium,

titanium etc. Investigations have revealed that ionic liquids can reserve 30% to 50% energy

use in contrast to conventional methods, receiving great support in industrial technology

(Guo-cai et al., 2010).

Investigations have demonstrated the discriminatory solubility potential of metal oxides in

ionic liquids, thus providing a novel technique for obtaining target metals in ionic liquids

advancing recovery and separation processes (Table 2.17). Ionic liquids could potentially be

used in the green leaching of low grade ores and refractory oxide ores. The 2nd generational

ILs consisting of 1-ethyl and/or butly-3methylimidazolium cation and distinct inorganic

anion components are gaining importance for their use in metal leaching (Davris et al., 2014).

In conventional methods, metallic cations remain in the liquid phase of the mixture. Hence

for metal removal from the liquid state into the hydrophilic ionic liquid, chelating or ligand

agents are required to produce a complex that will increase the metals hydrophobic nature.

The thermodynamics of the solubility power of the crown ether complex in ionic liquids is

more favoured in ILs compared to traditional solvents, which is one of the main advantages

of employing ionic liquids for mineral extraction (Zhao et al., 2005).

Factors affecting optimum recovery proficiency (Revised from Zhao et al., 2005):

a) Manipulating the ionic liquid arrangement in order to alter the hydrophobic properties has

the potential to advance the partition coefficient of the metal/s

b) The forms of extractant e.g. crown-ether can be adapted to accomplish optimum selection

for particular applications

c) The recovery effectiveness is also dependent on pH conditions

38

Table 2-17: Metal studies employing ionic liquids

Ionic liquid Metal studied Reference

Imidazolium chloroaluminate melt UO3 and V2O5 Davris et al., 2014

methyltrioctylammonium chloride, [MTOA+][Cl-],

Zn2+, Cd2+ , Fe3 and Cu2+,

Hernandez et al., 2010

1-methyl-3-octylimidazolium tetrafluoroborate [omim+][BF4-],

Zn2+, Cd2+ Hernandez et al., 2010

2.12 Choline/cholinium derived ionic liquids (ILs)

Synthesis of choline ILs has been a common theme in several investigations. Choline

chloride (2-hydroxyethyl trimethyl ammonium chloride also known as vitamin B4, an

inexpensive organic nutrient salt commonly used as a supplement in animal food) melts at

elevated temperature from 298 °C-204 °C (Lopes et al., 2013). In addition, choline chloride

derived ILs have been reported to be easily synthesised, demonstrative of high water/air

stability and affordable for up scaling (Haerens et al., 2009).

Eutectic solvents synthesized from choline acetate/choline chloride glycerol have been

reported as excellent biodegradables complemented by industrially immobilized Candida

antartic lipase B (Lopes et al., 2013). Choline is imperative for effective cell function and

has systems in place that regulate biosynthesis and hydrolysis reactions. Choline hydroxide

has reportedly been used as a catalyst in aldol condensation reactions. Eutectic

combinations of choline chloride as well as zinc chloride have been utilized as substitutes

for alkyl imidazolium and aluminium chloride Ionic Liquids facilitating O-acetylation on

monosaccharide’s and cellulose (Pernak et al., 2007).

More recently bioenergetics studies based on choline lactate nutrient source for the

development of Staphylococcus lentus have been reported to increase bacterial growth rates

in comparison to traditional carbon nutrients. Furthermore, the metabolic pathway of lactate

was directed by the consumption of this IL used as an energy source by the developing

bacteria. Heat production as a result of lactate utilization and oxygen compared to that of

glucose clearly indicated the preference of choline lactate by Stapylococcus lentus over

glucose (Sekar et al., 2013).

39

Figure 2-10: Metabolic activity of choline lactate by Stapylococcus lentus (Sourced from Sekar et al.,

2013).

2.13 Concluding remarks

Ionic liquids have proven to be the focal points in numerous research areas, mainly organic

chemistry, electrochemistry, nanotechnology, biotechnology, and process engineering and

separation applications. Specific applications in the extraction and the retrieval of unique

products from fermentation media or pharmaceutical remains have also been researched,

with upcoming research on the benefits of ionic liquids as “greener” alternatives. Studies on

the utilization of organometallic complexes in hydrogenation reactions so as to produce

valuable compounds and intermediates have also been investigated. Considering the

novelty of this media, the understanding of IL effects on cells and their biochemistry can

thus be useful towards the application of these products in the manipulation of proteins and

metabolic processes of importance in biotechnology (Martins et al., 2013). Ionic liquid thus

offer a novel media with catalytic properties that could potentially improve the bioleaching

kinetics, making it an attractive alternative technology to conventional hydrometallurgy

methods.

40

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CHAPTER THREE

CHARACTERIZATION OF HALOPHILIC BACTERIA FROM GOLD1 MINE

EAST RAND, SPRINGS JOHANNESBURG, SOUTH AFRICA FOR

MICROBIAL MINING APPLICATIONS

Letlhabile Moyaha1, Sudharshan sekar1, Rufaro Archibald Bhero1, Sibusiso

Sidu2, Antoine F. Mulaba-Bafubiandi3, Vuyo Mavumengwana1

1Department of Biotechnology and Food Technology, University of Johannesburg,

Johannesburg, South Africa – 2094

2Gold One International Limited, Springs, South Africa

3Mineral Processing and Technology Research Center, Department of Metallurgy, School of

Mining, Metallurgy and Chemical Engineering University of Johannesburg, South Africa,

Johannesburg, P.O. Box 17011, Doornfontein 2028, Gauteng, South Africa

*Corresponding author:[email protected]; Tel: +27115596915

Abstract

The South African gold mines represent extreme environments harbouring an ecosystem of

diverse microorganisms. The unique gold mine conditions attributed to extreme pH, pressure

temperature and/or salinity and elevated concentrations of heavy metals have created an

ecological niche for potential novel gene sequences that could be of biotechnological

importance. The microbial diversity of Gold 1 Mine (aquifer), East Rand, Springs

Johannesburg, South Africa was studied to investigate potential microorganisms that could be

applicable in the optimization of bioleaching processes. Sampling was conducted at five

mines sites namely: NRW Ramp, W2E19, N2W2, UK9 Raw and E2 Reef drive south. A sum

of six halophilic bacteria were isolated and characterized according to colony pigmentation,

form, margin, elevation, opacity, texture, gram nature and shape. The majority of bacterial

strains (60 %) were established to be gram positive bacillus and the remaining (30 %) were

gram negative bacillus. The bacteria were subsequently characterized by employing

biochemical tests, revealing their biological diversity. Due to the limitations of conventional

phenotypic characterization in identifying bacterial isolates, 16S rRNA was employed using

the conserved region in species of the same genus and thus used for identifying bacteria at

species level. The identification of the bacterial isolates at species level was conducted by

employing 16S rRNA sequencing and the GenBank database. The 16S rRNA gene fragments

of the bacterial isolates were amplified by PCR. The 27F and1492 universal primers were

52

subsequently employed to amplify the 16S target region. The nucleotide BLAST was then

conducted and the phylogenetic tree constructed using the determined sequences. The

molecular studies identified the two bacterial strains to be, Raoultella ornithinolytica and

Pseudomonas moraviensis. The remaining three strains 2KL, 12KL and 16KL were

discovered to belong to the Bacillus genus. The phylogenetic tree confirmed 12KL and 16KL

to be closely related and members of the Bacillus cereus group. According to research strain

2KL identified as Bacillus aryabhattai are characterised as salt, metal and UV radiation

tolerant bacteria suggesting their potential in metal dissolution. Raoultella sp. were found to

be characterized as ecological bacteria commonly identified in soil and water samples but

with no accounts of Raoultella ornithinolytica isolated from mine water samples. With the

exception of Bacillus sp. to one’s knowledge there have been no accounts of the application

of bacterial isolates Raoultella ornithinolytica and Pseudomonas moraviensis in bioleaching

process.

Keywords: Optimization, bioleaching processes, bacteria, characterized, halophilic bacteria, 16S rRNA, phylogenetic tree

3.1 Introduction

In the mid-1940s bacterial isolates Acidithiobacillus ferrooxidans (formerly Thiobacillus

ferrooxidans) were unearthed and by 1958 the Kennecott mine company had licenced the

usage of Acidithiobacillus ferrooxidans for the purpose of copper (Cu+) recovery and bio-

hydrometallurgy processes from run-of-mines /low grade copper ores located in the Bingham

Canyon Mines, USA (Córdoba et al., 2008). Since then microbial leaching of ore minerals

has grown significantly. Unlike conventional hydrometallurgy and pyrometallurgy practices,

biological leaching presents more advantageous properties for treating low grade ores,

including the extraction of minerals from acid leachate waste and flotation sites (Córdoba et

al., 2008). Microorganisms like bacteria and fungi perform a principal role in solubilizing,

transporting and depositing minerals in nature, functioning as biological catalysts of leaching

reactions (Brandl et al., 2008; Brierley et al., 2008).

Due to the depletion of high-grade mineral reserves, power price hikes and the negative

environmental impact of traditional technologies, biotechnologies offer an economic

advantage for current mine, mineral and waste water treatment projects (Baba et al., 2011).

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Drawbacks in the conventional ecological study of these microorganisms have been the result

of challenges associated with bacterial isolation of non-cultivable microorganisms,

identification and the enumeration of individual strains in environments concentrated with

microbes competing for the same metabolites. Literature studies assessing these

environments have also been uncommon and are perpetually conditioned by restrictions

associated with the culturing of microbes e.g. slow growing microorganisms. Cases have also

been identified with microorganisms displaying biochemical characteristics contradictory to

any pattern or recognised genus/species (Goebel et al., 1994; Pailan et al., 2015). Since then

the implementation of 16S rRNA sequencing has transformed ecological and phylogenetic

studies of microorganisms (Goebel et al., 1994; Takai et al., 2001). An improved

comprehension of these methods has furthered the characterization of microbial leaching of

minerals and advanced biotechnologies for metal recovery (Baba et al., 2011).

However there is a necessity for improved insight into the mine ecology and bacterial

evolution. Prior to future developments and implementation strategies regarding bioleaching

approaches, the responsible microbes need to be identified and their interactions in these

environments, establishing the conditions that activate the development of these microbes in

extreme acid mine drainage systems. Increasing the quantity of responsible microbes will

potentially provide additional knowledge on the geobiology of these areas, thus providing a

scientific basis for concrete and operative remedial action (Ramesh et al., 2014).

3.2 Methodology

3.2.1 Study area of interest for sampling

Figure 3-1: Gold 1 mine East Rand mine sites under study NRW Ramp water, W2E19, N2W2, UK9 and

E2 Reef drive south

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Water samples were aseptically collected from Gold 1 Mine East Rand, Springs

Johannesburg, South Africa, (26°15’17’’28°26’34’’E) at five points in the gold mine process

namely NRW Ramp water,W2E19,N2W2,UK9 and E2 Reef drive south. The pH, DO and

conductivity of the samples were examined at the respective sampling sites.

3.2.2 Collection of samples

Water samples were aseptically collected in triplicates from the 5 mine sites in sterile blue

cap bottles and marked accordingly. The samples were then stored at 4 ºC and transported to

the University of Johannesburg Biotechnology and Food technology laboratory for further

analysis.

3.2.3 Isolation and enrichment of moderately halophilic bacteria

Bacteria were isolated from mine water samples by employing a membrane filtration

technique. Membranes with a pore size of 0.22 µm were employed and enriched with

Himedia nutrient broth. One millilitre of bacterial culture from individual enrichment broth

flasks was inoculated into 9 ml of sterile phosphate buffered saline (PBS) and subsequently

spread onto moderate halophilic agar media (15 % of w/v). Once successful bacterial growth

was established the cultures were distinguished according to morphology characteristics and

sub cultured to attain pure colonies. The pure cultures were then preserved in 70 % glycerol

stock at -80 ºC

3.2.4 Phenotypic classification of bacteria: Microbiological identification of bacteria

Colony and cell morphology studies were conducted utilising phenotypic data of known

organisms described in Bergy’s Manual of Determinative Bacteriology (Bergey, 1934). The

macroscopic characterization of bacterial colonies was conducted by observing the nature of

the colony. Microscopic characterization of bacterial isolates was then conducted by Gram

staining to determine the cell wall morphology and observed under a high power Olympus

CX21 light microscope.

3.2.5 Biochemical characterization of bacteria API 20E® strips

Biochemical profiles for isolates were generated using API 20E® strips (bioMérieux Inc.,

Durham, NC). API 20E® strips included enzymatic tests for fermentation or oxidation of

glucose, mannitol, inositol, sorbitol, rhamnose, saccharose, melibiose, amygdalin, and

arabinose, along with nitrate reduction to nitrite and nitrate reduction to nitrogen gas. API

20E® strips also tested for the presence of b-galactosidase, arginine dihydrolase, lysine

55

decarboxylase, ornithine decarboxylase, citrate utilization, H2S production, urease,

tryptophan deaminase, indole production, acetoin production (Voges - Proskauer), and

gelatinase. API 20E® tests were performed according to the manufacturer’s instructions. The

number and types of positive tests were then tabulated for the isolates and used to construct

biochemical phenotype profiles of the cultures which were compared amongst the isolates.

3.2.6 Genotypic classification of bacteria: 16S rRNA sequencing

The ZR Fungal/ Bacterial DNA KITS TM were used to extract DNA from the pure bacterial

cultures. Universal primers 27F: AGAGTTTGATCMTGGCTCAG and 1492R:

CGGTTACCTTGTTACGACTT were used to amplify the 16S target region. The PCR

products were gel extracted, purified and sequenced using the ABI PRISM TM 3500xl Genetic

Analyser. The CLC Main Workbench 7 was then employed to analyse the sequences

followed by a BLAST search assisted by NCBI.

3.2.7 Phylogenetic tree

Using the 16S rRNA gene sequence data the MEGA-6 tool was employed to determine the

evolutionary relationship of the bacterial strains, which was used to construct a phylogenetic

tree.

3.3 Results and Discussion

3.3.1 Microbiological characterization of bacterial isolates

Figure 3-2:Streak plates of Springs Gold1 mine bacterial isolates after 24hrs incubation at 37°C to attain

pure cultures for further characterization studies: A) Strain 2KL, B) Strain 9KL, C) Strain 12KL, D)

Strain 16KL and E)Strain 19KL.

56

Table 3-1: Characteristics of five bacterial isolates obtained from mine water samples

Strain

code

Colony Characteristics

Cell

Characteristics

Colony

pigmentation

Nature of colony

Form Margin Elevation Opacity/Texture

Gram

Nature

+ve/-ve

Shape

2KL Cream white

to yellowish

Circular Entire Raised Translucent +ve Bacilli

9KL Cream Irregular Undulate Flat Translucent halo

-ve Bacilli

12KL White Irregular Undulate Flat Opaque +ve Bacilli

16KL White Irregular Undulate Flat Opaque +ve Bacilli

19KL Yellow Circular Entire Raised Translucent

mucoid

-ve Bacilli

Colony morphology of the bacterial isolates was conducted after 24 hr. incubation at a

temperature range of 37 °C (Table 3.1). The studied characteristics included pigmentation,

form, margin, elevation, opacity, texture etc. It was observed that bacteria strains displaying

an irregular form (9KL, 12KL and 16 KL) were undulate and flat in their morphology.

Similarly circular bacteria (2KL and 19KL) displayed a common pattern of entire and raised

morphology. While most of the bacteria displayed a cream or white pigmentation, strain 2KL

displayed a cream to yellowish colour and 19KL expressed yellow pigmentation. The opacity

of the bacteria was predominantly opaque or translucent with one strain, 9KL expressing a

translucent halo and 19KL expressing a mucoid texture. The cell morphology of the bacterial

isolates was characterized using gram staining. Upon gram staining, retention of crystal violet

resulting in a purple stain would be indicative of a thick peptidoglycan wall identified as

gram positive bacteria. Retention of counterstain safranin resulting in a red/pink stain would

be indicative of a thin peptidoglycan wall identified as gram negative bacteria. Of the

bacterial strains 60 % were gram positive bacillus and 30 % gram negative bacillus. All the

bacteria strains grew and thrived at a temperature range of 30-37 °C, though strain 19KL

demonstrated varying growth rates.

