Simultaneous Biotransformation of Carbonaceous Matter and Sulfides in Double Refractory Gold Ores Usin The Fungus

The Pennsylvania State University
The Graduate School

Department of Energy and Mineral Engineering
SIMULTANEOUS BIOTRANSFORMATION OF CARBONACEOUS MATTER AND

SULFIDES IN DOUBLE REFRACTORY GOLD ORES USING THE FUNGUS,
PHANEROCHAETE CHRYSOSPORIUM
A Dissertation in
Energy and Mineral Engineering
by
Grace Ofori-Sarpong
2010 Grace Ofori-Sarpong
Submitted in Partial Fulfillment

of the Requirements
for the Degree of
Doctor of Philosophy
August 2010
The dissertation of Grace Ofori-Sarpong was reviewed and approved* by the following:
Kwadwo Osseo-Asare
Distinguished Professor of Materials Science and Engineering,
and of Geo-Environmental Engineering
Dissertation Advisor
Chair of Committee
Ming Tien
Professor of Biochemistry and Molecular Biology
Dissertation Co-Advisor
Harold Schobert
Professor of Energy and Mineral Engineering
Dissertation Committee Member
Jennifer Macalady
Assistant Professor of Geosciences and Geomicrobiology
Yaw Yeboah
Head of the Department of Energy and Mineral Engineering
Robert Larry Grayson

Chair of Energy and Mineral Engineering Graduate Program
*Signatures are on file in the Graduate School
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Abstract
Double refractory gold ores (DRGO) contain metal sulfides that encapsulate gold and prevent its
dissolution by cyanide, and carbonaceous matter (CM) that adsorbs gold cyanide complex.
Pretreatment is therefore required to decompose the sulfides and liberate gold before cyanidation, and
to deactivate CM and prevent it from adsorbing gold during cyanidation. In this research, the fungus
Phanerochaete chrysosporium, has been used to effectively reduce the gold adsorption ability of CM
on one hand, and decompose metal sulfides on the other, to ultimately enhance gold extraction.
Different ranks of coal (lignite, subbituminous, bituminous and anthracite) were used as surrogates to
investigate the capability of the fungus to reduce the gold adsorption ability of CM. The extent of
biotransformation was assessed primarily by the difference in the ability of CM to adsorb gold before
and after the treatment. It was realized that relative to the other coals, anthracite possessed about 10
fold gold adsorption power in the as-received state, and after biotransformation, reduction in gold
adsorption was more pronounced in anthracite. P. chrysosporium reduced the ability of anthracite to
adsorb gold cyanide complex by over 90% in different growth media including glucose, millet and
wheat bran, and within a wide range of processing periods (up to 21 days), pulp densities (15-75 %),
temperatures (25 oC and 37 oC), pH (4 and 6.5), and level of agitation (0 and 150 rpm). A processing
time of 5-7 days in a mixture of millet and wheat bran (MWB) medium at 37 oC and pH 4 constituted
the best conditions for optimum biotransformation. The best pulp densities for stationary and shake
culturing respectively were 60 % and 25 %. Using the non-cyanide gold complexes; thiourea and
thiosulfate, biotransformation by P. chrysosporium in MWB medium led to reduction of 50-92 % in
gold adsorption by the carbonaceous surrogates and carbon-containing gold concentrates.
Analysis of as-received and processed anthracite using Raman, FTIR and XANES spectroscopies
indicated that fungal-treatment led to a reduction in carbon and a boost of oxygen-containing groups
such as carboxyls and carbonyls. In addition, surface area and pore volume reduced by about 75 % and
80 % respectively, whereas average pore diameter increased by 65 %. The results suggest that P.
chrysosporium biotransforms anthracite by surface oxidation, which leads to a decrease in C/O ratio
and disruption of the continuous nature of graphitic planes, thus decreasing the active sites necessary
for adsorption. Another factor is reduction in surface area via plugging of pores possibly by fungal
hyphae and spores, and biofilms formed through fungal interaction with the substrate, and hence
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decreasing accessibility of gold to the adsorption sites. Fungal action also led to pore enlargement, thus
rendering the pores relatively unsuitable for adsorption of aurocyanide ions, which are generally
known to adsorb preferentially into micropores.
P. chrysosporium, was also assessed for its ability to decompose sulfides in DRGO. Using surrogate
pyrite and arsenopyrite, and sulfide-containing gold concentrate, biotransformation led to high
concentrations of iron, sulfur and arsenic in incubation solutions, and reduction in sulfide sulfur in the
residual solids. Overall, decomposition of sulfide sulfur in the samples was 15 wt%, 35 wt% and 57
wt% respectively for pyrite, arsenopyrite and the gold concentrate after 21 days of treatment. Changes
in sulfide sulfur concentration and formation of oxide phases during fungal treatment were confirmed
using Raman spectroscopy and X-ray diffraction analysis. Oxide phases formed included hematite and
ferric arsenate.
Fungal treatment of flotation concentrate (FC) of DRGO and bacteria-oxidized FC (BFC) resulted in
enhancement of cyanidation gold recovery. An increase of 38 % and 13 % for FC and BFC
respectively over the as-received samples was realized. The single stage fungal treatment of FC
recorded 78 % in gold recovery which compares very well with the 81 % obtained from the
commercial biooxidation treatment of FC. Preliminary investigations on the use of cell-free extracts
from P. chrysosporium to effect biotransformation of sulfides and CM in DRGO led to 26 % increase
in gold recovery during cyanidation over the as-received, after a 3-day bioprocessing period.
The results suggest that P. chrysosporium is a promising microorganism for oxidative decomposition
of metal sulfides associated with refractory gold ores and for deactivation of CM. In addition, it
demonstrates an innovative and a potentially alternative pretreatment process for DRGO using either
whole cells (in vivo) or cell-free extracts (in vitro) of P. chrysosporium to simultaneously biotransform
sulfides and CM, and consequently enhance gold extraction.
iv
Table of contents
Table of contents ...............................................................................................................v
List of figures ............................................................................................................ix
List of tables ..............................................................................................................xiv
Acknowledgements ...........................................................................................................xv
Chapter 1 General Introduction ................................................................................................1
1.1 Problem definition........................................................................................................1
1.1.1 Gold ores ..........................................................................................................1
1.1.2 Relevance of microbial pretreatment of double refractory gold ores (DRGO).....3
1.1.3 Phanerochaete chrysosporium...........................................................................4
1.2 Hypothesis ...................................................................................................................5
1.3 Specific research objectives ..........................................................................................6
1.4 Approach to accomplish experimental objectives ..........................................................6
1.5 Scope of research .........................................................................................................7
References ........................................................................................................................9
Chapter 2 Mycohydrometallurgy: Application of fungi in hydrometallurgy ...............................13
Abstract ............................................................................................................................13
2.1 Introduction .................................................................................................................14
2.2 Fungi ...........................................................................................................................15
2.3 Sorption of metals by fungi ..........................................................................................16
2.4 Leaching of metals by fungi .........................................................................................21
2.5 Biotransformation of sulfur compounds by fungi ..........................................................25
2.6 Biotransformation of organic carbon-containing materials ............................................27
2.6.1 Biotransformation of rubber and rubber products ...............................................27
2.6.2 Biotransformation of coal ..................................................................................27
2.6.3 Microbial transformation of carbonaceous matter in gold ores ...........................28
2.7 Biotransforming action of white rot fungi ....................................................................29
2.8 Conclusions .................................................................................................................34
References ........................................................................................................................35
Chapter 3 Mycohydrometallurgy: Coal model for potential reduction of preg-robbing capacity
of carbonaceous gold ores using the fungus, Phanerochaete chrysosporium.......................43
Abstract ............................................................................................................................43
3.1 Introduction .................................................................................................................44
3.1.1 Mycohydrometallurgy .......................................................................................44
3.1.2 Preg-robbing effect of carbonaceous matter on gold extraction ..........................44
3.1.3 Biomodification of carbonaceous matter ............................................................45
3.2 Phanerochaete chrysosporium .....................................................................................47
v
3.3 Experimental investigations .........................................................................................47