57

3.3.2 Biochemical characterization of bacterial isolates

Table 3-2: Biochemical characterisation of bacterial isolates

Figure 3-3: API (Analytical Profile index) Colour change: A) 9KL before incubation at 37 °C for 24 hrs

and B) 9KL after incubation (addition of TDA, James, VP1, VP2 Reagents) C) 19KL before incubation at

37 °C for 24hrs and B) 19KL after incubation (addition of TDA, James, VP1, VP2 Reagents)

The gram negative bacteria exhibiting non fastidious growth were suspected to be

Enterobacteriacea strains and thus further studied using the API 20E identification system

Biochemical Tests Strain code

Test Substrate 9KL 19KL

Gram +ve/-ve Gram reaction -ve Rod

-ve Rod

ONPG Ortho-nitro-phenyl-β-D-

galactopyranoside isopropylthiogalacto-pyranosides

+ -

ADH Arginine - -

LDC Lysine + -

ODC Ornithine - -

CIT Sodium citrate - +

H2S Sodium thiosulfate - -

URE Urea - -

TDA Tryptophan - -

IND Tryptophan - -

VP Creatine sodium pyruvate + -

GEL Kohns gelatin - -

Glucose Glucose + -

MAN Mannitol + -

INO Inositol + -

SOR Sorbitol + -

RHA Rhamnose + -

SAC Sucrose + -

MEL Melibiose + -

AMY Amygdalin + -

ARA Arabinose + -

58

(Table 3.2). Subsequent to the incubation of the API 20E strips at 37 °C for 24 hrs and the

addition of reagents TDA to TDA test, James reagent to IND test and VP1/VP2 reagent to the

VP test, the colour changes (Figure 3.3) (indicative of a reaction taking place) were read and

interpenetrated according to the reading table. The API 20E tests suggested strain 9KL to be

members of bacteria family Enterobacteriacea and 19KL not to be Enterobacteriace. The API

20E test indicated that unknown strain19KL has the ability to utilise citrate as its sole source

of carbon.

3.3.3 Genotypic characterization of bacterial isolates

Table 3-3: Identification of bacterial sequence

Sample Band I.D GenBank

no.

Blast forecast

of closest

relative

%

Similarity

Phylogenetic

affiliation

2KL KYX3YDEC01R KM675970.1 Bacillus aryabhattai

99 Firmicutes

9KL KYZ4WZAD01R KP192770.1 Raoultella

ornithinolytica

99 Proteobacteria

12KL MPMUGDGP014 HM566903.1 Bacillus sp. 99 Firmicutes

16KL KYZ4WZAD01R KP192770.1 Bacillus thuringiensis

99 Firmicutes

19KL KYZ4WZAD01R KP192770.1 Pseudomonas moraviensis

99 Proteobacteria

The NCBI BLAST of strain 2KL revealed a 16S rRNA gene sequence similarity of 99 % to

Bacillus aryabhattai 16S ribosomal RNA gene sequence, which were reported to be

culturable bacteria isolated from tobacco rhizospheric soil. They are scientifically classified

under the Domain: Bacteria; Pylum: Firmicutes; Class: Bacilli; Order: Bacillales; Family:

Bacillaceae; Genus: Bacillus and species name B. aryabhattai (You & Zhang, 2014). Bacillus

aryabhattai (named after a distinguished Indian astrophysicist of the 5th century AD)were

initially isolated from stratosphere air samples (Shivaji et al., 2009) and successively

identified in diverse environments including desolate lands, the rhizospheres of halophytic

plants and in South China ocean water samples (Lee et al., 2012; Ray et al., 2012). They are

characterised as promoters in plant development and as salt, metal and UV radiation tolerant

bacteria (Pailan et al., 2015), which explains the presence of these microorganisms in

extreme mine water conditions.

Thus far there have been accounts of B. aryabhattai strains in mine water samples. Bacillus

colonies were reported to be entire, circular and plane (Shivaji et al., 2009). The Bacillus

aryabhattai mine bacteria colonies in this study proved to be raised, expressing a rather

59

cream to yellowish pigmentation which could be attributed to the production of intermediate

products between primary and secondary metabolites; or delayed secondary products

resulting from primary metabolism. Contrary to these finding Ray et al. (2012) reported

colonies to be brown later producing a peach pigmentation attributing this loss of

pigmentation to continual sub-culturing on nutrient-agar.


Raoultella ornithinolytica ribosomal RNA gene sequence. Raoultella species (named after

Didier Raou a French bacteriologist) are interrelated with Klebsiella. They are described as

gram-negative, capsule forming, non-motile bacillus scientifically classified under Domain:

Bacteria; Phylum: Proteobacteria; Class: Gammaproteobacteria; Order: Enterobacteriales;

Family: Enterobacteriaceae; Genus Raoultella; Species: R. ornithinolytica (Aziz et al., 2014;

Bhatt et al., 2015). The NCBI blast confirmed the API 20E test results of strain 9KL.

Raoultella species were initially reported to be ecological bacteria commonly identified in

soil and water samples Bhatt et al. (2015), which explain their presence in mine water

samples. However, though uncommon causes of human disease R. ornithinolytica, R.

planticola and R. terrigena have been associated with soft-tissue and blood-stream infection.

As a result, research on Raoultella ornithinolytica has predominantly been focused on the

isolation and characterisation of theses bacteria from clinical specimens. Of recent, genus

Raoultella species were distinguished from Klebsiella using molecular techniques. Pending

the 1990s Raoultella strains were incorrectly classified under genus Klebsiella (Bhatt et al.,

2015). This was due to the discovery that biochemical analysis commonly utilised in medical

laboratories were incapable of differentiating between Raoultella spp. and Klebsiella spp., as

a result there has been insufficient literature on the phenotypic classification of Raoultella

species (Park et al., 2011). As far as ones research, this is one of the first accounts describing

the colony morphology of Raoultella species from gold mine water samples, specifically

Raoultella ornithinolytica which were found to be cream coloured, irregular shaped, flat

colonies expressing a translucent halo.


Bacillus sp. 16S ribosomal RNA gene sequence. Bacillus species are gram positive rod

shaped bacteria and members of Phylum: Firmicutes; Class: Bacilli; Order: Bacillales;

Family: Bacillaceae and Genus: Bacillus (Yu et al., 2011). Bacillus isolates have been

reported from soil samples from the Elmadau savannahs (Aslim et al., 2000).The species

60

have also been identified in polluted acid drainage water and soil, displaying potential as a

bio-sorbent for bioremediation metal removal (Zondo et al., n.d.). Thus explaining the

presence of Bacillus sp. in mine water samples and also eluding to its potential metal

tolerance and applications in metal recovery processes.


Bacillus thuringiensis 16S ribosomal RNA gene sequence. They are scientifically classified

under the Phylum: Firmicutes, Class: Bacilli, Order: Bacillales, Family: Bacillaceae, Genus:

Bacillus, Species: B. thuringiensis (Liu et al., 2014). Bacillus thuringiensis are described as

aerobic, gram positive, soil bacteria and are reported to synthesize parasporal toxic proteins

in the course of sporulation, which are employed as bioinsecticides (Hofte et al., 1989). A

study was conducted on the bio-sorption of copper and manganese from Sarcheshme copper-

mine waste water employing locally isolated bacterium B. thuringiensis. The bacterium was

reported to be of high resistance and biocompatibility to toxic metals .Thus making it a model

microorganism for bioleaching applications (Marandi et al., 2011).


Pseudomonas moraviensis 16S ribosomal RNA gene sequence. They are scientifically

classified under Domain: Bacteria, Phylum: Proteobacteria, Class: Gammma Proteobacteria,

Order: Pseudomonadales, Family: Pseudomonadaceae, Genus: Pseudomonas, Species: P.

moraviensis (Xia et al., 2015). Pseudomonas moraviensis (moraviensis referring to Moravia

of the Czech Republic, the region microorganism 1B4T was located) was initially recovered

from soil. They have been reported to be gram-negative, non- spore forming bacilli

demonstrating spherical colony morphology (Tvrzova et al., 2006), supporting the

investigation findings.

3.3.4 Phylogenetic study of bacterial strains

Figure 3-4: Phylogenetic tree of consensus sequence 2KL

61





A phylogenetic tree is defined as a branching figure representing the evolutionary

relationships amongst numerous biological species based on their similarities and

dissimilarities in their genetic/physical makeup. Taxa that are connected in the tree are

assumed to be decedents of a common ancestor (Maher, 2002). Both strain 12Kl (Figure 3.5)

and 16KL (Figure 3.6) were confirmed to be categories of Bacillus cereus group. This group

of bacteria Bacillus species are no stranger to extreme mine conditions and have been

isolated from lead/zinc mines ( Hu et al., 2007) and gold mines( Bahari et al., 2013). Bacillus

cereus strains have also demonstrated high tolerance for metals such as Co, Cu, Au, Ag, Zn

(Nikovskaya et al., 2002), implying their potential as vehicles for metal recovery.

62

3.4 Conclusion

It can be concluded from the study that five bacteria strains (2KL, 9KL, 12KL, 16KL, 19KL)

were isolated from Gold 1 mine East Rand mine sites NRW Ramp water, W2E19, N2W2,

UK9 and E2 Reef drive south. The microorganisms were characterized and their identity was

further confirmed by analytical profile indexing, 16S rRNA sequencing, and phylogenetic

studies. The bacteria were identified to be Bacillus aryabhattai, Raoultella ornithinolytica,

Pseudomonas moraviensis and Bacillus cereus group (Bacillus sp., Bacillus thuringiensis).

The Gold1 mine East Rand, Springs Johannesburg, South Africa has proven to be an

ecosystem harbouring a diversity of microorganisms. Further research is required to

authenticate the novelty of microorganisms in the South African mine waters, potentially

yielding novel genomic data.

Acknowledgement

This work was supported by a research grant sponsored by the National Research

Foundation.

63

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67

CHAPTER FOUR

SYNTHESIS AND CHARACTERIZATION OF BIOCOMPATIBLE CHOLINE

DERIVED IONIC LIQUIDS FOR BIOTECHNOLOGY APPLICATIONS

Letlhabile Moyaha1, Sudharshan sekar1, Rufaro Archibald Bhero1, Sibusiso Sidu2,

Antoine F. Mulaba-Bafubiandi3, Vuyo Mavumengwana1






Johannesburg, P.O. Box 17011, Doornfontein 2028,Gauteng, South Africa

*Corresponding author:[email protected]; Tel: 27115596915

Abstract

Traditional organic solvents are toxic, volatile and highly flammable resulting in oxidant

smog and ozone destruction. This has sparked an interest in alternative, environmentally

friendly solvents and reaction media. Ionic liquids are regarded as potential contenders due to

their minimal toxicity and environmentally friendly potential. A great advantage of ILs is

their characteristic as “designer solvents”. As a result cation and anion combinations can be

manipulated to synthesis greener Ionic liquids with unique characteristics suitable for specific

processes. Cholinium/choline is a potential cation contender due to quaternary ammonium

incorporated with a polar hydroxyl functional group resulting in decreased levels of toxicity.

Choline is also a naturally prevalent and vital trace element that has the potential to break

down in aerobic environments.

The current study suggests that choline (cation) based ionic liquids can serve as an alternative

carbon source to conventional glucose sources, catalysing the growth of microorganisms for

further biotechnology applications. Keeping the cation component constant six ionic liquids

were synthesised employing a neutralization reaction and distillation processes. The ionic

liquids included choline lactate, choline dihydrogen phosphate, choline citrate, choline

tartarate, choline chloride, and choline levulinate. The ionic liquids were then characterized

by employing 1H NMR, 13C NMR and FT-IR techniques. Preliminary growth studies of

bacteria in the presence of ionic liquids were conducted by employing direct and indirect

colony counts. Shaker growth studies proved 80% of the bacterial strains to have a greater

68

preference for ionic liquids than the conventional glucose source, with the exception of

Bacillus aryabhattai. Choline chloride, choline lactate, choline dihydrogen phosphate and

choline levulinate were proven to be the most biocompatible ionic liquids across bacterial

strains Raoultella ornithinolytica, Bacillus sp., Bacillus thuringiensis and Pseudomonas

moraviensis.

Keywords: Ionic liquids (ILs), choline, choline lactate, choline dihydrogen phosphate,

choline citrate, choline tartarate, choline chloride, choline levulinate, 1H-NMR, 13C NMR,

and FT-IR

4.1 Introduction

Throughout the history of humankind biotransformations have been of economic and social

significance. Biocatalysis has been the most effective method of generating valuable

chemical products. To date, numerous chemicals including pharmaceuticals, esters, amino

acids, vitamins, saccharides and polysaccharides have been developed by enzymatic bio-

transformations (Habulin et al., 2011). A lot of research has been done on the benefits of

ionic liquids as “greener” alternatives. Studies on the utilization of organometallic

complexes in hydrogenation reactions so as to produce valuable compounds and

intermediates have also been investigated. Recent studies have shown the cation

component, more importantly the head group to be a major player in the toxicity of an ionic

liquid (IL). As an example, ILs with quaternary ammonium and alicyclic cations

demonstrate minimal toxicity compared to aromatic cations like imidazolium and

pyrimidinum. The inclusion of a polar hydroxyl, ether and a nitrile in the alkyl chain has the

potential to lower toxicity levels. As a result, cholinium is a potential cation IL contender

due to quaternary ammonium incorporated with a polar hydroxyl functional group resulting

in decreased levels of toxicity. Choline is a naturally prevalent and vital trace element that

has the potential to break down in aerobic environments. In recent times a selection of

choline based ionic liquids have been produced and reportedly showed minimal toxicity.

The ionic liquids synthesized also proved to be readily biodegradable. The ionic liquid

anion component has been known to add to the IL toxicity, though its role has been

disregarded (Guo-cai et al., 2010; Hou et al., 2013; Silva et al., 2014). Investigations

involving catalytic processes concerning metal complexes in ionic liquid have been noted,

with an average of 300 IL screened. Reduced enzyme turnover rates observed in organic

media have also ignited interest in ILs as alternative media (Rantwijk et al., 2003).

However minimal research has been done on the characterization of choline based ionic

69

liquids including their biodegradation and bioaccumulation (Ferlin et al., 2013). The

understanding of choline based IL effects on cells and their biochemistry can be useful

towards the application of these products in the manipulation of proteins and metabolic

processes of importance in biotechnology (Martins et al., 2013). The present paper thus

aims to uncover the understanding of choline derived ionic liquids relative to glucose as

carbon sources for catalysing microbial growth.

4.2 Methodology

4.2.1 Choline hydroxide properties

For the synthesis of choline based ionic liquids, choline hydroxide was employed as the

cation component of the ionic liquid.

N

CH3

H3C CH3

OHOH

Figure 4-1: Structure of choline hydroxide

Choline hydroxide solution was sourced from a reputable manufacturer, Sigma-Aldrich.

The properties of the solution are stated as,

Table 4-1: Properties of choline hydroxide solution

Stabilizer 0.5 % Paraformaldehyde as stabilizer

Concentration 45 wt. % in methanol

Refractive index n20/D 1.4186

Density 0.945 g/ml at 25 °C

70

4.2.2 Acids

High purity analytical reagents (Sigma-Aldrich) were employed for the preparation of choline

derived ionic liquids. The subsequent acids were utilized for this study:

Table 4-2: Ionic liquids reagents

Acid Structure Concentration

Lactic acid

OH

O

OH

85 %

Orthophosphoric

acid

P

O

HO

OH

OH

99.9 %

Citric acid

HO

O

OH

O OH

O

HO

99.5 %

Tartaric acid OH

OHO

HO

O

OH

99.5 %

Hydrochloric acid

H Cl 98 %

Levulinic acid

O

O

OH

98 %

To avoid potentially high exothermic reactions releasing immense amounts of heat, acid of

47.5 wt% was neutralized in water. The 47.5 wt% solution was prepared by the careful

71

addition of measured acid to measured double distilled water while stirring. To reduce the

heat released, the diluted mixture was placed in a water bath and cooled to room temperature.

4.2.3 Ionic liquid synthesis

The choline based ionic liquids were prepared by employing a neutralization reaction which

involves the combination of corresponding acid (Table 4.2) with choline hydroxide (Table

4.1). A classic method for the synthesis of choline based ionic liquids reads as follows:

choline hydroxide– methanol solution was gradually added drop by drop into equimolar acid

that was placed in an ice-bath with simultaneous stirring action. The reaction was left stirring

at room temperature overnight. Evaporation was employed to remove the solvent and the

final product is dried under vacuum at 80 °C for 2 days.

4.2.4 Evaporation process

Considering how dilute the chemistry of the end product is: choline hydroxide (45 % solution

in methanol) and acid (47.5 % solution in water) as a result of water and methanol. In order to

attain a concentrated product water and methanol have to be removed. This was executed by

rotary evaporation. Rotary evaporation was applied until the expected weight of pure product

was achieved. The volume of the diluted product after synthesis was 600 ml and came down

to 200 ml of pure product, after rotary evaporation. The final product was retained in a

vacuum oven at 80 °C for two days to ensure the complete removal of water.

Figure 4-2: Synthesised ionic liquids namely choline levulinate, choline chloride, choline citrate, choline

lactate, choline tartarate and choline dihydrogen phosphate

4.2.5 Characterization of the synthesised ionic liquids

In order to confirm the structure of the choline derived ionic liquid synthesized 1H NMR, 13C

NMR and FT-IR studies were employed.

72

4.2.5.1 1H NMR Spectra

High-resolution 1H-NMR spectra were recorded on a Bruker 400 MHz FT-NMR

spectrometer at room temperature using 10-15 % (w/v) solutions of deuterated water (D2O).

Tetramethylsilane (TMS) was used as the internal reference.

4.2.5.2 13C NMR Spectra

High-resolution 13C-NMR spectra were recorded on a Bruker 100 MHz FT-NMR

spectrometer at room temperature using 10-15 % (w/v) solutions of deuterated water (D2O).

Tetramethylsilane (TMS) was used as the internal reference.

4.2.5.3 FT-IR

Infrared spectra were recorded on a Nicolet iS10 FT-IR spectrophotometer by the standard

KBr disc method in a range of 4000 cm-1 to 650 cm-1.

Table 4-3: Nicolet iS10 FT-IR instrument specifications

Parameter Condition

Detector DTGS KBr

Beam splitter KBr

Source IR

Window Diamond

Recommended range

Max range limit

Min range limit

4000-525

4000

650

Gain 2

Optical velocity 0.4747

4.2.6 Preliminary growth studies of halophilic bacteria in the presence of choline

based ILs

The study was based on using five choline based ionic liquids as alternative carbon sources to

conventional glucose namely: Choline lactate, choline dihydrogen phosphate, choline citrate,

choline tartarate, choline chloride and choline levulinate

4.2.6.1 Inoculum preparation for halotolerant bacteria strains

For individual bacterial strains, the nutrient broth was inoculated with optimized seed culture

of 1 % (v/v).

73

4.2.6.2 Media preparation for growth plate studies (solid media)

Himedia nutrient agar media was employed in the study. The pH of the media was then

adjusted to pH 7.0 using 0.1 N HCL and 0.1 N Na OH. The media was inoculated with 5 g/L

of the respective ionic liquid before autoclaving at a temperature of 121°C for 15 minutes. A

1/10 serial dilution of the cultivated bacterial strains (Bacillus aryabhattai, Raoultella

ornithinolytica, Bacillus species, Bacillus thuringiensis and Pseudomonas moraviensis) was

prepared, employing the pour plate technique. The cultures were then incubated at 37 °C

followed by daily sampling conducted from day one.

4.2.6.3 Direct colony count

Using the Astor 20 Colony counter the number of colony (CFUs) forming units on agar plates

(solid media) were recorded.

4.2.6.4 Media preparation for shaker growth studies (liquid media)

Himedia nutrient broth media was employed in the study. The pH of the media was adjusted

to pH 7.0 using 0.1 N HCL and 0.1 N Na OH solution. The media was inoculated with 5g/L

of the respective ionic liquid and sterilized by autoclaving at a temperature of 121 °C for 15

minutes. A batch culture format was used in all shake flask studies and the cultures were

placed in the IncoShake rotary shaker incubator at 37 °C with a constant agitation of 120

rpm. Daily sampling was conducted, starting from day one.

4.2.6.5 Indirect colony count

An indirect colony count study was then conducted on the liquid media(shaker flasks) by

employing the Thermo Spectronic BioMate 3UV-Visable spectrophotometer at a

wavelength of 600 nm, as a measure of optical density(OD)/ the developing turbidity (an

index of increasing cell mass which is assumed to correlate with a rise in the cell

population).

74


4.3.1 NMR

Although a number of articles do not go to the extent of using NMR to characterise resultant

ionic liquids (including some of the ones described herein), it was deemed appropriate to

confirm the nature of the ionic liquids synthesised in this study. Upon NMR analysis, choline

lactate, choline dihydrogen phosphate, choline citrate, choline tartarate, choline chloride and

choline levulinate spectral data were found to be consistent with results obtained by Isahak et

al. (2011); Mota-Morales (2011); Wang et al. (2012) and Yaacob et al. (2011) as shown in

Figures 4.3 to 4.14.

4.3.1.1 Characterization of Choline Lactate 1H NMR

Figure 4-3: 1H NMR of choline lactate

OHN+

CH3

H3C

H3C

C

OH

CH3

O

-O

EA

A

B

A

C

D

F

D

75

1H NMR: 2-Hydroxy-N, N, N-trimethylethanaminium 2-hydroxypropanoate (400 MHz,

D2O, 20 °C) δ (ppm): 1.09 (3HE, m, CH3); 2.97 (9HA, d, N-CH3); 3.10 (1HD, s, C-H); 3.30

(2HB, t, N-CH2); 3.84 (2Hc, m, CH2); the last peak at 4.70 ppm, is characteristic of residual

water.

4.3.1.2 Characterization of Choline Lactate 13C NMR

OHN+

CH3

H3C

H3C

C

OH

CH3

O

-OE

A

B C

D

F

A

A

Figure 4-4: 13C NMR of choline lactate

13C NMR: 2-Hydroxy-N,N,N-trimethylethanaminium 2-hydroxypropanoate (100 MHz, D2O,

20 °C) δ (ppm): 20.28(CF); 53.82 (CA); 55.52 (CB), 67.36(CE); 68.38(CC) and peak 181.93

(CD)

76

4.3.1.3 Characterization of Choline Dihydrogen Phosphate 1H NMR

OHN+

PO

OH

OH

-O

A

A

A

B C

D

E

E

Figure 4-5: 1H NMR of choline dihydrogen phosphate

1H NMR: 2-Hydroxy-N,N,N-trimethylethanaminium dihydrogen phosphate (400 MHz, D2O,

20 °C) δ (ppm): 3.00 (9HA,m ,N-CH3); 3.64 (2HB,s,N -CH2); 3.80 (2Hc,s, CH2) ; the OH

groups of phosphate and choline predicted to be at the 2.90 ppm region is overlapped by the

choline peak and the last peak at 4.70 ppm is characteristic of residual water.

77

4.3.1.4 Characterization of Choline Dihydrogen Phosphate 13C NMR

OHN+

PO

OH

OH

-O

A

A

A

B C

Figure 4-6: 13C NMR of choline dihydrogen phosphate

13C NMR: 2-Hydroxy-N, N, N-trimethylethanaminium dihydrogen phosphate (100 MHz,

D2O, 20 °C) δ (ppm): 53.76 (CA); 55.46 (CB) and 67.23 (CC).

78

4.3.1.5 Characterization of Choline Citrate 1H NMR

OH

N+

HO

O

O-

O O-

O

-O E

E

A

AA

BC

D

F

Figure 4-7: 1H NMR of choline citrate

1H NMR: 2-Hydroxy-N,N,N-trimethylethanaminium 3-carboxy-2-(carboxymethyl)-2-

hydroxypropanoate (400 MHz, D2O, 20 °C) δ (ppm): 2.62 (4HE,m,CH2); 2.740 (9HA,m,N-

CH3); 3.26 (2HB,d,CH2); 3.82 (2Hc,s,CH2) and peak 4.71 ppm is characteristic of residual

water. The OH groups of choline and citrate are predicted to be overlapped by the one of the

peaks.

79

4.3.1.6 Characterization of Choline Citrate 13C NMR

OH

N+

HO

O

O-

O O-

O

-O E

A

A

B

A

C

D

F E

G

D

Figure 4-8: 13C NMR of choline citrate

13CNMR:2-Hydroxy-N,N,N-trimethylethanaminium 3-carboxy-2-(carboxymethyl)-2-

hydroxypropanoate (100 MHz, D2O, 20 °C) δ (ppm): 43.63 (CE); 53.80 (CA); 55.53 (CB);

67.32 (CF); 73.33 (CC); 174.63(CD); 178.48 (CG).

80

4.3.1.7 Characterization of Choline Tartarate 1H NMR

OH

N+

OH

OHO

-O

O

O-

A

A

A

BC

D

F

E

F

E

Figure 4-9: 1H NMR of choline tartarate

1H NMR: 2-Hydroxy-N, N, N-trimethylethanaminium 3-carboxy-2, 3 dihydroxypropanoate

(400 MHz, D2O, 20 °C) δ (ppm): 2.02 (2OHF ,s, OH); 3.00 (9HA,s,CH3 ); 3.13 (2HG,d,H);

3.32 (2HB,t,CH2); 3.85 (2HC ,d,CH2) and peak 4.70 is characteristic of residual water.

81

4.3.1.8 Characterization of Choline Tartarate 13C NMR

OH

N+

OH

OHO

-O

O

O-

A

A

B

A

C

D DEE

Figure 4-10: 13C NMR of choline tartarate

13C NMR: 2-Hydroxy-N, N, N-trimethylethanaminium 3-carboxy-2, 3-dihydroxypropanoate

(100 MHz, D2O, 20 °C) δ (ppm): 53.83 (CA); 55.53 (CB); 67.38 (CC); 81.91 (CD); 180.84

(CE)

82

4.3.1.9 Characterization of Choline Chloride 1H NMR

OHN+Cl-

A

A

A

B C

D

Figure 4-11: 1H NMR of choline chloride

1H NMR: 2-Hydroxy-N,N,N-trimethylethanaminium chloride (400 MHz, D2O, 20 °C) δ

(ppm): 3.09 (9HA,s,CH3); 3.23 (1HD,s,OH); 3.41 (2HB,m,CH2); 3.94 (2HC,t,CH2) and 4.70

ppm is characteristic of residual water.

83

4.3.1.10 Characterization of Choline Chloride 13C NMR

OHN+

Cl-

A

A

A

B C

Figure 4-12: 13C NMR of choline chloride

13C NMR: 2-Hydroxy-N, N, N-trimethylethanaminium chloride (100 MHz, D2O, 20 °C) δ

(ppm): 53.90 (CA); 55.70 (CB); 67.50 (CC).

84

4.3.1.11 Characterization of Choline Levulinate 1H NMR

OH

N+

O

O

-O

E

E

A

A

A

BC

D

F

Figure 4-13: 1H NMR of choline levulinate

1HNMR :2-Hydroxy-N,N,N-trimethylethanaminium 4-oxopentanoate (400 MHz, D2O, 20

°C) δ (ppm): 2.08(3HF,s,CH3); 3.047 (9HA,s,N-CH3); 3.37 (4HE,m,CH2); 3.91 (2HB,m,H2);

4.36(2HC,s,H2) and peak 4.67 ppm is characteristic of residual water.

85

4.3.1.12 Characterization of Choline Levulinate 13C NMR

OH

N+

O

O

-O

A

A

A

BC

D

G

EF

FE

Figure 4-14: 13C NMR of choline levulinate

13C NMR: 2-Hydroxy-N N, N-trimethylethanaminium 4-oxopentanoate (100 MHz, D2O, 20

°C) δ (ppm): 23.62 (CG); 53.83 (CA); 55.56 (CF); 67.35 (CB); 72.83 (CC); 176.42 (CE).

4.3.2 FTIR

Fourier transform infrared spectroscopy (FTIR) was crucial in analysing the identity and

relationship of different structural groups that constitute an ionic liquid. Characteristic

frequency shifts, band widths and absorbance quantities were a reference point for studying

the interaction between different groups; for analysing and identifying the relevant structures

as shown in Figure 4.15 to 4.20.

86

4.3.2.1 FTIR: 2-Hydroxy-N, N, N-trimethylethanaminium 2-hydroxypropanoate

Characterization using FTIR (Figure 4.15) confirmed the functional groups occurring in

region 4000 cm-1 to 600 cm-1: The absorptions in the region 1478 cm-1 (C-H Scissoring

vibration), 1350 cm-1(C-H methyl rock vibration), and 2961cm-1 (C-H stretch vibration) were

the result of the methyl group appearing in choline and lactate. The OH group observed in the

region 3278 cm-1 and C-O in region 1086 cm-1 appeared in choline and lactate, and 1119

cm-1 C-N in choline. The absorptions’ in the region 1588 cm-1 and 1406 cm-1 were the result

of the COO- group appearing in Lactate.

Figure 4-15: FTIR of choline lactate

87

4.3.2.2 FTIR: 2-Hydroxy-N, N, N-trimethylethanaminium dihydrogen phosphate


region 4000 cm-1 to 600 cm-1 : The absorptions in the region 1478 cm-1 (C-H scissoring

vibration) and 2980 cm-1 (C-H stretch vibration) were the result of methyl group appearing

in choline. The OH group appearing in choline and dihydrogen phosphate was observed in

the region 3250 cm-1. The C-O in region 1073 cm-1 and C-N in region 1131 cm-1 appeared in

choline. Absorption in region 1131 cm-1/1200 cm-1 is also indicative of the presence of

phosphate in dihydrogen phosphate.

Figure 4-16: FTIR of Choline dihydrogen phosphate

4.3.2.3 FTIR:2-Hydroxy-N,N,N-trimethylethanaminium 3-carboxy-2-(carboxymethyl)-

2-hydroxypropanoate




in choline. The OH group appearing in choline and citrate was observed in the region 3383

cm-1, and C-O in region 1083 cm-1. The C-N group in region 1207 cm-1 appeared in choline.

Absorption in regions 1588 cm-1 and 1708 cm-1 are the result of COO- groups appearing in

citrate.

88

Figure 4-17: FTIR of choline citrate

4.3.2.4 FTIR:2-Hydroxy-N,N,N-trimethylethanaminium3-carboxy-2,

3dihydroxypropanoate.



vibration) 1346 cm-1(C-H methyl rock vibration) and 2969 cm-1 (C-H stretch vibration) were

the result of methyl group appearing in choline and tartarate. The OH group appearing in

choline and tartarate was observed in the region 3433 cm-1, and C-O in region 1087 cm-1. The

C-N group in region 1207 cm-1 appeared in choline. Absorption in regions 1597 cm-1 and

1708 cm-1 are the result of COO groups appearing in choline and tartarte.

Figure 4-18: FTIR of choline tartarate

89

4.3.2.5 FTIR: 2-Hydroxy-N, N, N-trimethylethanaminium chloride


region 4000 cm-1 to 600 cm-1: The absorptions occurring at 1477 cm-1 (C-H scissoring

vibration) and 2969 cm-1 (C-H stretch vibration) were the result of a choline methyl group.

Whereas, the OH group was observed at around 3382 cm-1 with the C-O and the C-N groups

at 1083 cm-1 and 1132 cm-1 respectively as expected.

Figure 4-19: FTIR of choline chloride

4.3.2.6 FTIR: 2-Hydroxy-N, N, N-trimethylethanaminium 4-oxopentanoate


region 4000cm-1 to 600cm-1 : The absorptions in the region 1479 cm-1 (C-H scissoring


in choline and levulinate. The OH group appearing in choline was observed in the region

3215 cm-1, and C-O in region 1088 cm-1. The C-N group in region 1132 cm-1 appeared in

choline. Absorption in regions 1574 cm-1 and 1705 cm-1 are the result of COO- groups

appearing in choline and levulinate.

90

Figure 4-20: FTIR of choline levulinate

4.3.3 Preliminary growth studies:

Preliminary qualitative studies (Table 4.4 to 4.9) were essential for screening biocompatible

ILs and establishing quantitative data (Figure 4.21 to 4.26) related to the biocompatibility of

these ILs on bacterial growth and in addition, refining the process parameters for further

studies.