3.3.1 Materials ...........................................................................................................47
3.3.2 Media preparation and incubation ......................................................................48
3.3.3 Incubation and harvesting of carbonaceous samples...........................................50
3.3.4 Gold adsorption experiments .............................................................................51
3.4 Results and Discussion ................................................................................................51
3.4.1 Effect of carbonaceous characteristics on biotransformation ..............................52
3.4.2 Effect of growth media ......................................................................................57
3.5 Biotransforming action of P. chrysosporium ................................................................59
3.6 Conclusions .................................................................................................................61
References ........................................................................................................................63
Chapter 4 Fungal biotransformation of anthracite-grade carbonaceous matter: Effect on gold
cyanide uptake ..................................................................................................................68
Abstract ............................................................................................................................68
4.1 Introduction .................................................................................................................69
4.2.1 Materials, medium preparation and incubation...................................................70
4.2.2 Characterization of anthracite ............................................................................71
4.2.3 Photomicrographs of P. chrysosporium .............................................................72
4.2.4 Analysis of data ................................................................................................72
4.3 Results and discussions................................................................................................73
4.3.1 Reduction in gold adsorption following fungal-treatment ...................................73
4.3.2 Effect of pulp density ........................................................................................74
4.3.3 Effect of temperature.........................................................................................77
4.3.4 Effect of pH ......................................................................................................79
4.3.5 Effect of sterilization .........................................................................................81
4.3.6 Effect of growth medium...................................................................................82
4.4 Investigations of surface biomodification of anthracite .................................................83
4.4.1 Surface area and pore characterization of anthracite...........................................83
4.4.2 Photomicrograph of P. chrysosporium ...............................................................84
4.4.3 Modification of graphitic structure ....................................................................86
4.4.4 Modification of surface functional groups .........................................................87
4.5 Proposed mechanism responsible for reduction in gold adsorption after
biotransformation of anthracite by P. chrysosporium..................................................90
4.6 Conclusions .................................................................................................................92
References ........................................................................................................................94
Chapter 5 Gold adsorption on carbonaceous matter following fungal treatment: Comparison of
cyanide and non-cyanide gold complexes ..........................................................................98
Abstract ............................................................................................................................98
5.1 Introduction .................................................................................................................99
5.1.1 Lixiviants in gold extraction ..............................................................................99
5.1.2 Adsorption of gold complexes on carbon ...........................................................99
5.1.3 Application of P. chrysosporium in reducing adsorption of gold complexes on
carbon ................................................................................................................101
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5.2.2 Gold adsorption experiments .............................................................................102
5.2.3 Petrographic analysis of gold concentrate ..........................................................103
5.3 Results and Discussion ................................................................................................103
5.3.1 Analysis of gold concentrates ............................................................................103
5.3.2 Effect of carbonaceous characteristics on gold adsorption from cyanide,
thiourea and thiosulfate solutions onto surrogate CM ..........................................104
5.3.3 Effect of fungal biotransformation on the adsorption of gold complexes onto
gold concentrates................................................................................................110
5.4 Fungal biotransforming action on the adsorption of gold thiourea and thiosulfate on
carbonaceous materials ..............................................................................................112
5.5 Conclusions .................................................................................................................114
References ........................................................................................................................115
Chapter 6 Biotransformation of sulfides using the fungus, Phanerochaete chrysosporium: A
potential pretreatment process for refractory sulfidic gold ores...........................................118
Abstract ............................................................................................................................118
6.1 Introduction .................................................................................................................119
6.1.1 Refractory sulfidic gold ores and bacteria oxidation ...........................................119
6.1.2 Fungal-biotransformation of sulfides and carbonaceous matter ..........................121
6.2.1 Materials ...........................................................................................................122
6.2.2 Medium preparation, incubation and harvesting of treated material ....................122
6.2.3 Determination of dissolved components and sulfide sulfur analysis of fungaltreated material...................................................................................................123
6.2.4 Characterization of flotation concentrate ...........................................................124
6.2.5 Analysis of data ................................................................................................124
6.3 Results and discussion .................................................................................................125
6.3.1 Transformation of pyrite by P. chrysosporium ...................................................125
6.3.2 Transformation of arsenopyrite by P. chrysosporium .........................................129
6.3.3 Transformation of flotation (sulfidic gold) concentrates by P. chrysosporium ....132
6.3.4 Characterization of flotation concentrate ...........................................................135
6.4 Conclusions .................................................................................................................138
References ........................................................................................................................139
Chapter 7 Biotransformation of double refractory gold ores by the fungus, Phanerochaete
chrysosporium...................................................................................................................143
Abstract ............................................................................................................................143
7.1 Introduction .................................................................................................................144
7.2.2 Cyanidation .......................................................................................................147
7.2.3 Fire Assaying ....................................................................................................147
7.2.4 Analysis of data ................................................................................................148
7.3.1 Fungal-biotransformation of sulfides in flotation concentrate (FC).....................149
7.3.2 Fungal-biotransformation of sulfides in bacterial-treated FC (BFC) ...................150
vii
7.3.3 Effect of fungal-treatment on cyanide amenability of FC and BFC ....................151