4.3.3.1 Growth plate studies

Table 4-4: Growth plate studies of Bacillus aryabhattai (CFU) in ionic liquids

Day Nutrient Agar Supplement Dilution factor (DF)

10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8

1 Glucose 301 76 0 0 0 0 0 0

Choline Lactate 556 128 0 0 0 0 0 0

Choline Dihydrogen Phosphate 249 13 0 0 0 0 0 0

Choline Citrate 0 0 0 0 0 0 0 0

Choline Tartarate 0 0 0 0 0 0 0 0

Choline Chloride 800 230 15 0 0 0 0 0

Choline Levulinate 800 219 21 0 0 0 0 0

2 Glucose 320 101 0 0 0 0 0 0





Choline Chloride NC 236 20 0 0 0 0 0

Choline Levulinate NC 259 21 0 0 0 0 0

3 Glucose 322 101 0 0 0 0 0 0






91

Where NC indicates too numerous to Count

Table 4-5: Plate growth studies of Raoultella ornithinolytica (CFU) in ionic liquids




4 Glucose NC 101 0 0 0 0 1 0

Choline Lactate NC 153 0 0 0 0 0 0

Choline Dihydrogen Phosphate NC 27 0 0 1 0 0 0






10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8

1 Glucose NC NC 515 600 273 112 30 5

Choline Lactate NC NC NC NC 526 226 57 6


Choline Citrate NC NC 515 340 143 55 1 0

Choline Tartarate NC NC NC NC 0 0 0 0

Choline Chloride NC NC NC NC 465 207 42 0

Choline Levulinate NC NC NC NC 520 246 75 6

2 Glucose NC NC NC NC 346 162 62 5


Choline Dihydrogen Phosphate NC 600 NC 600 346 152 63 8

Choline Citrate NC NC NC 400 220 136 60 0


Choline Chloride NC NC NC NC NC 225 45 0

Choline Levulinate NC NC NC NC NC 250 84 7



Choline Dihydrogen Phosphate NC 632 NC NC 526 256 71 8

Choline Citrate NC NC NC NC 524 243 53 2





Choline Lactate NC NC NC NC NC 570 66 8

Choline Dihydrogen Phosphate NC NC NC NC 548 264 76 8

Choline Citrate NC NC NC NC 548 255 65 6

Choline Tartarate NC NC NC NC NC 0 28 0

Choline Chloride NC NC NC NC NC NC 52 1

Choline Levulinate NC NC NC NC NC NC 104 11

92

Table 4-6: Growth plate studies of Bacillus sp. (CFU) in ionic liquids


Day Nutrient Agar Supplement Dilution Factor(DF)

10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8

1 Glucose NC NC 524 10 2 1 0 0

Choline Lactate NC NC NC 110 8 0 0 0

Choline Dihydrogen Phosphate NC NC 460 2 2 0 0 0



Choline Chloride NC NC NC 550 10 0 0 0

Choline Levulinate NC NC NC 550 10 0 0 0

2 Glucose NC NC 540 11 2 1 0 24







3 Glucose NC NC NC 11 2 1 0 34


Choline Dihydrogen Phosphate NC NC NC 2 2 0 0 19












93

Table 4-7: Growth plate studies of Bacillus thuringiensis (CFU) in ionic liquids


Day Nutrient Agar Supplement Dilution Factor (DF)

10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8

1 Glucose NC NC 524 220 24 69 1 0














3 Glucose NC NC NC 255 32 110 1 0







4 Glucose NC NC NC 259 32 118 2 0







94

Table 4-8: Plate growth studies of Pseudomonas moraviensis


Day Nutrient Agar Supplement Dilution factor(DF)

10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8

1 Glucose NC 450 0 0 0 0 0 0

Choline Lactate NC NC 0 0 0 0 0 0




Choline Chloride NC NC 55 52 0 0 0 0

Choline Levulinate NC NC 8 0 0 0 0 0

2 Glucose NC 544 0 0 0 0 0 0






Choline Levulinate NC NC 150 24 0 0 0 0

3 Glucose NC NC 0 0 0 0 0 0







4 Glucose NC NC 0 0 0 0 0 0







95

Table 4-9: Plate growth studies -Control

Day Nutrient agar supplement Dilution Factor(DF)

10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8

1 Glucose 0 0 0 0 0 0 0 0







2 Glucose 0 0 0 0 0 0 0 0







3 Glucose 0 0 0 0 0 0 0 0







4 Glucose 0 0 0 0 0 0 0 0






Choline Levulinate 0 0 0 0 0 0 0 0 Where NC indicates too numerous to Count

Colony forming unit (CFU) measurements proved Raoultella ornithinolytica (Table 4.5) to be

biocompatible with ionic liquids choline levulinate, choline lactate, choline dihydrogen

phosphate, choline chloride, choline citrate and choline tartarate. The above mentioned ionic

liquids also demonstrated competitive growth to conventional glucose source. The Raoultella

ornithinolytica indicated the least viability in ionic liquid choline tartrate.

Colony forming unit (CFU) measurements proved Bacillus sp. (Table 4.6) to be

biocompatible with ionic liquids choline dihydrogen phosphate, choline lactate, choline

chloride and choline levulinate. The above mentioned ionic liquids also demonstrated

competitive growth to conventional glucose source. Bacillus species were not viable in ionic

96

liquids choline citrate and choline tartrate, which was indicated by the absence of CFUs,

suggesting choline citrate and choline tartrate to be unfavorable nutrient sources for Bacillus

species.

Colony forming unit (CFU) measurements proved Bacillus aryabhattai (Table 4.4) , Bacillus

thuringiensis (Table 4.7), and Pseudomonas moraviensis (Table 4.8) strains to be

biocompatible with ionic liquids choline chloride, choline lactate, choline dihydrogen

phosphate and choline levulinate. The above mentioned ionic liquids also demonstrated

competitive growth to conventional glucose source; on the other hand poor growth was

cultivated in choline citrate. Furthermore Bacillus aryabhattai, Bacillus thuringiensis and

Pseudomonas moraviensis were not viable in choline tartrate which was indicated by the

absence of CFUs, suggesting unfavorable nutrient sources.

As predicted, growth was entirely absent in the control (Table 4.9), verifying the sterility and

reliability of the experiment.

4.3.4 Shaker growth studies

Figure 4-21: Shaker growth studies of Bacillus aryabhattai in choline based ionic ILs vs. glucose as an

alternative carbon source at a speed of 120 rmp and temperature of 37 °C (5days)

In descending order, Bacillus aryabhattai as shown in Figure 4.21 demonstrated the highest

growth in glucose (1.722 nm), choline lactate (1.638 nm), choline dihydrogen phosphate

97

(1.489 nm), choline levulinate (1.486 nm), choline chloride (1.438 nm), choline citrate (0.371

nm) and the least growth in choline tartarate (0.014 nm).

Bacillus aryabhattai in the presence of conventional glucose source demonstrated the highest

exponential phase in the shortest period, alluding to the rapid adaption of Bacillus

aryabhattai in glucose carbon sources. A log phase followed by a stationery phase was a

common pattern noted amongst the other ionic liquids which could be the result of the rapid

competition for nutrients resulting in the depletion of the carbon source and production of

toxic waste material/secondary products.

The bacterial growth of Bacillus aryabhattai was least favoured in choline citrate followed by

choline tartarate. The lack of adaption of the bacteria to choline tartrate is demonstrated by

the bacteria predominantly remaining in the lag phase alluding to the potential toxicity of

choline tartarate to Bacillus aryabhattai.

Figure 4-22: Growth curve of Raoultella ornithinolytica in choline ILs vs. glucose as an alternative carbon

source at speed of 120 rpm and temperature of 37 °C

In descending order Raoultella ornithinolytica as shown in Figure 4.22 demonstrated the

highest growth in the presence of choline dihydrogen phosphate (2.184 nm), choline lactate

(2.090 nm), choline citrate (2.075 nm), choline chloride (2.010 nm), choline levulinate (1.707

nm), glucose (1.297 nm) and the least growth in choline tartarate (0.223 nm).

98

As observed in the log phase, Raoultella ornithinolytica adapted rapidly in the presence of

choline lactate and choline chloride compared to conventional glucose source. In contrast to

the other ionic liquids glucose and choline levulinate demonstrated a rapid death phase.

Choline tartarate predominantly displayed minimal bacterial growth alluding to the potential

toxicity of choline tartarate to Raoultella ornithinolytica.

Figure 4-23: Growth curve of Bacillus sp. in choline based ILs vs. glucose as an alternative carbon source

at speed of 120 rpm and temperature of 37 °C.

In descending order Bacillus sp. as shown in Figure 4.23 demonstrated the highest growth in

the presence of, choline chloride (1.715 nm), choline dihydrogen phosphate (1.607 nm),

choline levulinate (1.593 nm), choline lactate (1.526 nm), glucose (1.396 nm), choline citrate

(0.301 nm), and the least growth in choline tartarate (0.009 nm). Choline chloride, choline

dihydrogen phosphate and choline lactate demonstrated a steady exponential growth rate

pattern, which implies that the Bacillus species was thriving in this environment.

Bacillus sp. demonstrated rapid adaption to glucose sources on the first day but went into

death phase within the third day and choline levulinate within the fourth day of the shaker

flask growth studies, indicating the depletion of the carbon sources at a competitive rate

resulting in a sharp death phase. On the other hand, Bacillus sp. demonstrated poor growth in

choline citrate and had challenges adapting to this environment. Bacillus sp. could not

99

develop in choline tartrate, alluding to the possible toxicity of this carbon source to the

bacteria.

Figure 4-24: Growth curve of Bacillus thuringiensis in choline based ILs vs. glucose as an alternative

carbon source at speed of 120 rpm and temperature of 37 °C.

In descending order Bacillus thuringiensis as shown in Figure 4.24 demonstrated the highest

growth in the presence of choline chloride (1.943 nm), choline lactate (1.860 nm), choline

dihydrogen phosphate (1.761 nm), choline levulinate (1.550 nm), glucose (1.241 nm), choline

citrate (0.325 nm) and the least growth in choline tartarate (0.029 nm). Choline lactate and

choline dihydrogen phosphate demonstrated steady exponential growth, which implies that

the Bacillus thuringiensis species was thriving in this environment.

Choline chloride started showing a decline in the Bacillus thuringiensis population on the

fourth day; while choline lactate and choline dihydrogen phosphate demonstrated consistent

growth. Bacillus thuringiensis demonstrated rapid adaption to glucose sources on the first day

but went into death phase within the third day and choline levulinate within the fourth day of

the shaker flask growth studies, indicating the depletion of the carbon sources at a

competitive rate resulting in a sharp death phase. Bacillus thuringiensis demonstrated

minimal growth in choline citrate and battled to adapt to this environment. Bacillus

thuringiensis could not develop in choline tartarate, alluding to the possible toxicity of this

carbon source to the bacteria.

100

Figure 4-25: Growth curve of Pseudomonas moraviensis in choline based ionic liquids vs. glucose as an

alternative carbon source at speed of 120 rpm and temperature of 37 °C.

In descending order Pseudomonas moraviensis as shown in Figure 4.25 demonstrated the

highest growth in the presence of choline lactate (2.058 nm), choline chloride (1.995 nm),

choline levulinate (1.438 nm), choline dihydrogen phosphate (1.304 nm), glucose (0.744 nm),

choline citrate (0.401 nm) and the least growth in choline tartarate (0.019 nm).

Pseudomonas moraviensis demonstrated rapid adaption in choline lactate choline chloride

environments and choline levulinate, reaching rapid exponential growth within the first day

of the growth studies followed by a gradual death phase. Choline dihydrogen phosphate

gradually went into the exponential phase followed by a stationery phase which was

gradually followed by a death phase, indicating nutrient depletion during which the rate of

Pseudomonas moraviensis cell growth was equivalent to the rate of bacterial death.

Pseudomonas moraviensis demonstrated poor growth in choline citrate. Contrary to

expectations, the glucose source also demonstrated poor growth of Pseudomonas moraviensis

bacteria, with the bacteria battling to adapt to the conventional glucose carbon source.

The bacteria could not develop in choline tartarate, alluding to the possible toxicity of this

carbon source to the bacteria.

101

Figure 4-26: Growth curve in the absence of bacteria in choline based ionic liquids vs. glucose as an

alternative carbon source at speed of 120 rpm and temperature of 37 °C

In the absence of bacteria as shown in Table 4.26 there was no growth, indicating the absence

of contamination and proving the sterilization technique effective.

4.4 Conclusion

The applications of 13C NMR, 1H NMR and FT-IR were successful in confirming the

presence of methyl groups, alcohol and amine functional groups in all the cation components

of choline derived ionic liquids. The functional groups methyl, hydroxyl, ether and carbonyl

were common denominators in the anion component of choline lactate, choline tartarate and

choline citrate. Choline levulinate anion compounds were confirmed to have functional

groups methyl, ether and carbonyl. The choline dihydrogen phosphate anion component was

confirmed by FTIR analysis to have the phosphorous functional group, phosphate.

Biocompatibility studies of bacterial sp. isolated from Gold1 Mine, East Rand Springs

Johannesburg confirmed the biocompatibility of ionic liquids choline dihydrogen phosphate,

choline lactate, choline chlorite, choline citrate, choline levulinate with poor biocompatibility

noted across ionic liquids, choline citrate and choline tartarate. Bacterial growth among all

the strains was noted to be insignificant in ionic liquid choline tartarate alluding to its poor

biocompatibility. Due to the presence of the quaternary- ammonium cation integrating a polar

102

OH it was projected that all choline based ILs would not be of a toxic nature, however

choline tartarate indicated poor biocompatibility indicating the influence of the anion

component on the total toxicity.

Bacillus sp. and Bacillus thuringiensis demonstrated a similar growth curve and

biocompatibility pattern across the ionic liquids, confirming their phylogenetic relationship.

The ionic liquids choline chloride, choline lactate, choline dihydrogen phosphate and choline

levulinate were the preferred source of carbon across all five bacteria strains. Choline lactate,

choline chloride, choline dihydrogen phosphate and choline levulinate in agreement with both

plate and shaker flask studies were concluded to be the most biocompatible ionic liquids

across all five bacterial strains, and selected for further studies.

Acknowledgement


Foundation.

103

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Biodegradability of Cholinium amino Acids Ionic Liquids. Primary Library of Science, 8, 3.

Isahak, W., Ismail, M., Mohd Jahim, J., Salimon, J. & Yarmo, M. (2011). Transesterification

of Palm Oil by using Ionic Liquids as a New Potential Catalyst. Trends in Applied Sciences

Research. doi: http://dx.doi.org/10.3923/tasr.2011.

Martins, I., Hartmann, D., Alves, P., Planchon, S., Renaut, J., Leitão, M., Rebelo, L. &

Pereira, C. (2013). Proteomic alterations induced by ionic liquids in Aspergillusnidulans and

Neurosporacrassa. Journal of Proteomics, 9 4, 2 6 2 – 2 7 8.

Mota-Morales, J., Gutiérrez, M., Sanchez, I., Luna-Bárcenas, G. & Monte, F. (2011). Made

Deep-Eutectic Solvents Suitable for Frontal Polymerization. The Royal Society of Chemistry.

Rantwijk, F., Lau, R. & Sheldon, R. (2003). Biocatalytic transformations in ionic liquids.

Tends in Biotechnology, 21, 3.

Silva, F., Siopa, F., Figueiredo, B., Gonçalves, A., Pereira, J., Gonçalves, F., Coutinho, J.,

Afonso, C. & Ventura, S. (2014). Sustainable design for environment-friendly mono and

dicationiccholinium-based ionic liquids. Ecotoxicology and Environmental Safety, 108, 302 –

310.

Wang, H., Jla, Y., Wang, X., Ma, J. & Jing, Y. (2012). Physico-chemical Properties of

Magnesium Ionic Liquid Analogous. Journal of Chilean Chemical Society, 57, 3.

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Esterification of Lauric Acid. The Malaysian Journal of Analytical Sciences, 15(1), 46-53.

105

CHAPTER FIVE

THE VALORISATION OF SLAG FROM BCL USING BIOCOMPATIBLE

CHOLINE BASED IONIC LIQUIDS AS A SUPPORT

Letlhabile Moyaha1, Sudharshan sekar1, Antoine F. Mulaba-Bafubiandi3, Sibusiso

Sidu2, Vuyo Mavumengwana1






Johannesburg, P.O. Box 17011, Doornfontein 2028,Gauteng, South Africa

*Corresponding author:[email protected]; Tel: 0712839617

Abstract

Slags often contain significant volumes of base metals and can potentially be exploited as

reliable secondary resources if treated in an environmentally friendly fashion or potential

pollutants if not processed correctly. Considering the environmental and economic

implications of these pollutants, it was imperative to establish an efficient and feasible

method for the optimal recovery of these metal values and slag waste disposal. As a result a

novel bioleaching method was developed for the valorisation of slag from BCL using choline

based ionic liquids as a support. The study was indicative of choline ILs, specifically choline

lactate having a metabolic advantage over glucose carbon sources, which was validated by

the optimal leaching kinetics represented by significant iron recoveries ranging from 65 % to

97% by Bacillus aryabhattai, Raoultella ornithinolytica and mixed cultures; with the highest

dissolution efficiency facilitated by Bacillus species and Bacillus thuringiensis. Moderate

zinc recoveries ranging from 26% to 31% were also indicated in IL choline dihydrogen

phosphate. Thus, suggesting the selective bioleaching of base metals by ILs choline lactate

and choline dihydrogen phosphate, used as a substrate for the valorisation of slag from BCL.

Keywords: Slags, valorisation, bioleaching, choline, ILs, base metals

106

5.1 Introduction

Slags are formed in bulk quantities as by-products of ferrous / non-ferrous smelting processes

comprising of metal oxides which can either be recovered or re-used. Slags also contain base

metals namely Co, Ni, Zn etc. that can potentially pollute the atmosphere if not recovered

accordingly (Yang et al., 2010). Slag minerals can thus be valorised by the value of their

minor constituents; by the iron-oxides and silicates occurring in large concentrations

(Kalinkin et al., 2012; Maweja et al., 2010; Wang et al., 2015). Investigations on

hydrometallurgy methods for the treatment of slag have been extensively conducted. Such as

investigations on the dissolution rate of 95 % cadmium, nickel and zinc from zinc plant

residues, by employing 1.22 mol/L of sulphuric acid at a temperature of 25 °C for 60

minutes, though with insignificant extraction of Lead (Kai-qi et al., 2012). However

hydrometallurgy processes have indicated limitations, favouring the recovery of metals

occurring in high concentrations and have also been discovered to produce contaminants in

the form of excess hazardous leaching agents. As a result, bioleaching processes provide an

alternative approach for slag wastes with low quantities of metal values (Hocheng et al.,

2014).