7.4 Assessment of fungal- and bacterial- treatment of gold concentrates: Single-stage
against two-stage processes ........................................................................................155
7.5 Conclusions .................................................................................................................157
References ........................................................................................................................159
Chapter 8 In vitro transformation of carbonaceous matter and sulfides in refractory gold ores
using cell-free extracts of Phanerochaete chrysosporium ...................................................162
Abstract ............................................................................................................................162
8.1 Introduction .................................................................................................................163
8.2.1 Materials ...........................................................................................................164
8.2.2 Preparation of cultures and extraction of cell-free components ...........................165
8.2.3 Preparation of reagents for the treatment of materials ........................................165
8.2.4 Post-incubation experiment ...............................................................................166
8.2.5 Surface area and porosity analysis .....................................................................167
8.2.6 Transmission electron microscopy (TEM) .........................................................167
8.3.1 Effect of cell-free extracts processing on gold adsorption behavior and surface
modification of anthracite ...................................................................................168
8.3.2 Effect of cell-free extracts and hydrogen peroxide processing on
biotransformation of sulfides ..............................................................................172
8.3.3 Biotransformation of flotation concentrate by cell-free extracts .........................174
8.3.4 Cyanidation of flotation concentrate ..................................................................176
8.4 Proposed mechanism of biotransformation of sulfides by P. chrysosporium .................178
8.5 Conclusions .................................................................................................................180
References ........................................................................................................................182
Chapter 9 General conclusions, recommendations and processing proposal ...............................186
9.1 General Conclusions ....................................................................................................186
9.2 Recommendations .......................................................................................................188
9.3 Processing proposal .....................................................................................................190
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List of figures
Fig. 2.1. Photomicrograph of Web-like hyphal network of Phanerochaete chrysosporium
(Sachs et al., 1989) ............................................................................................................. 30
Fig. 2.2. Structure of lignin peroxidase (A), and manganese peroxidase (B); enzymes
generated by P. chrysosporium (Banci et al., 1992)............................................................. 31
Fig. 3.1. Incubation of P. chrysosporium in millet and wheat bran. Autoclaved millet (A),
autoclaved MWB (B), 1-week culture of fungus and millet (C), 1-week culture of fungus
and MWB (D), 2-week incubated culture of P. chrysosporium with anthracite and millet
(E), and 2-week incubated culture of P. chrysosporium with anthracite and MWB (F) ........ 49
Fig. 3.2. Growth of P. chrysosporium on anthracite coal after 2-week incubation in MWB
medium .............................................................................................................................. 50
Fig. 3.3. Effect of fungal-treatment time on gold adsorption by lignite, subbituminous,
bituminous and anthracite coals. The growth medium was glucose broth ............................ 52
Fig. 3.4. Effect of two-week fungal-treatment on the preg-robbing ability of lignite,
subbituminous, bituminous and anthracite coals. The growth medium was moist glucose
agar .................................................................................................................................... 53
subbituminous, bituminous and anthracite coals. The media used were millet and MWB .... 54
Fig. 3.6. Correlation between the atomic ratios of carbon to oxygen and gold adsorption by
lignite, subbituminous, bituminous and anthracite coals ...................................................... 56
Fig. 3.7. Effect of different media utilized on gold adsorption behavior of anthracite; asreceived (AR), glucose broth (GB), glucose agar (GA), millet + wheat bran (MWB),
millet (M). The biotransformation period was 2 weeks. ...................................................... 58
Fig. 3.8. A comparison of the gold adsorption behavior of controls and fungal-treated
anthracite. .......................................................................................................................... 59
Fig. 4.1. Reduction in gold adsorption by anthracite after fungal treatment at 60 % solids
density for 14 days at 37 oC and pH 6.5. For adsorption tests, 0.1 g of anthracite was
contacted with 25 mL of 5 g/mL gold solution and agitated at 150 rpm for 24 hours ......... 73
Fig. 4.2. Effect of pulp density on biotransformation of anthracite by P. chrysosporium in
stationary culturing (37 oC, pH 6.5; 14 days) ...................................................................... 74
Fig. 4.3. Effects of pulp density on biotransformation of anthracite by P. chrysosporium in
shake culturing (37 oC, pH 6.5; 14 days) ............................................................................. 75
Fig. 4.4. Effect fungal-treatment time on biotransformation of anthracite at pulp densities of
60 % and 75 % solids under stationary culturing (37 oC, pH 6.5) ........................................ 76
ix
Fig. 4.5. The effects of incubation temperature as a function of time in stationary culturing
(60 % solid, pH 6.5) ........................................................................................................... 77
Fig. 4.6. The effects of incubation temperature as a function of time in shake culturing (25 %
solid, pH 6.5) ..................................................................................................................... 78
Fig. 4.7. Effect of pH on biotransformation of anthracite as a function of time in shake
culturing (25 % solid, 37 oC) .............................................................................................. 80
Fig. 4.8. Effect of sterilizing on the time dependence of the biotransformation action of P.
chrysosporium (37 oC, pH 6.5; 60 % solids; stationary) ...................................................... 81
Fig. 4.9. Combined effect of MWB and fungus in the biotransformation of anthracite (37 oC,
pH 6.5; 60 % solids; stationary; up to 14 days) ................................................................... 82
Fig. 4.10. A comparison of surface area and pore characteristics of as-received and biotreated
anthracite. Anthracite was treated for 14 days in MWB mixture at 60 % solids, 37 oC and
pH 4. .................................................................................................................................. 84
Fig. 4.11. Photomicrograph of P. chrysosporium. The fungus was maintained on MWB
medium for 2 weeks. .......................................................................................................... 85
Fig. 4.12. Raman spectra of anthracite; as received and 14-day fungal treated at 37 oC and
pH 4. .................................................................................................................................. 86
Fig. 4.13. Infrared spectra of both as-received and biotreated anthracite. Anthracite was
treated for 14 days at 60 % solids, 37 oC and pH 4. ............................................................. 88
Fig. 4.14. A comparison of the as-received and biotreated anthracite spectra from oxygen
XANES. Anthracite was treated for 14 days, at 60 % solids density, 37 oC and pH 4. ......... 89
Fig. 4.15. Interaction between the gold cyanide complex, Au(CN)2-, and the graphitic
structure of carbon (after Jones et al. 1989; Klauber, 1991; Ibrado and Fuerstenau; 1992;
1995; Poinen and Thurgate, 2003) ...................................................................................... 91
Fig. 5.1. Effect of one-week fungal-treatment on the gold adsorption ability of lignite, subbituminous, bituminous and anthracite coals in cyanide (CN) solution (37 oC; pH 6.5; 60
% solids) ............................................................................................................................ 105
Fig. 5.2. Effect of one-week fungal-treatment on the gold adsorption ability of lignite, subbituminous, bituminous and anthracite coals in thiourea (TU) solution. (37 oC; pH 6.5;
60 % solids) ....................................................................................................................... 106
Fig. 5.3. Effect of one-week fungal-treatment on the gold adsorption ability of lignite, subbituminous, bituminous and anthracite coals in thiosulfate (TS) solution. (37 oC; pH 6.5;
60 % solids) ....................................................................................................................... 107
Fig. 5.4. Reduction in cyanide (CN), thiourea (TU) and thiosulfate (TS) gold adsorption on
lignite, subbituminous, bituminous and anthracite due to surface biotransformation by P
chrysosporium (37 oC; pH 6.5; 60 % solids; 7 days)............................................................ 108
Fig. 5.5. Comparison of gold adsorption on anthracite from cyanide, thiourea and thiosulfate
solutions as a function of fungal-treatment time. (37 oC; pH 6.5; 60 % solids; up to 21
days) .................................................................................................................................. 109
Fig. 5.6. Gold adsorption on as-received, control and treated anthracite from cyanide, thiourea
and thiosulfate solutions (37 oC; pH 6.5; 60 % solids; 7 days). ............................................ 109
Fig. 5.7. Comparison of gold adsorption on flotation concentrate (FC) from cyanide, thiourea
and thiosulfate solutions before and after fungal-treatment. (37 oC; pH 6.5; 60 % solids;
7 days) ............................................................................................................................... 110
Fig. 5.8. Comparison of gold adsorption on bacterial-oxidized flotation concentrate (BFC)
from cyanide, thiourea and thiosulfate solutions before and after fungal-treatment. (37
o
C; pH 6.5; 60 % solids; 7 days) ......................................................................................... 111
Fig. 5.9. Petrographic analysis of double refractory flotation gold concentrate ............................ 112
Fig. 6.1. Two-week biotransformation of pyrite in the absence and presence of P.
chrysosporium. The experiment was conducted at 37 oC for 14 days at pH 4 and 30 %
solids ................................................................................................................................. 126
Fig. 6.2. Accumulation of dissolved sulfur and iron during the incubation of pyrite with P.
chrysosporium. The experiment was conducted at 37 oC for up to 21 days at pH and 30
% solids ............................................................................................................................. 127
Fig. 6.3. Biotransformation of sulfide sulfur in pyrite as a function of fungal-treatment. The
experiment was conducted at 37 oC for up to 21 days at pH 4 and 30 % solids .................... 128
Fig. 6.4. Two-week biotransformation of arsenopyrite in the absence and presence of P.
solids ................................................................................................................................. 129
Fig. 6.5. Dissolution of iron, sulfur and arsenic from arsenopyrite as a function of incubation
time. The experiment was conducted at 37 oC for up to 21 days at pH 4 and 30 % solids..... 130
Fig. 6.6. Eh-pH diagram for Fe-As-S-H2O system (Osseo-Asare, 1984) ..................................... 132
Fig. 6.7. Two-week biotransformation of flotation concentrate in the absence and presence of
P. chrysosporium. The experiment was conducted at 37 oC for 14 days at pH 4 and 30 %
solids ................................................................................................................................. 133
Fig. 6.8. Dissolution of sulfides in flotation concentrate by P. chrysosporium as a function of
time for 21 days. The experiment was conducted at 37 oC for up to 21 days at pH 4 and
30 % solids......................................................................................................................... 134
xi
Fig. 6.9. Biotransformation of sulfide sulfur in the flotation concentrate (FC) as a function of
incubation time. The experiment was conducted at 37 oC for up to 21 days at pH 4 and
30 % solids......................................................................................................................... 135
Fig. 6.10. Raman spectrogram of as-received and fungal-treated flotation concentrate.
Treatment was conducted for 14 days at 37 oC and pH 4 ..................................................... 136
Fig. 6.11. X-ray diffractogram of as-received and fungal-treated flotation concentrate.
Treatment was conducted for 14 days at 37 oC and pH 4 ..................................................... 137
incubation time. The experiment was conducted at 37 oC for up to 21 days at pH 4 and
30 % solids......................................................................................................................... 149
Fig. 7.2. Two-week fungal-treatment of BFC in the absence and presence of P.
solids ................................................................................................................................. 151
Fig. 7.3. Gold recovery from fungal-treated and control FC and BFC. Cyanidation was
conducted at 30 % pulp density for 24 h at pH 11 and cyanide strength of 10 g/L ............... 152
Fig 7.4. The effect of fungal treatment time on gold extraction from Sample FC. Cyanidation
was conducted at 30 % pulp density for 24 h at pH 11 and cyanide strength of 10 g/L......... 152
Fig. 7.5. Variation of sulfur transformation and gold extraction as a function of fungaltreatment time. Cyanidation was conducted at 30 % pulp density for 24 h at pH 11 and
cyanide strength of 10 g/L .................................................................................................. 153
Fig 7.6. Gold extraction from BFC as a function of fungal incubation time. Cyanidation was
Fig. 7.7. Comparison of gold extraction from flotation concentrate (FC), fungal-treated FC
(FFC), bacterial-treated FC (BFC) and fungal-treated BFC (FBFC). Cyanidation was
Fig. 7.8. Changes in sulfide sulfur content, gold adsorption by carbonaceous matter and gold
extraction from FC following 14-day fungal treatment. Cyanidation was conducted at 30
% pulp density for 24 h at pH 11 and cyanide strength of 10 g/L. ........................................ 156
Fig. 8.1. A comparison of the gold adsorption behavior of treated anthracite (15 % solids,
37 oC, 24 hr, pH 4) ............................................................................................................. 168
Fig. 8.2. Surface area and porosity analysis of anthracite samples before and after treatment
using whole cell and cell-free extracts of P. chrysosporium (15 % solids, 37 oC, 24 hr,
pH 4).................................................................................................................................. 169
Fig. 8.3a. TEM analysis of as-received anthracite samples showing a very structured surface ..... 170
xii
Fig. 8.3b. TEM analysis of anthracite samples after treatment using whole cells of P.
chrysosporium showing relatively more amorphous surface (37 oC, pH 4, 60 % solids, 7
days) .................................................................................................................................. 171
Fig. 8.3c. TEM analysis of anthracite samples after treatment using cell-free extracts of P.
chrysosporium showing very amorphous surface (37 oC, pH 4, 15 % solids, 3 days) ........... 171
Fig. 8.4. In vitro dissolution of constituents during pyrite transformation as a function of time
(15 % solids, 37 oC, up to 72 hr) ......................................................................................... 173
Fig. 8.5. Comparison of constituents in solution after pyrite treatment with H 2O2 and fungal
extract (15 % solids, 37 oC for 24 hr) .................................................................................. 173
Fig. 8.6. In vitro dissolution of constituents during transformation of flotation concentrate as
a function of time (15 % solids, 37 oC, up to 72 hr) ............................................................. 175
Fig. 8.7. Decomposition of sulfide sulfur in flotation concentrate with fungal extracts (15 %
solids, 37 oC, up to 72 hr) ................................................................................................... 175
Fig. 8.8. Cyanide amenability of flotation concentrate (as-received) and after 24-hr treatment
with H2O2, and cell-free extracts of P. chrysosporium. Both samples were processed at
15 % solids and 37 oC at pH 4 ............................................................................................ 176
Fig. 8.9. Cyanide amenability of flotation concentrate (as-received) and after treatment with
whole cell and cell-free extracts of P. chrysosporium (15 % solids, 37 oC, 72 hr). ............... 178
Fig. 9.1. Proposed flow sheet for bioheap leaching of flotation concentrate using P.
chrysosporium.................................................................................................................... 191
Fig 9.2. Proposed flowsheet for fungal biotreatment of flotation concentrate using a
continuously stirred tank reactor (CSTR) ............................................................................ 192
xiii
List of tables
Table 1.1. Summary of the methodology required to accomplish the objectives ..................... 7
Table 1.2. Brief description of the dissertation write-up......................................................... 8
Table 2.1. Heavy metal biosorption by fungi ......................................................................... 18
Table 2.2. Heavy metal leaching with fungi .......................................................................... 22
Table 2.3. Fungal transformation of sulfur compounds .......................................................... 26
Table 5.1. Partial chemical analysis of as-received gold concentrates .................................... 104
xiv
Acknowledgements
Inter-disciplinary researchers like me normally owe a great deal to many people, and I wish to convey
my gratitude to the major contributors to this study, though this list may not be exhaustive. I wish to
express my profound appreciation to my dissertation advisors, Professors K. Osseo-Asare and Ming
Tien for their outstanding guidance through the project. I also thank my committee members,
Professors Yaw Yeboah, Harold Schobert and Jenn Macalady for accepting to serve on the committee,
and also for their readiness to assist in various ways whenever I called on them. Professor Yaw
Yeboah deserves special mention for his time, mentorship and financial assistance.
The organizations: Ghana Education Trust Fund and Scholarship Secretariat; University of Mines and
Technology, Ghana; Embassy of Ghana, USA; PEO International Peace Scholarship, USA; and
Faculty for the Future Fellowship, Netherlands; are acknowledged for offering financial assistance.
Golden Star Prestea-Bogoso Resources (GSPBR), Ghana, is also acknowledged for providing gold
concentrates for the research and for assisting with the analysis.
I wish to thank Dr. Yongsheng Chen for his help with the XANES analysis, Irene Schaperdoth for
assisting with photomicrographs of the fungus, and also Prashanti Lyer, Camille Stephens and other
lab colleagues for giving me insight into microbial work. Other individuals who have contributed
greatly in acquiring characterization results include Henry Gong, Gary Mitchell, Nichole Wonderling,
Joe Stitt, Magda Salama, John Cantolina and Trevor Clark, all of the Materials Characterization
Laboratory, Penn State University. Others are Ahmed Adam and Amon Asiedu of GSPBR, Ghana.
My heartfelt appreciation goes to my mentors, Professors Richard Amankwah and Elias Asiam of the
University of Mines and Technology, Ghana, for their immeasurable support. Dr. Levi Yafetto of
Havard University, USA, is also appreciated for his time and great suggestions. Many loved ones,
family and friends who have loved me and stood by me through all challenging moments include
Pastor Ben Abbey, Pastor and Mrs Benefo, Madam Alice Ansomah, Mr. Ben Boamah, Mrs. Monica
Osei all of Ghana, and Mr. Samuel Obeng and Ms. Ruth Yidana of the USA. Finally, I am very
thankful to God for his grace and mercy. He was always in when I called, and He always made a
way, leading me by road in the wilderness and showing me rivers in the desert.
xv
Chapter 1
General Introduction
1.1
Problem definition
1.1.1 Gold ores

A mineral is basically a naturally occurring solid that has a distinct chemical composition and
structure, and an aggregate of minerals constitutes a rock. An ore is a type of rock mass from
which a mineral of interest, such as gold can be recovered economically, given the available
level of technology (Kesse, 1985; Boyle, 1987). Gold ores can be classified broadly as nonrefractory (alluvial and free-milling) and refractory from a metallurgical standpoint depending
on the mineralogy and the ease of gold extraction. In alluvial ore, gold exists as descrete
particles naturally liberated by weathering. Gold in alluvial ores may be recovered by
scrubbing and concentration processes, where the high specific gravity of gold is exploited for
separation. Free-milling ores are those from which about 95 % of the gold can be recovered
by gravity concentration and/or simple cyanidation (dissolution via aurocyanide, Au(CN) 2-,
formation) after milling (grinding) to 80 % passing 75 m (Marsden and House, 2006).
Refractory gold ores are more difficult to treat and depending on the degree of refractoriness,
recovery could be below 50 % (Hausen, 2000; Vaughan and Kyin, 2004). Since gold ores are
fixed resources, continuous mining and subsequent depletion of non-refractory gold ores have
generated profound interest in research on gold recovery from refractory ores. These hard-totreat ores occur in several gold mining regions throughout the world (Baako, 1972; Boyle,
1979; Kesse, 1985; Brierley, 1995; Vaughan, 2004). The annual world gold production
increased by 47% from 1677 t in 1989 to over 2464 t in 2004. Within this period, gold
production from refractory gold ores increased by 165 % from 94 t to 249 t, which is
approximately 10% of global production of the precious metal (Marsden 2006; Yen et al,
2008).
In refractory ores, gold usually exists as extremely fine particles (< 1 m) (Marsden and
House, 2006) in intimate association with gangue (waste) materials, and the major problem of
refractoriness centres on the presence of metal sulfides and/or carbonaceous matter. In sulfidic
refractory gold ores, tiny gold particles may be highly disseminated and locked up within the
grain boundaries or fractures of sulfides such as pyrite and arsenopyrite. For this reason,
decomposition of the sulfides is required to liberate the gold (Boyle, 1979; Guay, 1981;
Arriagada and Osseo-Asare, 1984; Cabri et al., 1989; Marsden and House, 2006; Benzaazoua
et al, 2007). The presence of carbonaceous matter (CM) in gold ores leads to two main
deleterious effects: (a) confinement of gold with attendant difficult release from the CM
matrix and (b) loss of dissolved metal via the ability of CM to adsorb gold from goldimpregnated solution. Adsorption of gold by CM leads to gold losses due to the fineness of
the carbon (0.0022 m) which does not permit it to be screen-separated for gold recovery,
and this phenomenon is termed as preg-robbing (Osseo-Asare et al., 1984; Hausen and
Bucknam, 1985; Hutchins et al., 1988; Afenya, 1991; Kohr, 1994; Adams and Burger, 1998;
Pyke et al, 1999). Due to the similarities between CM and the various coal ranks, coal is
normally used as a surrogate in the characterization of this carbonaceous material (Ibrado and
Fuerstenau, 1992; Van Krevelen, 1993; Van Vuuren et al., 2000).
If the same refractory ore contains sulfides and carbonaceous matter, the ore is referred to as
double refractory (Nyavor and Egiebor, 1992; Amankwah et al., 2005). Recovery of gold
from double refractory ores can be less than 50 % when subjected to the conventional
cyanidation process due to (a) the inability of cyanide to permeate sulfides and dissolve the
encapsulated gold, and (b) the uptake of dissolved gold by CM (Osseo-Asare et al., 1984;
Abotsi and Osseo-Asare, 1986).
1.1.2 Relevance of microbial pretreatment of double refractory gold ores (DRGO)