While thorough investigations have been conducted into the architecture and engineering-

technology of biological mining processes, the microbiology aspect has been neglected,

lacking critical studies and clear parameters (Kai-qi et al., 2012; Kaksonen et al., 2011;

Rawlings et al., 2007). The main challenge presented in the commercialization of bioleaching

processes lies in the slow bacterial growth rates impeding the leaching kinetics; as a result

further research is required for improving the growth kinetics of these microorganisms.

Contrary to common industrial companies that employ microorganisms such as fermenting

technology industries, biopharmaceuticals etc. the collection, regulation and supervision of

microbes in bio-mining has been minimum or completely absent. This poses a question

whether commercially used microbes are the most competent consortium of microbial species

for diverse mineral processing. Supposing a supreme microbial consortium was discovered in

lab studies, there remains the question, as to how the population of mineral oxidizers can be

improved and monitored, specifically in heap-leaching processes. Furthermore there are

significant debates referring to how optimized microbial consortiums used in the bio-

oxidation of specific minerals in fixed operating conditions can be developed e.g.

temperature/pH. The debates also argue whether there are advantages in the transfer of

107

bacterial consortiums from foreign locations in comparison to the selection of native

microbes identified within the vicinity of the deposits (Rawlings et al., 2007).

To date ionic liquids have grown popular in different fields namely organic synthesis,

catalysis , bio-catalysis and biomass pre-treatment , which is owed to their thermal stability,

diverse solubility and their properties as designer solvents (Guo-cai, Jian & Yi-xin, 2010;

Samori et al., 2010). Furthermore choline chloride derived ionic liquids (ILs) were reported

to be advantageous due to their simple prep, good water/air stability and inexpensive nature,

thus qualifying their application at an industrial scale (Haerens et al., 2009).

Choline is a crucial component for healthy cellular function with an existing mechanism for

regulating its biological synthesis and hydrolysis. Recently an investigation on the efficiency

of choline lactate for the growth of Staphylococcus lentus evaluating the fate of the cation

and anion component was conducted, establishing favourable biochemistry and bioenergetics

in choline lactate medium (Sekar et al., 2013). Choline hydroxide has also been employed for

the catalysis reaction of aldol condensation (Pernak et al., 2007). Even though numerous

papers have been published on the subject of ionic liquids; pertinent investigations in the

hydrometallurgical field have proven unsuccessful. As a result, the application of ionic

liquids in this field is still in its infancy and requires great attention (Pernak et al., 2007).

Furthermore is the research gap in the use of these ionic liquids as catalysts for biological

processes.

With the aim of minimising the loss of valued product from bio-mining operations and

reducing the impact of process waste on the environment, an investigation was conducted to

determine optimal base metal recovery from BCL slag employing choline based ionic liquids

as an alternative substrate to conventional glucose sources. The ionic liquids of interest

included choline levulinate (CLe), choline lactate (CLa), choline dihydrogen phosphate

(CDP) and choline chloride (CCh) vs. conventional glucose (G) sources.

108

5.2 Methodology

5.2.1 Sampling of the processing

Figure 5-1: Schematic of BCL slag (Adapted from Malema & Legg, 2006)

The slag was obtained from BCL Limited which operates a nickel copper flash smelting

furnace (Figure 5.1) at Selebi-Phikwe yielding high-grade sulphide matte, consisting of

nickel (Ni), copper (Cu) and cobalt (Co) for further refining; and large volumes of slag which

pass through a cleaning furnace before being crushed and discarded (Malema & Legg, 2006).

Slag was sampled in triplicates and stored at room temperature.

5.2.2 Particle size distribution

Due to the large size of the particles, the slag was initially pulverised. The particle size

distribution of BCL slag sample was then determined using the Microtrac S3500 laser

diffraction analyser.

5.2.3 Scanning electron microscopy (SEM)

The surface morphology of the BCL slag before and after leaching was established using

scanning electron microscopy (SEM) and the elemental distribution was determined by

energy dispersion microanalysis (EDS). For the sample preparation method, BCL slag

samples were treated with a carbon coat and subsequently studied in a scanning electron

microscope TESCAN equipped with EDX software; and operating conditions as shown in

Table 5.1.

109

Table 5-1: SEM Tescan EDX measurement specification

Parameter Condition

Resolution In high vacuum mode (SE0

In medium, low vacuum

mode BSE

3.0 nm at 30 kV 3.5 nm at 30 kV

Working vacuum High vacuum

Medium vacuum mode

Low vacuum mode

< 9 X10⁺ᶟ Pa

3 - 150 Pa 3 - 500 Pa

Electron optics working

modes

Resolution, Depth, Field, Wield Field, Channelling

Magnification

In low vacuum

1.5 to 1 000 000x in the continual wide field mode

6.5x to 1 000 000x

Maximum field of view

In high vacuum mode (SE0 In low vacuum mode

87.0 mm

19.9 mm

Accelerating voltage 200 V to 30 kV

Electron gun Tungsten heated cathode

Probe current 1 Pa to 2 uA

Scanning speed From 20 ns to 10 ms per pixel adjustable in steps or continuously

Image size Up to 8.192 x 8 192 pixel in 16-bit quality, size is adjustable separately for live image (in 3 steps)

and for saved images (in 10 steps), for square and rectangular 4:3

or 2:1 image shapes

5.2.4 Microwave digestion of raw slag for ICP-OES analysis

The Mars 6-Microwave digestion system was employed using the EasyPrep plus with the

operating conditions as stated in Table 5.2. The slag samples (triplicates) were dissolved by

acid digestion thus increasing the sample solubility, releasing the metals into solution for

quantification.

Table 5-2: Microwave digestion specification

Parameters Conditions

Power

Ramp time

Hold time

Temperature

Pressure

Stages

Weight

Volume of acids

400-1800 Watts

20 min

15 min

200 °C

800 bar/sec

1

0.5 g in 10 ml of acid

3 ml HNO3

2 ml HCL

5 ml HF

.

110

5.2.5 Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis

The ICP-OES (Spectro Acros) spectrometer was used to characterize and quantify the

chemical composition of the raw slag material before leaching; and the default operating

conditions as stated in Table 5.3. A concentration of 500 ppb, 100 ppb and 100 ppm multi-

element standards were prepared and made up to 50 ml in a volumetric flask using 1 % ACE

HNO3. The 500 ppb multi-element standard was the used to prepare standards 0.5 ppm, 100

ppb; which were then used to prepare 0.1 ppm and 100 ppm; which was finally used to

prepare 1 ppm, 5 ppm, 10 ppm, 20 ppm and 40 ppm. The blank was prepared using the acids

employed for digesting.

Table 5-3: ICP-OES measurement specifications for slag analysis


Generator parameters Plasma power

Pump speed

Coolant gas flow Auxiliary gas flow

Nebulizer gas flow

Global preflush parameters Fast Speed

Total

Normal speed

Rinse parameters

Rinse time Pump speed

Elements analysed

Analyte and wavelength

1400 Watts

30 Rpm

13 L/ min 2 L/min

0.95 L/min

63 Rpm

45 s

30 Rpm

10 s 30 s

Cu 224.700 Ni 227.021

Zn 202.613

Co 230.786 Fe 259.941

Al 176.641 Mg 285.213

Ca 315.887

Cr 205.618 Mn 257.611

Si 212.412

Ti 323.452

111

5.2.6 X-ray fluorescence (XRF) analysis

The chemical composition of the solid slag before and after bioleaching (residue) was

determined using X-ray fluorescence spectrometer (XRF) Rigaku ZXS Primus II with semi-

quantitative (SQX) analysis software by Scatter fundamental parameter (FP) method (Table

5.4).

Table 5-4: ZXS Primus II measurement specification

X-ray source X-ray tube End window type Rh Target 4kW or 3kW

High Voltage Generator High Frequency Inverter type Maximum Rating

4 kW, 60 kV-150 mA

Optics Maximum

sample Size

Ø51 mmx30 mm (H)

Primary Beam Filter 4 Filters (Al,Al-2,Cu,Zr)

Diaphragm 6 position Automatic Exchanger

(Ø 35, 30, 20, 10, 1, 0.5mm)

Crystal

Exchanger

10 position Automatic Exchanger

Counting Detector Heavy Elements: SC

Light Elements: F-PC

Components

analysed

Elements Na2O

MgO

Al2O3

Cr2O3

Fe2O3

Co2O3

SiO2

P2O5

K2O

TiO2

NiO

CuO

CaO

Zn

Mn

Pb

Rb

Cl

V

S

112

Sample preparation and analysis:

10g of the sample was pulverised to -38µm and mixed with a binder (30% Sasol wax)

The mixture was placed in an aluminium cup

Then placed on the pelletiser and pressed to a pressure of 20kpa

The pellet was placed in the oven for 1hr at 100°C to dry off prior to analysis

The calibration of the instrument was confirmed using certified machinery standards

The sample was then collected and loaded for analysis

5.2.7 X-ray powder diffraction (XRD) analysis

The mineralogical phase/s of the slag before and after bioleaching was determined using X-

ray Powder Diffraction spectrometer (XRD) RigakuUltimalV equipped with PDXL analysis

software. The specification used to run the experiment is displayed in Table 5.5.

Table 5-5: XRD RigakuUltimalV measurement specification

X-Ray 40 kV , 30 mA Scan speed /

Duration time

2.0000 deg./min.

Goniometer Step width 0.0200 deg.

Attachment - Scan axis 2theta/theta

Filter K-beta filter Scan range 3.0000 - 90.0000 deg.

CBO selection slit - Incident slit Open

Diffrected beam

mono.

Fixed Monochro.(U4) Length limiting slit -

Detector Scintillation counter Receiving slit #1 2/3deg.

Scan mode CONTINUOUS Receiving slit #2 Open

Sample preparation analysis:

The calibration of the instrument was confirmed using certified machinery standards

The sample was pulverised to -38µm

The sample was placed on the aluminium sample holder and flattened using a glass

slide

The sample was then mounted on the goniometer for analysis

The collected spectrum was analysed using the PDXL software loaded with ICDD

(International Centre for Diffraction Data) pdf card

113

5.2.8 Inductively coupled plasma mass spectrometry (ICP-MS)

To determine the concentration of elements in solution after leaching in addition providing a

broader range of data and isotopic information of the leachate, the Perkin Elmer SCIEX-

ELAN 6100 ICP-MS (Table 5.6) was employed (Table 5.7).

Table 5-6:Perkin- Elmer SCIEX-ELAN 6100 ICP-MS measurement specifications


RF power

Plasma gas flow

Nebulizer gas flow

Lense voltage

Pulse stage voltage

Discrimination threshold

AC rod offset

Sweeps/Reading

Replicates

Read Delay

Delay and analysis speed

Wash

Wash speed

1350 Watts

1.2-1.5 L/min

1.5 L/min

14 Voltage

1050 Volts

30m Volts

-12 Volts

20

3

15 sec

-24 (± rpm)

40 (sec)

-48 (± rpm)

Table 5-7: Elements of interest for ICP-MS analysis

Element Isotope mass

Mg

Fe

Fe

Co

Ni

Ni

Cu

Cu

Zn

Zn

S

Ca

Al

Ga

Lu

Bi

24

54

56

59

58

60

63

65

64

66

32

40

27

69

175

209

114

5.2.9 Standard solution preparation:

5.2.9.1 Internal standard

To minimize instrumental and signal intensity drifts internal standards were prepared. One

had to validate that elements used for the internal standard preparation were not naturally

occurring in the sample matrices. Subsequently, a concentration of 1000 ppm 69Ga, 175Lu and

209Bi elements was used to prepare 10 ppm internal standards in 1 % nitric acid. Considering

that internal standard elements should be as close as possible to the masses of the analytes of

interest, and that the ionization potential should be similar to that of the analytes of interest;

gallium (Ga69) corrections were used.

5.2.9.2 Calibration standards

Calibration standards were necessary for constructing a multipoint standard curve that would

cover the concentration scope of the analytes expected in the samples. Intermediate standards

were prepared by using 1000 ppm standard solution to prepare 1 ppm/1000 ppb intermediate

standards in 1 % nitric acid. Calibration standards (Table 5.8) were then conducted by

preparing dilutions of the intermediate standard with 1% nitric acid and 0.5 ml internal

standard to produce a final concentration of internal standards (0 ppb, 1 ppb, 5 ppb, 10 ppb,

20 ppb, and 40 ppb).

Table 5-8: Calibration standard preparation specification for ICP-MS

Standard St0 St1 St2 St3 St4 St5

Concentration 0 1 ppb 5 ppb 10 ppb 20 ppb 40 ppb

Intermediate Standard 0 0.05 ml 0.25 ml 0.5 ml 1 ml 2 ml

Internal Standard 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml

HNO3 49.5 ml 49.45 ml 49.25 ml 49 ml 48.5 ml 47.5 ml

Total 50 ml 50 ml 50 ml 50 ml 50 ml 50 ml

5.2.10 Atomic absorption spectroscopy (AAS)

As an alternative to the ICP-MS method, due to polyatomic interferences of different isotopes

produced by oxygen, argon and calcium on the iron signal the Thermo Scientific ICE 3300

Series Atomic Absorption Spectrometer was employed to determine the concentration of the

iron elements in solution(leachate). Acetylene gas was used as a fuel while the oxygen

present in air was blown using a volumetric pump to produce the flame for atomisation. For

the analysis in order to accommodate the iron concentrations, standards were prepared from

1.25 ppm to 5 ppm for low concentrations and 62.5 ppm to 1000 ppm for high concentrated

115

solutions. The wavelength used for analysis was Fe 238.2047 while the calibration was the

normal linear least squares with the fitting set to R2 of 0.995.The standards used were of high

purity and supplied by associated chemical enterprises (ACE).

5.2.11 Bioleaching experiment of mineral sample (BCL slag)

A pre-growth approach was implemented, which entailed the cultivation of microbial cultures

in mineral salt media in the absence of choline based ionic liquids / glucose and slag material,

enabling the bacterial cultures to initiate and sustain optimum leaching conditions. A standard

method was employed where mineral salt media was prepared as per Table 5.9 with 5 g/l of

the respective ionic liquid and adjusted to pH 2 (0.1N HCL/0.1N NaOH) before sterilizing at

121 °C for 15 minutes. Employing 250 ml scotts bottles, a concentration of 9 g/l of sterilized

BCL slag was prepared in 150 ml of mineral salt media. The cooled media was then

inoculated with 10 % v/v of the corresponding pre-grown culture, with the exception of the

control which comprised of mineral salt media without bacteria. The flasks were incubated in

an orbital shaker at temperature of 37 °C and agitation speed of 120 rpm, maintaining a

homogenous mixture. Sampling was conducted daily using disposable sterilized pipettes.

Throughout the experiment pH and absorbance at 600 nm (Thermo Spectronic BioMate

3UV-Visable spectrophotometer) was measured. The pH range monitored was from pH 2 to

pH10. Upon the conclusion of the experiment the quantity of metal release/bioleached in the

culture medium was quantified by ICP-MS and AAS. The experiment was concluded on the

onset of the cell death phase. The solid residue before and after bioleaching was characterised

by SEM, XRF and XRD. The study was conducted in triplicates to obtain representative

figures of the samples.

Table 5-9: Formulation of mineral salt medium

Reagent Formula Quantity(g/l)

Dipotassium hydrogen phosphate

Potassium dihydrogen phosphate

Magnesium sulphate heptahydrate

Sodium chloride

Ferrous sulphate heptahydrate

Ammonium nitrate

Calcium chloride

Choline lactate

K2HPO4

KH2PO4

MgSO4· 7H2O

NaCl

FeSO4 · 7H2O

NH4NO3

CaCl2 · 2H2O

C5H14NO(+) + C3H5O3(-)

1.73

0.68

0.1

4

0.03

1

0.02

2

116


5.3.1 Particle size distribution

10 100 1000

0

20

40

60

80

100

% P

ass

ing

Size (Um)

Figure 5-2: Particle size distribution of BCL slag before bioleaching process

The particle size distribution curve revealed that 80 % passing particle size using the present

experiment were 100 μm.

5.3.2 Slag morphology

Figure 5-3: SEM of raw BCL slag (Before bioleaching process) indicative of an amorphous structure

117

Figure 5-4: EDS spectrum of BCL slag as directed in Figure 5.3

118

Figure 5-5: Scanning electron photomicrograph of BCL slag after bioleaching in mixed culture

Figure 5-6: Scanning electron photomicrograph of BCL slag after bioleaching in mixed culture

119

The SEM micrograph of raw BCL slag (Figure 5.3) directed according to the EDS spectrum

(Figure 5.4) revealed that the material was composed of heterogeneous particles consisting of

different size, shape and texture; and a surface chemistry consisting of high amounts of

sulphur and iron. The morphology of individual bacterial strains in each IL was studies but to

give an overview of the general morphology of bioleaching activity only the leaching activity

of slag in mixed cultures will be mentioned. Subsequent to bioleaching the slag surface in

mixed cultures indicated spherical ball shaped structures and thin lath needle like structures

(Figure 5.5) also displaying platys shaped structure with fractures and crevices (Figure 5.6)

suggestive of bioleaching activity. The rod-like structures were indicative of bacilli shaped

microorganisms and flaked particles on the slag surface characteristic of bioleaching end

products. It was evident from the surfaces of BCL slag material that samples had transformed

from an amorphous structure prior to bioleaching to a crystalline structure after bioleaching

activity.