For gold locked up in the refractory components, especially sulfides, to be amenable to
cyanidation, pretreatment is required to decompose these shielding minerals, thus liberating
the gold. The processes that have been used commercially to pretreat refractory gold ores
include roasting, pressure oxidation and bacteria oxidation (Arriagada and Osseo-Asare, 1984;
Berezowsky et al., 1988; Komnitsas and Pooley, 1989; Afenya, 1991; Rawlings et al., 2003).
Biooxidation employs the use of iron and sulfur chemolithoautotrophic bacteria such as
Acidithiobacillus
ferrooxidans,
Acidithiobacillus
thiooxidans
and
Leptospirillum
ferrooxidans, to catalyze the oxidation of metal sulfides, thus liberating gold for subsequent
cyanidation (Livesey-Goldblatt et al, 1983; Brierley, 1995; Sand et al., 1995 ; Hackl, 1997).
Since the bacteria are not organotrophs they do not utilize the organic carbon (Silver, 1970;
Madigan and Martinko, 2006) which is consequently routed into the cyanidation circuit where
it preg-robs dissolved gold.
Current research efforts aimed at improving gold extraction from double refractory gold ores
have been focused on two-stage processes to first oxidize sulfides and then deactivate the CM
(Portier, 1991; Brierley and Kulpa, 1992; Amankwah et al., 2005). Microorganisms employed
in deactivating CM include the bacteria Pseudomonas sp., Achromobacter sp. and
Streptomyces setonii, and fungi Aspergillus bruneio, Penicillium citrinum and Trametes
versicolor (Portier, 1991; Brierley and Kulpa, 1993; Amankwah and Yen, 2006; Afidenyo,
2008; Yen et al., 2008). It is expected, however, that a single-stage microbial process which
simultaneously deactivates the CM and solubilizes the sulfides will be less time-consuming
and more cost-effective.
Available literature indicates that microbial degradation of both sulfides and CM in a singlestage has thus far not achieved much success (Portier, 1991; Brierley and Kulpa, 1993;
Amankwah and Yen, 2006; Yen et al., 2008). The limitation to single-stage processes has
been attributed to the inability of the heterotrophic carbon deactivating microorganisms which
work at relatively higher pH range (4-8) to coexist and function effectively at the optimum
3
conditions within which the acidophilic (pH, 1-2) sulfide oxidizing bacteria work (Amankwah
et al., 2005; Madigan and Martinko, 2006).
Most of the investigations have recorded substantial degradation for bituminous-grade CM

with little success in reducing preg-robbing for anthracite-grade CM, which incidentally
constitutes about 50% of CM in refractory gold ores (Osseo-Asare et al., 1984; Sibrell et al.,
1990; Pyke et al., 1999; Stenebraten et al., 2000). In addition, anthracite grade CM has a far
higher capacity to adsorb gold than bituminous and lignite coals due to its maturity and hence
well-developed graphitic planes (Jones et al, 1989; Ibrado and Feurstenau, 1992; Van Vuuren
et al., 2000; Amankwah and Yen, 2006; Afidenyo, 2008). Consequently, using a
microorganism that can decompose metal sulfides, and simultaneously deactivate CM and
reduce its preg-robbing nature significantly, will be of immense benefit in the treatment of
double refractory gold ores.
1.1.3 Phanerochaete chrysosporium

The white rot basideomycete, Phanerochaete chrysosporium, is a prominent lignin-degrading
fungus from which the first lignin-degrading enzyme, lignin peroxidase (LiP), was isolated
and characterized by Tien and Kirk (1983; 1984). The fungus has hence been studied in many
laboratories as a model organism as it grows quickly and degrades lignin rapidly. P.
chrysosporium also secretes manganese peroxidase (MnP), and these oxidative enzymes
catalyze the biodegradation of lignin and a variety of aromatic carbonaceous materials
including low rank coals, wood chips and environmental pollutants (Glenn et al., 1983;
Bumpus and Aust, 1986; Ralph and Catcheside, 1997; Tunde and Tien, 2000; Martin and
Petersen, 2001). Since the carbonaceous matter (CM) in refractory gold ores is characterized
to be similar to coal, it is expected that the oxidative enzymes can biotransform the CM.
P. chrysosporium also secretes a H2O2-generating enzyme, glyoxal oxidase (Kersten and Kirk,
1987), and hydrogen peroxide is known to solubilize pyrite and arsenopyrite (McKibben and
4
Barnes, 1986; Antonijevic, et al., 1997; Jennings et al., 2000). The strong oxidizing
environment produced by the oxidative enzymes of P. chrysosporium is also known to be
responsible for the oxidative removal of sulfur from coal (Schreiner et al., 1988; Gonsalvesh
et al., 2008; Aranda et al., 2009). It is therefore possible that P. chrysosporium can catalyze
the biotransformation of carbonaceous matter and sulfides, and hence enhance gold extraction
in the subsequent cyanidation step.
This dissertation thus describes research aimed at utilizing a single-stage fungal process to
achieve simultaneous deactivation of carbonaceous matter and decomposition of metal
sulfides in double refractory gold ores. Analysis of the data obtained from this study would
contribute to the pool of scientific knowledge regarding processing of refractory gold ores and
hence be of immense benefit to academia. In addition, the results could also find application
in gold production communities.
1.2
Hypothesis
On the basis of the above considerations, the following hypotheses were built:
1. The extra-cellular oxidative enzymes and hydrogen peroxide generated by P.
chrysosporium can;
a. biotransform the native carbonaceous matter in double refractory gold ores and
thus reduce its activity in gold adsorption, and
b. solubilize sulfides and hence liberate the locked-up gold for direct contact with
cyanide solution,
2. The fungal-treatment will ultimately increase gold extraction via cyanidation.
1.3
Specific research objectives
This study sought to investigate the simultaneous fungal-degradation of carbonaceous matter

(CM) and sulfides in double refractory gold ores (DRGO) with the following specific
interests:
Investigate the extent of biotransformation of CM by P. chrysosporium with
subsequent reduction in gold adsorption capabilities;
Investigate the extent of biotransformation of sulfides ultimately to sulfate by P.
chrysosporium;
Assess the effect of process variables (time, pH, pulp density, temperature, and level
of agitation) on biomodification of CM and sulfides;
Examine the simultaneous degradation of CM and sulfides in DRGO by P.
chrysosporium; and
Provide understanding of the mechanism for biotransformation of DRGO by P.
chrysosporium.
1.4
Approach to accomplish experimental objectives
The objectives set out for this study were realized by obtaining the dependent variables as a
function of the independent variables using the techniques listed in Table 1.1 as a guide.
Table 1.1. Summary of the methodology required to accomplish the objectives

Material
Independent variables
Dependent variables
Technique
1. Carbonaceous
Coal type (lignite, sub-bituminous,
Gold adsorption test of
matter
bituminous and anthracite)
treated solids
Media (glucose, millet and wheat bran)
Degree of graphitization
Raman
Time (1-21 days)
Degree of surface
Infrared, XANES
Pulp density (15-75 % solids)
oxidation
Temperature (25 oC and 37 oC)
Micro-porosity
BET
pH (4 and 6.5)
Structural analysis
TEM
Sulfides (pyrite and arsenopyrite)
Measurement of dissolved
ICP-AES
Time (1-21 days),
constituents in incubation
Pulp density (15-60 %)
solution
2. Sulfides
ICP-AES
Temperature (37 C)
Sulfur speciation and
XRD, LECO
pH (4 and 6.5)
surface oxidation products
combustion methods
of treated solids
3. Gold ores
Gold concentrate (flotation concentrate
Maturity of CM
Petrographic analysis
(FC) and bacterial-oxidized FC)
Measurement of dissolved
ICP-AES
Time (1-21 days)
constituents in incubation
Pulp density (15-60 %)

o
solution
Temperature (37 C)
Sulfur speciation and
XRD, LECO
pH (4 and 6.5)
surface oxidation products
combustion methods
of treated solids
1.5
Gold preg-robbing
ICP-AES
Gold cyanidation
ICP-AES, fire assay
Scope of research
This dissertation is presented in nine chapters including the introductory and final concluding
chapters. The various titles with brief descriptions of the chapters are shown in Table 1.2.
Table 1.2. Brief description of the dissertation write-up