5.3.3 XRF of BCL slag before and after leaching

The chemical composition of raw BCL slag (before bioleaching) and bioleaching residue

(after bioleaching) was studied (Tables 5.10 -5.16) to determine changes in the chemistry of

the slag as a result of bioleaching processes.

Table 5-10: XRF of slag before and after leaching with Bacillus aryabhattai in ILs vs. glucose

Component Na2O MgO Al2O3 SiO2 P2O5 S Cl K2O

Mass

perc

en

tag

e

Feed 0.8 1.9 6.2 24.0 0.04 2.0 0.01 0.9

Residue CLe(2) 3.1 0.8 5.0 14.3 15.1 1.5 1.3 1.6

CLa(2) 1.0 0.4 6.8 13.8 20.0 2.1 1.23 1.8

CDP(2) 1.0 0.3 5.3 8.7 27.7 1.5 0.8 2.6

CCh(2) 1.3 0.7 5.2 11.9 19.4 1.4 0.8 2.2

G(2) 1.3 0.4 5.8 11.3 21.3 1.5 1.0 2.2

Component TiO2 Cr2O3 Fe2O3 Co2O3 NiO CuO CaO

Mass

perc

en

tag

e

Feed 0.3 0.1 59.9 0.2 0.3 0.4 2.8

Residue CLe(2) 0.2 0.1 54.1 0.1 0.3 0.4 2.0

CLa(2) 0.3 0.1 50.50 0.1 0.2 0.3 1.4

CDP(2) 0.3 0.1 49.7 0.1 0.2 0.4 1.2

CCh(2) 0.2 0.1 54.3 0.1 0.2 0.5 1.7

G(2) 0.2 0.1 52.5 0.1 0.3 0.4 1.6

120

Table 5-11: XRF of slag before and after leaching with Raoultella ornithinolytica in ILs vs. glucose

Table 5-12: XRF of BCL slag after leaching with Bacillus sp. in ILs vs. glucose


Mass

Perc

en

tag

e

Feed 0.8 1.9 6.2 24.0 0.04 2.0 0.01 0.9

Residue CLe(12) 2.9 0.8 5.0 14.3 15.2 1.5 1.2 1.6

CLa(12) 0.9 0.4 7.2 13.3 21.2 2.4 1.0 1.8

CDP(12) 1.0 0.3 5.5 9.0 28.0 1.6 0.8 2.5

CCh(12) 1.2 0.6 4.9 11.5 21.0 1.2 0.9 2.4

G(12) 1.2 0.6 5.6 12.9 19.8 1.5 0.8 2.1


Mass

perc

en

tag

e

Feed 0.3 0.1 59.9 0.2 0.3 0.4 2.8

Residue CLe(12) 0.2 0.1 55.0 0.1 0.2 0.4 2.0

CLa(12) 0.2 0.1 49.1 0.1 0.2 0.4 1.3

CDP(12) 0.2 0.1 49.0 0.1 0.2 0.4 1.2

CCh(12) 0.2 0.1 53.6 0.1 0.2 0.4 1.7

G(12) 0.3 0.1 52.7 0.1 0.2 0.4 1.7

Table 5-13: XRF of BCL slag before and after leaching with Bacillus thuringiensis in ILs vs. glucose


Mas

s

Per

centa

ge

Feed 0.8 1.9 6.2 24.0 0.04 2.0 0.01 0.9

Residue CLe(16) 2.7 0.7 4.9 14.6 14.8 1.6 1.1 1.6

CLa(16) 0.8 0.4 7.3 14.1 21.1 2.1 1.0 1.8

CDP(16) 0.9 0.3 5.5 7.7 29.6 1.5 0.8 2.7

CCh(16) 1.4 0.7 5.1 12.4 19.5 1.4 0.9 2.2

G(16) 1.0 0.5 5.4 10.4 22.3 1.4 0.8 2.3


Mas

s

per

centa

ge

Feed 0.3 0.1 59.9 0.2 0.3 0.4 2.8

Residue CLe(16) 0.0 0.1 55.0 0.1 0.3 0.4 2.0

CLa(16) 0.4 0.1 48.8 0.1 0.3 0.4 1.3

CDP(16) 0.3 0.1 48.8 0.1 0.2 0.4 1.1

CCh(16) 0.3 0.1 53.3 0.2 0.2 0.4 1.8

G(16) 0.4 0.2 53.1 0.2 0.3 0.4 1.4


Mass

perc

en

tag

e

Feed 0.8 1.9 6.2 24.0 0.04 2.0 0.01 0.9

Residue CLe(9) 1.3 0.7 3.7 13.6 7.7 1.8 0.8 1.2

CLa(9) 1.1 0.5 7.7 17.8 16.0 2.3 1.1 1.5

CDP(9) 0.8 0.2 4.7 6.6 32.7 1.3 0.8 3.0

CCh(9) 1.3 0.6 4.7 11.1 21.6 1.3 0.8 2.3

G(9) 1.2 0.6 5.2 12.7 19.0 1.5 0.6 1.9


Mass

perc

en

tag

e

Feed 0.3 0.1 59.9 0.2 0.3 0.4 2.8

Residue CLe(9) 0.2 0.2 65.7 0.2 0.3 0.5 2.2

CLa(9) 0.3 0.1 49.0 0.1 0.2 0.4 1.6

CDP(9) 0.3 0.1 47.9 0.00 0.2 0.4 0.9

CCh(9) 0.2 0.1 53.6 0.11 0.2 0.4 1.8

G(9) 0.2 0.1 54.6 0.1 0.3 0.5 1.6

121

Table 5-14: XRF of BCL slag before and after leaching with Pseudomonas moraviensis in ILs vs. glucose


Mass

Perc

en

tag

e

Feed 0.8 1.9 6.2 24.0 0.04 2.0 0.01 0.9

Residue CLe(19) 4.0 0.7 4.7 13.5 17.4 1.4 1.8 1.8

CLa(19) 0.9 0.4 6.7 14.9 19.1 2.3 1.1 1.7

CDP(19) 1.1 0.2 4.6 7.0 31.1 1.1 0.9 2.8

CCh(19) 1.3 0.5 4.6 11.0 22.4 1.3 0.9 3.7

G(19) 0.8 0.6 4.9 13.2 20.1 1.7 0.7 1.7


Mass

perc

en

tag

e

Feed 0.3 0.1 59.9 0.2 0.3 0.4 2.8

Residue CLe(19) 0.3 0.1 51.9 0.0 0.2 0.3 1.7

CLa(19) 0.3 0.1 50.3 0.1 0.3 0.4 1.3

CDP(19) 0.2 0.1 49.2 0.0 0.2 0.4 1.0

CCh(19) 0.3 0.1 52.5 0.1 0.2 0.4 1.6

G(19) 0.3 0.2 53.8 0.0 0.2 0.3 1.5

Table 5-15: XRF of BCL slag before and after leaching with a mixed culture in ILs vs. glucose


Mass

Perc

en

tag

e

Feed 0.8 1.9 6.2 24.0 0.04 2.0 0.0.1 0.9

Residue CLe(All) 3.2 0.7 4.6 12.9 16.7 1.4 1.4 1.7

CLa(All) 1.1 0.5 7.7 14.7 19.9 2.1 1.4 1.8

CDP(All) 1.1 0.2 5.1 7.7 29.8 1.3 0.9 2.7

CCh(All) 1.2 0.7 5.0 11.9 19.9 1.5 1.0 2.2

G(All) 1.3 0.5 5.2 10.7 21.0 1.4 0.9 2.2


Mass

perc

en

tag

e

Feed 0.3 0.1 59.9 0.2 0.3 0.4 2.8

Residue CLe(All) 0.3 0.1 54.3 0.1 0.2 0.4 2.0

CLa(All) 0.3 0.1 48.1 0.1 0.3 0.4 1.5

CDP(All) 0.0 0.1 49.1 0.0 0.2 0.4 1.1

CCh(All) 0.0 0.1 53.9 0.1 0.3 0.4 1.7

G(All) 0.2 0.1 54.1 0.1 0.3 0.4 1.7

122

Table 5-16: XRF of BCL slag before and after leaching with no bacteria in ILs vs. glucose

Component Na2O MgO Al2O3 SiO2 P2O5 S Cl K2O M

ass

Per

centa

ge

Feed 0.8 1.9 6.2 24.0 0.04 2.0 0.01 0.9

Residue CLe(0) 3.3 0.7 5.1 13.5 17.0 1.5 1.5 1.7

CLa(0) 1.0 0.4 7.0 11.6 22.6 2.6 1.3 1.8

CDP(0) 1.1 0.4 6.0 8.5 29.7 1.7 0.8 2.6

CCh(0) 1.1 0.8 5.5 14.3 15.1 1.6 0.7 1.7

G(0) 1.2 0.5 5.3 12.5 20.00 1.5 0.9 2.1


Mas

s

per

centa

ge

Feed 0.3 0.1 59.9 0.2 0.3 0.4 2.8

Residue CLe(0) 0.2 0.2 52.3 0.1 0.3 0.4 2.0

CLa(0) 0.3 0.1 49.1 0.1 0.2 0.3 1.3

CDP(0) 0.2 0.1 47.0 0.1 0.2 0.4 1.1

CCh(0) 0.3 0.2 55.7 0.2 0.2 0.5 2.1

G(0) 0.2 0.1 53.0 0.2 0.3 0.4 1.6

The presence of metals Zn, Mn, V, Pb, Rb elements were also detected in the XRF analysis

but were not reported due to low concentrations. In agreement with Hocheng et al. (2014)

iron characterized as Fe2SiO3 was a major component of BCL slag, indicative of a non-

ferrous smelting process producing an iron-silicate based slag. The profusion of iron in BCL

slag was suggestive of the potential of BCL slag to function as energy source for iron

oxidizing bacteria in the bioleaching process (Isildar et al., 2016). The changes in elemental

concentrations before and after bioleaching were also indicative of bioleaching activity.

5.3.4 ICP-OES raw slag elemental analysis

Table 5-17: ICP-OES Characterization of raw BCL slag

Analyte Wavelength Average concentration (mass %) Standard deviation

Cu 224.700 0.0985 0.00041

Ni 227.021 0.0598 0.00019

Zn 202.613 0.00601 0.00001

Co 230.786 0.0343 0.00016

Fe 259.941 9.47 0.00025

Al 176.641 0.280 0.00075

Mg 285.213 0.0230 0.00014

Ca 315.887 0.297 0.00118

Cr 205.618 0.0101 0.00013

Mn 257.611 0.0106 0.00010

Si 212.412 0.00263 0.00023

Ti 323.452 0.0380 0.00016

Using the XRF results as a qualitative study, the ICP-OES study (Table 5.10) was conducted

to determine the true concentrations of the elements of interest, namely base metals and

123

associated metals in raw slag. In correlation with the XRF results, the elemental analysis of

BCL slag (Table 5.17) indicated Iron (Fe) to be in the highest concentration.

Additional elements confirmed in the sample included calcium (Ca), aluminium (Al), copper

(Cu), nickel (Ni), titanium (Ti), cobalt (Co), magnesium (Mg), manganese (Mn), chromium

(Cr) , zinc (Zn) and silicone (Si).

5.3.5 XRD analysis of BCL slag before and after leaching

The mineralogical study was employed, obtaining qualitative data on the effects of leaching

on raw BCL slag (Figure 5.7) with Bacillus aryabhattai (Figure 5.8), Raoultella

ornithinolytica (Figure 5.9), Bacillus sp. (Figure 5.10), Bacillus thuringiensis (Figure 5.11),

Pseudomonas moraviensis (figure 5.12), mixed cultures (Figure 5.13) and in the absence of

bacteria (Figure 5.14) in choline based ionic liquids vs. conventional glucose nutrient source.

Figure 5-7: Raw BCL slag sample before bioleaching process (feed)

124

Figure 5-8: BCL slag subsequent to bioleaching with Bacillus aryabhattai (2) in choline based ILs vs.

glucose

Figure 5-9: BCL slag subsequent to bioleaching with Raoultella ornithinolytica (9) in choline based ILs vs.

glucose

125

Figure 5-10: BCL slag subsequent to bioleaching with Bacillus sp. (12) in choline based ILs vs. glucose

Figure 5-11: BCL slag subsequent to bioleaching with Bacillus thuringiensis (16) in ILs vs. glucose

126

Figure 5-12: BCL slag subsequent to bioleaching with Pseudomonas moraviensis (19) in choline based ILs

vs. glucose

Figure 5-13: BCL slag subsequent to bioleaching with a mixed culture (All) of bacteria in choline based

ILs vs. glucose

127

Figure 5-14: BCL slag subsequent to leaching in the absence (0) of bacteria in choline based ILs vs.

glucose

The XRD peaks of raw BCL slag before the bioleaching process (Figure 5.7) indicated the

presence of mineralogical phase fayalite (Fe2SiO4) a major iron carrier and additional phases

olivine ((MgFe)2SiO4) and quartz (SiO2) with sulphide minerals specified by chalcopyrite

(CuFeS2), pyrite(FeS2), pyrrhotite (FeS) and vaesite (NiS2).

This was suggestive of a nonferrous slag, described by Yang et al. (2010) as by-products of

pyro-metallurgy copper processing for concentrated copper sulphide products. Essentially

pyrite oxidation gives rise to iron-oxide occurring in silica, producing a crystal structured

fayalite (Fe2SiO4) slag occasionally containing a small amorphous silica (SiO2) phase. The

mineralogical studies were conclusive of slag material, characteristic of metal oxides and

silicon dioxide including metal sulphides (Bazan et al., 2015).

In contrast to raw BCL slag, a zinc silicate (Zn2SiO4) phase in the region 42.9Å was

identified, including the release of a Q-quartz (SiO2) phase in the region 27.9Å from the slag

matrix by Pseudomonas moraviensis (Figure 5.12) bacteria in choline levulinate (CLe),

signalizing the destruction of the slag mineral giving rise to new phases.

5.3.6 Bacterial growth studies

The growth curves of bacterial cultures namely Bacillus aryabhattai (Figure 5.15), Raoultella

ornithinolytica (Figure 5.16), Bacillus sp.(Figure 5.17), Bacillus thuringiensis (Figure 5.18),

128

Pseudomonas moraviensis (figure 5.19), mixed cultures (Figure 5.20) and in the absence of

bacteria (Figure 5.21) in choline based ionic vs. conventional glucose nutrient source were

investigated. This was an essential study for determining the effect of choline based ILs on

the growth rates of bacteria, required for optimal leaching.

Figure 5-15: Growth curve of Bacillus aryabhattai in choline based ILs vs. glucose for the recovery of base

metals at a speed of 120 rmp and temperature of 37 °C (3 weeks)

Figure 5-16: Growth curve of Raoultella ornithinolytica in choline based ILs vs. glucose for the recovery

of base metals at a speed of 120 rmp and temperature of 37 ⁰C (3 weeks)

129

Figure 5-17: Growth curve of Bacillus sp. in choline based ILs vs. glucose for the recovery of base metals

at a speed of 120 rmp and temperature of 37 °C (3 weeks)

Figure 5-18: Growth curve of Bacillus thuringiensis in choline based ILs vs. glucose for the recovery of

base metals at a speed of 120 rmp and temperature of 37 °C (3 weeks)

130

Figure 5-19: Growth curve of Pseudomonas moraviensis in choline based ILs vs. glucose for the recovery

of base metals at a speed of 120 rmp and temperature of 37 °C (3 weeks)

Figure 5-20: Growth curve of a mixed culture of bacteria in choline based ILs vs. glucose for the recovery

of base metals at a speed of 120 rmp and temperature of 37 °C (3 weeks)

131

Figure 5-21: Growth curve in the absence of bacteria in choline based ILs vs. glucose for the recovery of

base metals at a speed of 120 rmp and temperature of 37 °C (3 weeks)

With the exception of Bacillus aryabhattai (Table 5.15) which thrived in glucose nutrients,

all bioleaching bacteria indicated optimal growth in the presence of choline lactate (Figure

5.16-5.21). The growth pattern of bioleaching bacteria in the presence of choline lactate

presented logarithmic growth, thus indicating choline lactate as a preferred nutrient source to

the conventional glucose source for optimal bacterial growth during bioleaching processes.