Chapter
Title
General introduction
Problem statement, motivation, hypothesis and

objectives for the work
One
Two
Brief description
Mycohydrometallurgy: application of fungi in
Literature review of the diversity and application
hydrometallurgy
of fungi biosorption, bioleaching and degradation

of organic matter
Three
Four
Five
Mycohydrometallurgy: Coal model for potential
Experiments establishing the potential of P.
reduction of preg-robbing capacity of
chrysosporium to reduce gold adsorption (preg-
carbonaceous gold ores using the fungus,
robbing) capacity of carbonaceous matter in
Phanerochaete chrysosporium
refractory gold ores
Fungal biotransformation of anthracite-grade
Establishment of a set of incubation parameters
carbonaceous matter: effect on gold cyanide
that leads to maximum reduction in preg-robbing
uptake
of anthracite when using P. chrysosporium
Gold adsorption on carbonaceous matter
Ability of carbonaceous matter of different
following fungal treatment: comparison of
maturity to adsorb different gold complexes (i.e.,
cyanide and non-cyanide gold complexes
cyanide, thiourea, and thiosulfate), and reduction

in adsorption due to fungal-biotransformation
Six
Biotransformation of sulfides using the fungus,
Experiments establishing the ability of P.
Phanerochaete chrysosporium: A potential
chrysosporium to oxidize pyrite and arsenopyrite
pretreatment process for refractory sulfidic gold
in refractory sulfidic gold ores
ores
Seven
Biotransformation of double refractory gold ores
Experiments to establish the ability of P.
by the fungus, Phanerochaete chrysosporium
chrysosporium to simultaneously deactivate

carbonaceous matter and decompose sulfides in
double refractory gold ores (DRGO), thus
increasing gold extraction via cyanidation
Eight
In vitro biotransformation of sulfides and
Preliminary investigations to assess the possibility
carbonaceous matter in refractory gold ores
of simultaneous biotransformation of DRGO with
using cell-free extracts of Phanerochaete
cell-free extracts of P. chrysosporium.
chrysosporium
Nine
Conclusions and recommendations for future
General conclusions, recommendations for future
research
work and processing proposals
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12
Chapter 2
Mycohydrometallurgy: Application of fungi in hydrometallurgy
Abstract
Mycology is the study of the properties of fungi as well as their beneficial and adverse effects on
humans and the environment, whereas hydrometallurgy is a branch of extractive metallurgy that
focuses on the use of aqueous chemistry to recover metals from materials such as ores, concentrates,
scrap and residues. Mycohydrometallurgy is therefore adopted to define the connection between
mycology and hydrometallurgy, hence, the application of fungi in the field of metal extraction.
For decades, bacterial-based systems have been the focus of research efforts in biohydrometallurgy
whereas fungal-mediated hydrometallurgy has received relatively little attention. Bacteria are useful in
the oxidation of inorganic substances such as sulfide minerals and some organic materials like low
rank coals, while fungi are capable of biotransforming recalcitrant (difficult to degrade) organic
matter. Different types of fungi have been utilized in many areas where such organic matter is
encountered, including the paper and pulp industry, breweries, coal solubilization, environmental
clean-up and nutrient recycling in soils. In this study, the literature on the application of fungi in
hydrometallurgy is reviewed so as to provide a general background with respect to diversity, versatility
and hydrometallurgical applications in the areas of biosorption, bioleaching and biodegradation of
recalcitrant organic matter. The biotransforming activities of the fungus of interest, Phanerochaete
chrysosporium, are also highlighted.
Keywords:
Mycohydrometallurgy;
Mycology;
Hydrometallurgy;
Fungi;
Biosorption,
Bioleaching;
Biodegradation
13
2.1
Introduction
Microorganisms fall into five major groups which are bacteria, viruses, fungi, algae and
protozoa, and biotechnology exploits the spectacular metabolic versatility of these
microorganisms in the environment (Shuler and Kargi, 1992; Glazer and Nikaido, 1995). The
contributions of bacteria and fungi to microbial biotechnology far exceed those of the other
microorganisms, and these have been studied extensively (Stanier et al., 1986; Tortora et al.,
2004; James et al., 2006; Madigan and Martingo, 2006). Biohydrometallurgy, an extension of
biotechnology, is the branch of extractive metallurgy which focuses on the use of aqueous
chemistry and biological systems to recover metals from ores and metallurgical products
(Gupta and Mukherjee, 1990; Glazer and Nikaido, 1995; Madigan and Martinko, 2006).
Biohydrometallurgy has focused on bacterial-based systems for decades whereas fungalmediated hydrometallurgy has received relatively little attention (Sterflinger, 2000). Bacteria
are useful in the oxidation of inorganic substances such as sulfide minerals and some organic
materials like low rank coals (lignite) whereas fungi are capable of biotransforming
recalcitrant organic matter.
Fungi are classified as a kingdom that is separate from plants, animals and bacteria (Tortora et
al., 2004; James et al., 2006; Madigan and Martingo, 2006; Anon, 2009a). Several types of
fungi have been used in the degradation of organic matter in the biopulping industry,
breweries, coal solubilization, environmental clean-up and nutrient recycling in soils (Bumpus
and Aust, 1986; Glazer and Nikaido, 1995; Sterflinger, 2000). The metabolic versatility of
fungi and their application in hydrometallurgy is currently being recognized and
mycohydrometallurgy is adopted to define the link between mycology and hydrometallurgy,
thus the application of fungi in metal recovery.
Fungi are particularly effective in colonizing and degrading wood and other organic carboncontaining materials such as coal, by secreting powerful extracellular enzymes and nonenzymatic agents (Crawford et al., 1983; Cohen et al., 1987; Tien and Kirk, 1988; Quigley et
al., 1989; Hofrichter et al., 1997; Holker et al., 1999; Fakoussa and Hofrichter, 1999). The
oxidative enzymes include lignin peroxidase, manganese peroxidase and laccase, and
14
hydrolytic enzymes, mainly esterases, whereas the non-enzymatic agents include alkaline
metabolites and natural chelaters such as biogene amines and ammonium oxalate (Fakoussa
and Hofrichter, 1999). In this chapter, the literature on the application of fungi in
hydrometallurgy is reviewed so as to provide a general background with respect to diversity,
versatility and hydrometallurgical applications such as biosorption, bioleaching and
degradation of recalcitrant organic matter.
2.2 Fungi
Fungi are neither plants nor animals and exist as a different group of living organisms
(Martin, 2002). Mycology is the discipline of biology devoted to the study of fungi and is
often regarded as a branch of botany, even though genetic studies have shown that fungi are
more closely related to animals than to plants (Tortora et al., 2004; James et al., 2006;
Madigan and Martingo, 2006; Anon, 2009a).
The main difference between plants and fungi is that fungi lack chlorophyll and hence cannot
synthesize food from sunlight through the process of photosynthesis. Like animals, fungi
obtain food from other organisms, and as fruiting bodies the large ones may be used for food.
The fruit is supported by a vast network of extremely fine, hair-like filaments known as
hyphae with which they access soil, wood and leaf litter, breaking it down and feeding on the
nutrients. A mass of hyphae is known as the mycelium. Fungi reproduce by releasing large
numbers of small spores estimated at 200 million per hour (Martin, 2002; Madigan and
Martinko, 2006).
Fungi may be saprophytic (feed on dead plants and animal materials), parasitic (feed off a
living host) or symbiotic (share a mutually beneficial relationship with another organism).
Saprophytic fungi generate enzymes to soften the dead plant or animal. These enzymes
increase the decomposition rate of the dead material and the fungi thus digest food externally,
and reabsorb the nutrients. These fungi are recyclers and are responsible for the release of 85
% of carbon in dead plant and animal material, and free up the nutrients making them
15
available to other living organisms. Phanerochaete chrysosporium, Ceratocystis fagacearum