Choline lactate and glucose based bacteria were still growing optimally at three weeks

suggesting their potential for further growth past the three week time frame. This was

significant considering the study was based on a closed system where no continuous

inoculation was performed. Trailing behind choline lactate, ionic liquid choline levulinate

also supported the growth of the bioleaching bacteria, however bacterial strains reached the

death phase within two weeks of leaching (Figure 5.16-5.19). The absence of a lag phase in

choline lactate and choline levulinate was noteworthy, demonstrating the rapid adaption of

the bacterium in these ionic liquids, a clear indication of the success of the pre-growth

approach in establishing and maintaining favourable bioleaching conditions (Isildar et al.,

2016). In comparison to the application of mixed bacterial cultures (Figure 5.20), all the

bacterial strains with the exception of Bacillus arybhattai indicated more favourable growth

for bioleaching processes when applied individually. The poor growth in the mixed culture

can be attributed to interspecific competition resulting in the competition for nutrients.

132

No significant growth was indicated in the control (Table 5.21) however, the slight signals

detected could be the result of light refraction errors related to the slag particulates. Errors

due to light refraction, reflection and scattering are known to affect the absorption in the

absence of any true absorption.

5.3.7 pH studies

The pH profiles of bioleaching bacteria in choline based ILs vs. glucose sources were

investigated (Figure 5.22-5.28) for the elucidation of pH effects on bioleaching rates.

Figure 5-22: The pH profile of BCL slag bioleaching by Bacillus aryabhattai in choline based ILs vs.

glucose

Figure 5-23: The pH profile of BCL slag bioleaching by Raoultella ornithinolytica in choline based ILs vs.

glucose

133

Figure 5-24: The pH profile of BCL slag bioleaching by Bacillus sp. in choline based ILs vs. glucose

Figure 5-25: The pH profile of BCL slag bioleaching by Bacillus thuringiensis in choline based ILs vs.

glucose

134

Figure 5-26: The pH profile of BCL slag bioleaching by Pseudomonas moraviensis in choline based ILs vs.

glucose

Figure 5-27: The pH profile of BCL slag bioleaching by a mixed culture in choline based ILs vs. glucose

135

Figure 5-28: The pH profile of BCL slag bioleaching without bacteria in choline ILs vs. glucose

The pH of the solution was initially adjusted to pH 2 for optimal bioleaching conditions

(Olubambi et al., 2008; Shaikh et al., 2010). Within the first week of leaching across all the

bacterial strains, the samples indicated an increase in their pH values, with a maximum pH of

3.5 to 3.7 indicated in all choline levulinate (CLe) nutrient sources and pH 3.9 noted for the

Pseudomonas moraviensis strain in choline chloride (CCh). Week two introduced varying pH

profiles with some samples indicating further increase in pH and others initiating a decrease

in the pH value. With the exception of glucose (G), towards week three a decreasing pattern

was indicated across the pH profiles of all the bacterial strains, with the lowest pH values

noted in choline dihydrogen phosphate (CDP) nutrient sources, which indicated a pH range of

2.6 to 2.8. The increasing pH at the commencement of the bioleaching process may have

been the result of the alkalinity of the added slag material but also suggests proton

consumption as a result of acid-leaching and ferrous-oxidation, subsequently followed by

decreasing pH resulting from sulphuric acid production via the oxidation of sulphur by

bioleaching microorganisms (Ma et al., 2017).

5.3.8 Bioleaching Studies

The efficiency of halophilic bacteria in choline based ILs as an alternative nutrient source to

conventional glucose sources for bioleaching was investigated (Figure 5.29-5.35).

136

Figure 5-29: The recovery rates of base metals from BCL slag using Bacillus aryabhattai in choline ILs vs.

glucose

Figure 5-30: The recovery rates of base metals from BCL slag using Raoultella ornithinolytica in choline

ILs vs. glucose

137

Figure 5-31: The recovery rates of base metals from BCL slag using Bacillus sp. in choline based ILs vs.

glucose

Figure 5-32: The recovery rates of base metals from BCL slag using Bacillus thuringiensis in choline

based ILs vs. glucose

138

Figure 5-33: The recovery rates of base metals from BCL slag using Pseudomonas moraviensis in choline

based ILs vs. glucose

Figure 5-34: The recovery rates of base metals from BCL slag using a mixed culture in choline based ILs

vs. glucose

139

Figure 5-35: The recovery rates of base metals from BCL slag in choline based ILs vs. glucose (absence of

bacteria)

Choline lactate produced the most optimal base metal recovery, with the most efficient

bioleaching rates occurring at initial pH 2. According to Kaksonen et al. (2016) and

Sarcheshmehpour et al. (2009), the biochemical activity of acid on slag can be highly

efficient as sulphides can occur at insignificant levels in slag minerals, initiating the recovery

of ferric iron sulphates in solution. Conventional glucose sources produced iron recoveries of:

13 % (Bacillus aryabhattai), 13 % ( Raoultella ornithinolytica), 13 % (Bacillus species.), 14

% (Bacillus thuringiensis) and 20 % (mixed cultures) in comparison with IL choline lactate

which indicated significant iron recoveries of: 65 % (Bacillus aryabhattai), 69 % (Raoultella

ornithinolytica), 71 % (Raoultella ornithinolytica), 74 % (mixed cultures with) with the

highest dissolution efficiency of 97 % (Bacillus species) and 97 % (Bacillus thuringiensis).

Kaksonen et al. (2016) also reported high recoveries of iron due to the production of Fe3+

and Fe2+ ions stimulated by a sequence of biochemical reactions, initiated by the oxidation of

pyrite. In addition Štyriaková et al. (2003) reported high iron recovery rates, essentially

stating iron as a vital element for the metabolic activity of Bacillus species, which also

participate in the attack of Silicates.

Though favourable growth was not observed in choline dihydrogen phosphate (CDP),

choline dihydrogen phosphate indicated moderate zinc recovery rates, such as 26 %

140

(Pseudomonas moraviensis), 28 % (mixed cultures), 31 % (Bacillus species) and 31 %

(Bacillus thuringiensis). Ionic liquid choline levulinate also produced moderate zinc (Zn)

recoveries in Bacillus sp. and Bacillus thuringiensis. The zinc recoveries were validated by

the appearance of the zinc silica phase after bioleaching processes (Table 5.8-5.14).

In accord with Kaksonen et al. (2016) the initial dissolving of acid in solution at room

temperature increased the solubility of fayalite (Fe2SiO4) additional silica and metal oxide

phases. This is was due to the yield of ferrous (Fe2+) by fayalite which oxidised the oxygen in

the acid solution and thereafter was accelerated by iron oxidizing microbes. The fayalite

(Fe2SiO4) phase normally comprises of additional elements, which are dissolved in

association with fayalite (Kaksonen et al., 2016). The decrease in iron dissolution rates

towards the third week was due to ferric (Fe3+) ion precipitation (Abdollahi et al., 2014;

Córdoba et al., 2008).

The zinc recovery rates in the absence of optimal bacterial growth suggest the chemical

leaching of the zinc base metal by choline dihydrogen phosphate.

The low dissolution rates of the other base metal elements may be attributed to the

unavailability of sulphur in slag material, as slag minerals are inadequate as substrate material

for microorganisms thus requiring additional sulphur and ferrous (Kaksonen et al.,2016 ), and

the reduction of oxidizable ferrous (Fe2+). In addition Cordeba et al. (2008) suggested that

cumulative iron concentrations can cause minor drops in biological leaching kinetics related

to nucleating and precipitating jarosites. Hence the high iron dissolution rates caused

precipitating irons (ferric) in solution, ultimately leading to the passivation of the slag

minerals and affecting the dissolution rates of other elements. Alternatively solubilised

Silicates associated with fayalite (Fe2SiO4) and zinc silicate (Zn2SiO4), indicated by the

release of the quartz phase may have increased the leachate viscosity, affecting bioleaching

rates.

In comparison to the control (Table 5.35) representative of hydrometallurgical leaching,

metal recoveries increased slightly in some cases in the presence of bioleaching bacteria in

choline based ILs ;overall indicating competitive bioleaching rates. Furthermore, contrary to

common findings pure cultures indicated higher dissolution rates compared to mixed cultures.

The dissolution rates did not adhere to the conventional pattern of decreasing pH associated

with increasing dissolution rates, thus suggesting major extraction due to Fe3+ ions and other

contributing factors due to by-products formed by the metabolic break down of these ionic

141

liquids, possibly affecting bioleaching parameters namely pH associated with bio-

dissolution.

5.4 Conclusion

The BCL slag was confirmed to be an Iron-silicate-based slag typical of metal oxides and

silicon dioxides. Bioleaching activity by Raoultella ornithinolytica, Bacillus, Bacillus

thuringiensis, Pseudomonas moraviensis and mixed cultures was evident in the release of

quartz and structural changes confirmed by SEM analysis. With optimal bacterial growth and

pH conditions maintained at acidic levels, bacterial growth was catalysed in the presence of

choline ILs dramatically increasing the leaching efficiency of the major Iron element. In

comparison to the chemical leaching control, bioleaching in choline ILs indicated competitive

recoveries offering a novel, environmentally and economical friendly technology from Cu-Ni

smelter slag.

Acknowledgement


Foundation.

142

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CHAPTER SIX

6.0 GENERAL DISCUSSION AND CONCLUSION

6.1 GENERAL DISCUSSION

The volumes of metal value in slag are generally low and regarded to be uneconomical for

reprocessing using conventional extraction processes, hence the need for innovative

extraction technologies. The benefits of such methods lie in the prospective yields of metal

values of which the natural stockpiles are increasingly becoming depleted (Guezennec et al.,

2014). In contrast to traditional metal extraction methods, the commercialization of

bioleaching has progressed significantly, attributable to favourable processing economics and

reduced environmental challenges (Guezennec et al., 2014). As a result, the employment of

microorganisms for the recovery of metals has been researched as an eco-friendly and

economical alternate (Rawlings et al., 2007). One of the shortcomings impeding further

commercialization of these processes is the slow bioleaching kinetics related to metal

solubilisation. Thus, considerable research is required in improving the bioleaching kinetics

(Das, Ayyappan and Chaudhury, 1999). Considering the properties of ILs as designer

solvents and their applications as reaction media in catalytic applications, electrolytes for

metal extraction and bio-catalysis, ILs are regarded as potential contenders for improving the

slow bioleaching kinetics (Hou et al., 2013).

The selection of halophilic microorganisms was based on the unique biology of these

halophiles and their ability to produce enzymes that make them tolerant and adaptive to

heavy metal conditions, that would otherwise be toxic (Bahari et al., 2013; Hu et al., 2007;

Marandi et al., 2011; Pailan et al., 2015). Employing molecular biology techniques the study

undertaken to analyse the identification and characteristics of Gold 1 mine East Rand mine

sites revealed a microbial community of gram positive and gram negative bacillus

microorganisms, which were confirmed to be Bacillus aryabhattai, Raoultella

ornithinolytica, Pseudomonas moraviensis and Bacillus cereus group (Bacillus sp., Bacillus

thuringiensis). With the exception of Bacillus sp. as far as ones research there have been no

accounts of the application of bacterial isolates Raoultella ornithinolytica and Pseudomonas

moraviensis in bioleaching processes. Thus attesting to the fact that extreme environments

and/or waste sites do indeed harbour a diversity of microbes with unique properties.

The applications of 1H NMR ,13C NMR, and FT-IR techniques were vital for confirming the

chemistry of the synthesized choline derived ILs, which was required for establishing the

146

relationship between the structures and biological properties of these ILs. The study

confirmed the presence of methyl groups, alcohol and amine functional groups in all the

cation components of choline derived ionic liquids, confirming the presence of carbon and

nitrogen required for the biosynthesis of intermediate products for cell functioning in

developing microbial colonies (Pernak et al., 2007). Biocompatibility studies employing

these halophilic strains were indicative of exponential growth in ILs choline dihydrogen

phosphate, choline lactate, choline citrate, choline levulinate with poor growth noted across

choline citrate and choline tartarate. This was apparent in the lag phase of the growth curve in

choline tartarate impeding bacterial activity, thus alluding to its toxic nature. The anion

component and extended branching of the side chain of choline tartarate were found to have

contributed significantly to its toxicity thus producing poor biocompatibility (Gathergood,

Garcia & Scammels, 2014; Hou et al., 2013). However, with the exception of Bacillus

aryabhattai, halophilic bacterial strains Bacillus aryabhattai, Raoultella ornithinolytica,

Pseudomonas moraviensis and Bacillus sp. in choline based media were found to have a

metabolic advantage over conventional glucose. Ionic liquids choline dihydrogen phosphate,

choline lactate, choline citrate and choline levulinate were thus selected for further studies.

Slag characterization was valuable in producing information on the properties of the slag

material which was confirmed to mainly consist of metal oxides, silicon dioxides and metal

sulphides, further advancing the comprehension of the behavioural pattern of BCL slag under

bioleaching conditions. In addition chemical and phase analyses of BCL slag revealed the

heterogeneous nature of BCL slag material with the major element confirmed to be iron in

the form of fayalite (Fe2SiO4), olivine ((MgFe)2SiO4), chalcopyrite (CuFeS2), pyrite (FeS2)

and pyrrhotite (FeS).

The structural transformation of BCL slag was confirmed by the development of crystalline

particles typical of a quartz phase. The optimal growth of bioleaching bacteria, namely

Raoultella ornithinolytica, Bacillus species, Bacillus thuringiensis, Pseudomonas

moraviensis and mixed cultures suggested that choline lactate as carbon and nitrogen source

provides a metabolic advantage to conventional glucose sources, with the pre-growth

approach also contributing to maintaining optimal bioleaching environments and cell

viability. As a result, with optimal bacterial growth and pH conditions maintained at acidic

levels, 97% of iron recovery rates proved to be the highest recoveries in BCL slag which was

leached by Bacillus species and Bacillus thuringiensis catalysed by choline lactate as a

substrate. However, although optimal bacterial growth was not observed in choline

147

dihydrogen phosphates nutrients, its zinc recoveries were fairly significant. Thus alluding to

the selective nature of choline lactate and choline dihydrogen phosphate ILs for the recovery

of Fe and Zn metals. In comparison to the chemical leaching control, bioleaching in choline

ILs indicated competitive recoveries alluding to the potential of these IL based reactions as an

alternative to conventional hydrometallurgy methods.

6.2 CONCLUSION

The extraction of microbes from extreme environments presented a wealth of halophilic

microorganisms with unique properties and microbial activity. Choline base ionic liquid

choline dihydrogen phosphate, choline lactate, choline citrate and choline levulinate were

confirmed to be biocompatible substrates for the development of halophilic microorganisms;

with choline lactate substrates having a metabolic advantage over glucose carbon source.

Furthermore, the logarithmic growth and metabolic activity of bacteria species, namely

Raoultella ornithinolytica, Bacillus sp., Bacillus thuringiensis, Pseudomonas moraviensis

and mixed cultures in IL choline lactate was discovered to affect the bio-dissolution of Iron

atoms from the fayalite phase in BCL slag. The characterization of ILs for biocompatibility

studies was thus imperative for identifying process deficiencies which would assist in

optimizing bioleaching processes. It was thus considered key in designs of quaternary

ammonium based ILs for bioleaching processes, that limited branching of the side chain be

adhered to, promoting the attachment of liner alkyls to the amides . Furthermore, the addition

of an amide was suggested to increase the biocompatibility of the ILs.

Overall, the study could potentially contribute to the understanding of cell and biochemical

response to choline based ionic liquids, which could provide further direction and content on

the employment of these salts in the manipulation of proteins and metabolic pathways,

offering an economical and eco-friendly alternative for the optimal recovery of metal

values/base metals from Cu-Ni slags. Further studies to elucidate the leaching mechanism

should be conducted via the speciation of dissolved irons and characterization of by-products

from the metabolic breakdown of these ILs. Additional studies on the leaching parameters

such as contact time, temperature, particle size etc. could further contribute to the

optimization of this technology. Bioenergetics studies would provide vital data on the

biomass growth, substrate consumption (ILs), oxygen consumption and power time profiles

that would give insight into the metabolic action of the bacterial isolates in these ILs during

bioleaching processes.

148

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150

APPENDICES

Appendix 1: Bacillus aryabhattai strain 16S ribosomal RNA gene sequence

GTCGAGCGAACTGATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACC

TGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGA

TGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACG

GCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCA

GACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGA

GTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACGAGAGTAACTGCTCGTACC

TTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGC

GTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTC

AACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGG

TGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGC

GCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGT

TAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGA

CTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGA

AGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTG

ACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC

TTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGG

GGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGC

TGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTA

CATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACA

CCGCCCGKCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCTAGCCGCGTAA

G

Appendix 2: Raoultella ornithinolytica strain 16S ribosomal RNA gene sequence

GGGTGACGAGCGGCGGACGGGTGTAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAA

CGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACCTTCGGGCCTCATGCCATCAGATGTGC

CCAGATGGGATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGAT

GACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAAT

GGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGAG

GAGGAAGGCTTTAAGGTTAATAACCCTTGKYGATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGT

GCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCG

GTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCGAAACTGGCAGGCTAGAGTC

TTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAA

GGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG

TAGTCCACGCTGTAAACGATGTCGACTTGGAGGTTGTTCCCTTGAGGAGTGGCTTCCGGAGCTAACGCGTT

AAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGG

TGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAG

AGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATG

TTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTCGGCCGGGAACTCAAAGGA

GACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTAC

ACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCG

TAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCA

CGGTGAATACGTTCCCGGGCCTTGTACACACCGCCSGTCACACCATGGGAGTGGGYTGCAAAAGAAGTAGG

TAGCT

Appendix 3: Bacillus sp. 16S ribosomal RNA gene sequence

CGAGCGAATGGATTGAGAGCTTGCTCTCAAGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCA

TAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACTGCATGGTTCGAAATTGAAAG

GCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAA

CGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAG

CAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAARGCTTTCSGGTCGT

151

AAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGACCCCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTG

ACGGGAGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTT

CATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGTTTTATGAGATTAGCTCCACCTCGCGGTCTTGCA

GCTCTTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCAC

CTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTTAATGATGGCAACTAAGATCAAGGGTTGCGC

TCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCTCTCTGCTCCCG

AAGGAGAAGCCCTATCTCTAGGGTTTTCAGAGGATGTCAAGACCTGSTAAGGTTCTTCGCGTTGCTTCGAATTAA

ACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTC

Appendix 4: Bacillus thuringiensis strain 16S ribosomal RNA gene sequence

AGTCGAGCGATGGATTGAGAGCTTGCTCTCAWGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACC

TGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACTGCATGGTTCG

AAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACG

GCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCA

GACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGA

GTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCAC

CTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATAGTAGGTGGCAAGC

GTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTC

AACCGTGGAGGGTCATTGGAAACTGGGAGACTTGRRTGCMARAGAGGAAAGGGGAATTCCATGTGTAGCGG

TGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGC

GCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGT

TAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAMSSCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGG

CTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCTTGTGGTTTAATTCGAAGCAACGCGA

AGAACCTTACCAGGTCTTGACASTCCTCGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGAC

AGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTT

GATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGA

TGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGC

AAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACAT

GAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCG

CCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCC

Appendix 5: Pseudomonas moraviensis strain 16S ribosomal RNA gene, partial sequence

GTCGAGCGGATGAAGGAGCTTGCTCCTGGATTCAGCGGCGGACGGGTGAGTMATGCCTAGGAATCTGCCTGGTAG

TGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTT

GCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTG

GTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATAT

TGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAG

TTGGGAGGAAGGGTTGTAGATTAATACTCTGCAATTTTGACGTTACCGACAGAATAAGCACCCGGCTAACTCTGT

GCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTT

GTTAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCAAAACTGACAAGCTAGAGTATGGTAGAG

GGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTG

GACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAA

CGATGTCAACTAGCCGTTGGGAGCCTTGAGCTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGT

ACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAG

CAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACATTG

AGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTT

GTCCTTAGTTACCAGCACGTAATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATG

ACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCC

GCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAA

TCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCSGTCACACCATG

GGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTT

152

Appendix 6: Scanning electron photomicrographs of BCL slag leaching in the absence of bacteria

Appendix 7: Percentage recovery formulae

% 𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑦 =𝑀𝑎𝑠𝑠 𝑜𝑓 𝑒𝑙𝑒𝑚𝑒𝑛𝑡 𝑖𝑛 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

𝑀𝑎𝑠𝑠 𝑜𝑓 𝑒𝑙𝑒𝑚𝑒𝑛𝑡 𝑖𝑛 𝑓𝑒𝑒𝑑∗ 100

153

Appendix 8: The recovery concentrations of base metals from BCL slag using Bacillus aryabhattai (2) in choline ILs vs. glucose

Concentration (ppm)

Week 1 Week2 Week3

Element

Cu Ni Zn Co Fe Al Mg Cu Ni Zn Co Fe Al Mg Cu Ni Zn Co Fe Al Mg

G 10.83

828 290.7

654 31.54

374 138.3

166 10432

8.1 8.449

906 3629.

308 25.32

999 362.1

073 31.62

584 163.9

898 67120

.77 42.86

204 4052.

823 130.85

9611 450.6

746 36.286

7261 192.6

187 11230

8.1 436.1

006 4518.

696

Ion

ic l

iqu

ids

CLe 87.33

922 269.8

31 12.24

772 88.36

044 405.1

332

-3.743

58

3842.762

153.8576

302.7797

14.06825

94.74136

301.1155

-6.187

15

4025.742

500.699533

366.0985

17.1983214

101.4942

551.0731

23.95393

4059.867

CLa 11.84

272 419.1

627 53.16

986 313.8

314 55112

9.9 257.3

752 6833.

558 628.4

582 542.3

376 61.43

304 340.9

876 53254

0.9 1318.

224 7330.

97 1246.9

5403 746.5

818 70.603

1668 358.4

436 50122

4.2 1967.

979 7701.

127

CDP 45.00

123 390.6

883 36.62

114 248.8

636 10886

8.1 10.11

974 6917.

054 67.27

213 467.5

366 45.21

59 293.9

861 35337

.86 206.0

54 7874.

401 346.39

3701 540.9

564 57.814

0394 341.8

148 66954

.42 708.0

473 8365.

68

CCh 16.53

773 295.8

033 21.37

622 96.88

743 14690

.45 13.13

638 3528.

191 20.71

927 323.5

419 26.24

979 112.5

24 4011.

405 12.64

985 3480.

132 87.790

5552 294.6

46 58.281

7544 161.6

529 3470.

696 49.00

568 2821.

431

Appendix 9: The recovery concentrations of base metals from BCL slag using Raoultella ornithinolytica in choline ILs vs. glucose

Concentration (ppm)

Week 1 Week2 Week3

Element


G 14.49

599 351.7

058 28.61

14 163.5

533 95078

.93 11.15

451 4062.

514 61.08

908 407.5

206 32.04

805 185.1

024 11702

6.9 233.4

086 4454.

093 279.67

4922 485.0

536 34.344

3782 191.4

822 11721

8 569.4

427 4539.

45

Ion

ic l

iqu

ids

CLe 361.9

39 322.4

828 17.50

451 107.1

894 197.7

02 45.58

401 4265.

36 569.9

179 435.9

572 17.02

081 111.2

96 195.1

385 69.92

652 4233.

737 626.59

3983 561.8

506 21.830

9252 117.9

904 179.7

15 30.28

887 4416.

587

CLa 50.46

134

433.4

761

59.35

267

328.9

654

59088

6.1

537.4

744

7746.

005

621.6

417

561.1

669

61.94

432

343.1

203

52935

9.6

2038.

477

7938.

367

1239.7

392

776.6

368

63.896

6367

360.9

687

50926

5.1

2944.

93

8178.

493

CDP 9.429

332 379.5

608 37.16

768 252.9

467 12964

0 0.994

124 6866.

216 39.75

927 477.1

115 46.00

985 286.7

519 38668

.34 85.21

12 7483.

143 262.52

7864 558.6

876 53.945

0615 339.4

747 74757

.63 741.3

658 8646.

417

CCh 19.29

431 317.9

786 32.65

557 122.1

412 4920.

662 7.785

769 3616.

011 62.95

431 362.9

607 28.53

568 133.7

932 2678.

225 32.18

616 4385.

439 107.88

573 387.8

039 26.768

9683 134.6

966 373.6

263 37.27

06 4383.

263

154

Appendix 10: The recovery concentrations of base metals from BCL slag using Bacillus sp. in choline based ILs vs. glucose

Concentration (ppm)

Week 1 Week2 Week3

Elem

ent Cu Ni Zn Co Fe Al Mg Cu Ni Zn Co Fe Al Mg Cu Ni Zn Co Fe Al Mg

G 17.64

93

333.8

681

25.11

847

147.7

866

11260

9

14.04

415

4025.

315

49.39

447

383.1

085

33.18

437

177.8

238

73073

.88

134.1

907

4532.

82

179.53

543

451.0

923

35.813

3902

198.3

336

11453

0.6

620.7

003

4823.

077

Ion

ic l

iqu

ids

CLe 129.8

404 245.7

877 12.47

79 76.16

844 1491.

656

-7.744

06

3468.628

255.4494

333.3456

106.5499

93.12924

10206.14

3.765083

4188.736

347.573412

352.7051

20.7124402

95.05232

233.5473

14.35511

4103.639

CLa 71.78

753 472.4

526 66.13

239 349.9

452 82828

2.3 508.2

05 8102.

525 754.0

288 576.7

279 60.26

258 358.6

555 57531

9.3 1321.

65 8226.

034 1319.9

4519 808.2

044 84.667

0023 377.3

48 54111

3.3 1929.

138 8405.

139

CDP 8.241

812

393.1

913

136.1

102

249.9

343

15240

7.4

12.64

38

4590.

445

63.16

659

454.2

968

125.6

19

282.6

782 42537

132.4

563

4759.

25

351.30

8704

555.2

993

168.15

6061

334.4

304

56970

.82

579.2

741

5755.

469

CCh 11.07

674 280.9

356 22.70

822 89.40

105 35386

.83 7.516

386 3558.

666 17.66

848 301.3

45 27.98

267 90.54

164 3837.

269 16.51

119 3755.

984 60.013

8603 415.6

27 40.690

7858 134.4

564 2309.

376 45.32

099 3733.

507

Appendix 11: The recovery concentrations of base metals from BCL slag using Bacillus thuringiensis in choline based ILs vs. glucose

Concentration (ppm)

Week 1 Week2 Week3

Element


G 17.11

163 344.0

331 27.43

062 150.6

608 95816

.41 6.980

769 4070.

313 34.46

322 409.6

679 32.40

468 178.1

239 45735

.91 88.32

524 4653.

581 138.53

2246 480.3

595 51.767

8187 200.5

388 11071

6.8 482.9

674 4917.

822

Ion

ic l

iqu

ids

CLe 123.7

87 291.2

733 12.08

724 87.84

186 1050.

45

-7.325

39

4030.734

134.8947

345.7214

15.28939

96.34774

492.7643

-3.382

74

4141.689

446.15131

417.0922

16.0991207

101.6543

277.5831

19.56172

4091.013

CLa 75.55

542 423.9

348 56.68

605 333.7

119 59084

6.4 256.0

679 8456.

775 682.6

889 553.5

076 63.22

845 358.2

446 55769

2.3 1436.

537 8508.

492 1248.5

6651 744.1

928 62.710

8349 372.7

162 53872

0.2 2200.

619 8789.

322

CDP 19.04

232 395.8

352 109.2

306 256.0

114 13864

5.2 13.62

661 4505.

515 84.49

115 469.7

209 170.0

673 305.1

275 36869

.35 142.4

82 4905.

331 393.96

7176 542.5

329 159.57

9002 348.4

235 75175

.16 571.8

275 5692.

313

CCh 5.371

834 318.7

348 22.33

502 101.6

021 33740

.28

-1.958

6

3189.195

11.78632

357.7179

24.37404

117.0438

3140.595

6.810211

3546.561

62.2663154

396.691

34.2201834

133.7355

930.5213

36.95547

3815.077

155

Appendix 12: The recovery concentrations of base metals from BCL slag using Pseudomonas moraviensis in choline based ILs vs. glucose

Concentration(ppm)

Week 1 Week2 Week3

Element


G 247.4

368 393.2

819 32.78

474 189.0

467 37424

.52 801.6

565 4822.

154 694.7

463 626.2

301 43.36

164 211.1

972 25563

.56 1276.

806 5053.

27 844.21

9981 808.2

978 39.017

2478 219.7

983 27583

.96 1270.

584 5245.

924

Ion

ic l

iqu

ids

CLe 395.0

261

341.9

104

23.47

374

109.9

804

209.9

869

27.78

6

4425.

009

457.9

746

422.6

359

14.46

737

109.7

68

171.2

316

25.07

176

4371.

528

471.52

1608

491.2

756

18.637

7394

113.9

222

176.0

681

21.58

678

4446.

927

CLa 142.1

911 486.0

941 61.47

584 359.2

492 61054

9.7 1379.

226 8971.

574 802.5

054 624.8

518 62.93

84 366.6

257 52100

1.3 2588.

909 9173.

676 1452.2

2444 895.7

397 64.547

463 389.1

112 49397

8.3 3613.

428 9414.

088

CDP 8.716

801 399.0

311 98.03

626 269.5

771 13334

9.3 20.67

209 4653.

135 63.68

603 476.9

917 135.6

429 308.3

523 32526

.78 157.3

522 4824.

773 328.96

3637 566.3

734 140.43

8672 358.2

385 78700

.65 567.1

807 5936.

908

CCh 20.82

129 338.3

585 53.74

087 142.4

158 149.0

478 10.06

153 3912.

555 107.3

502 409.9

31 20.51

015 143.0

955 166.5

42 5.418

66 3928.

566 184.53

5823 482.3

348 20.396

5091 146.4

219 178.5

545 2.907

455 3890.

073

Appendix 13: The recovery concentrations of base metals from BCL slag using a mixed culture in choline based ILs vs. glucose

Concentration(ppm)

Week 1 Week2 Week3

Element


G 18.75

915 357.8

28.83

601

153.9

445

11333

9.8

24.92

913

4431.

833

36.02

206

416.8

954

29.20

886

173.1

085

17310

8.5

51.44

099

4916.

873

130.13

8653

479.4

071

44.659

139

201.9

393

12891

4.6

437.0

038

5306.

113

Ion

ic l

iqu

ids

CLe 69.85

457 289.2

279 13.34

152 93.30

235 550.6

84

-

4.4334

4125.553

166.3606

357.9736

17.64822

106.3799

249.377

30.65518

4257.705

377.172701

455.2794

20.2899012

138.5681

139.3728

175.1919

4285.991

CLa 86.31

661 478.9

489 65.09

079 362.4

123 63074

5.5 505.9

221 9473.

809 588.8

711 595.6

059 57.87

189 391.4

618 60509

1.8 1443.

563 6401.

743 1090.3

9648 727.5

483 65.855

9341 360.8

138 57736

5.5 1593.

786 7131.

623

CDP 34.84

658 392.6

86 136.4

86 266.7

019 15754

6.5 13.09

939 4838.

97 48.92

594 480.1

719 128.8

589 308.4

41 41059

.35 125.8

512 4790.

734 367.48

7203 612.3

416 150.83

1785 371.0

939 73006

.33 814.3

376 5961.

622

CCh 4.542

525

323.0

986

18.36

257

101.6

73

12215

.81

-

1.88851

3333.

294

29.74

855

370.5

461

31.77

512

122.0

778

2852.

648

17.03

831

3677.

268

94.970

0564

425.7

348

24.388

0018

138.8

261

1514.

593

54.37

535

4055.

51

156

Appendix 14: The recovery concentrations of base metals from BCL slag in choline based ILs vs. glucose (control-absence of bacteria)

Concentration(ppm)

Week 1 Week2 Week3

Elem

ent Cu Ni Zn Co Fe Al Mg Cu Ni Zn Co Fe Al Mg Cu Ni Zn Co Fe Al Mg

G 19.11

921 366.3

337 29.32

537 156.9

417 16142

6.9 26.30

206 4753.

899 34.08

419 426.1

487 51.55

099 181.1

999 13770

3.7 124.4

944 5423.

362 273.28

8454 507.0

404 43.435

057 215.6

083 15411

5.7 685.9

539 5823.

327

Ion

ic l

iqu

ids

CLe 154.2

882 311.5

767 15.90

288 92.70

13 478.4

679 4.815

693 4236.

783 222.5

924 383.1

338 21.58

135 100.1

766 529.7

849 0.540

785 4512.

537 332.10

535 441.6

439 34.812

1299 114.4

154 220.3

156 23.30

462 4782.

295

CLa 32.91

166 496.1

141 55.82

071 336.5

884 74680

2.4 499.6

034 7792.

182 839.7

615 696.1

564 62.87

546 368.2

287 69553

9.6 1451.

136 8397.

651 1479.1

4093 1086.

475 66.924

8997 396.4

696 65236

6.5 2302.

65 8689.

657

CDP 6.578

657

493.7

023

142.8

667

298.0

418

21811

9.9

23.28

216

5222.

868

37.94

386

552.4

185

130.4

424

324.5

97

99693

.72

146.2

233

5091.

044

268.15

6331

625.1

375

134.98

9711

370.8

696

10815

6.3

665.9

156

5379.

263

CCh 5.116

214

347.4

579

27.01

794

103.8

457

99899

.57

3.121

313

3506.

384

12.59

105

410.4

505

23.54

184

118.9

665

29124

.44

7.609

691

3844.

683

70.636

6986

453.8

08

24.956

9614

135.5

13

3920.

268

60.55

371

4152.

787

dissertation - uj ir

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