and Mycorrhizal are examples of saprophytic, parasitic and symbiotic fungi respectively.
Parasitic fungi are harmful to hosts while symbiotic fungi penetrate the roots of trees and feed
on the nutrients produced during photosynthesis. In response to the symbiotic relation, the
fungal hyphae acts as an extension of the roots of the plant, collecting essential resources such
as water, phosphorus and trace elements which are fed back into the host plant. In addition,
symbiotic fungi protect the host plant by attacking parasitic fungi (Martin, 2002; Madigan and
Martinko, 2006).
2.3
Sorption of metals by fungi
The need for cheaper and more efficient systems for the removal of heavy metals from
effluents and from contaminated environments has generated interest in the use of biological
systems, and these systems have been studied considerably in recent times. Available
literature indicates that the use of plant biomass and that of lower animals in the sorption of
heavy and toxic metals have several advantages over chemical techniques due to their
efficient removal of metal ions at lower concentrations (Brierley, 1990; Glazer and Nikaido,
1995; Volesky, 2001; Yalcinkaya et al., 2001).
Several fungi have been investigated for their metal sorption/bioaccumulation activities, and
the complexity of the fungal structure implies that there are many ways by which dissolved
metals can be captured by the cells. The sorption mechanisms are therefore diverse and in
some cases they are still not very well understood (Brierley, 1990; Glazer and Nikaido, 1995;
Volesky, 2001; Yalcinkaya et al., 2001; Madigan and Martinko, 2006). In general, however,
fungal sorption mechanisms can be divided into (1) metabolism dependent and (2) nonmetabolism dependent. Relative to where the metal removed from solution is found, sorption
may be classified as (a) extracellular accumulation, (b) cell surface sorption and (c)
intracellular accumulation (Volesky, 2001).
16
Biosorption can be accomplished either with only fungal biomass or fungal biomass
immobilized in other materials such as carboxymethyl cellulose (Lakshmanan et al., 1989;
Alibhai et al., 1993; Yetis et al., 2000; Blaudez et al., 2000; Yalcinkaya et al., 2001; Baldi et
al., 2007). Baldrian (2003) noted that the two white rot basidiomycetous fungi, Phanerochaete
chrysosporium and Trametes versicolor have the ability to bioaccumulate Cd, Cu, Hg, Ni, Pb,
Cr and Zn. Investigations by Pryfogle et al. (1989) shows that the fungi Rhizopus cohnii and
Rhizopus arrhizus bioaccumulate chromium, and Aspergillus niger, Pleurotus pulmonarius
and Schizophyllum commune sorb copper. The microorganisms, Aspergillus niger,
Penicillium simplicissimum, Aspergillus foetidus and Aspergillus carbonarius are known to
survive in environments with high metal ion concentrations and exhibit high metal tolerance
in the presence of Ni, Co, Fe, Mg and Mn up to metal ion concentrations of 2000 mg/L (Valix
et al., 2001; Valix and Loon, 2003).
A summarized compilation of biosorption of heavy metals by fungi in the literature is

presented in Table 2.1. As shown in the table, the two most tested fungi are Phanerochaete
chrysosporium and Trametes versicolor which demonstrated the ability to sorb Cr3+, Cd2+,
Ni2+, Pb2+, Cu2+ Zn2+ and Hg2+ from both wastewater and contaminated soils. From initial
metal ion concentrations of between 10 and 700 mg/L, sorption values of up to 100% were
recorded utilizing Phanerochaete chrysosporium in the sorption of Pb2+, Cu2+ and Zn2+.
17
Table 2.1a. Heavy metal biosorption by fungi

Fungus name and mode of
Metal sorbed
Experimental techniques
contact
Phanerochaete chrysosporium
Trametes versicolor, mycelia
Phanerochaete chrysosporium,
Quantitative estimates per
Authors
gram of biosorbents
Cr3+, Cd2+, Ni2+, Pb2+,
2+
2+
Cu and Hg
2+
2+
Review of sorption of metals by
N/A
Baldrian (2003)
135.3, 102.8, 50.9 mg/g of
Iqbal and Edyvean
1050 C
Pb, Cu and Zn
(2004)
60 mins
20-100 % removal
fungal biomass
2+
Pb , Cu and Zn
pH 6.0
o
immobilized biomass
Initial concentration 10-400 mg/L

Phanerochaete chrysosporium,
2+
2+
Pb and Zn
Polyporous versicolor,
Arca et al. (2003;
25 C
of Pb and 30, 37 and 48
2004)
Initial concentration 30600 mg /L
mg/g for Zn
N/A
57.5 and 110 mg Pb/g
Yetis et al. (2000)
15 and 45 oC
124 and 153 mg Cd/g
Yalcnkaya et al.
Initial concentration 30700 mg/L
68-84 % removal
(2002)
pH 4.0 and 6.0
1.51 -1.84 mmol/g Cu, 0.85 - Bayramoglu et al.
immobilized biomass
Phanarochaete chrysosporium,
230, 282 and 355 mg/g
pH 5.0 and 6.0
Cu2+, Cr3+, Cd2+, Ni2+ and

2+
Pb
mycelium
Trametes versicolor,
Cd2+
immobilized biomass
Trametes versicolor,
immobilized biomass
Cu2+, Pb2+ and Zn2+
15 and 45 C
1.11 mmol/g Pb and 1.33-
1 hr
1.67 mmol/g Zn
(2003)
1.0 g/L stock solution
18
Table 2.1b. Heavy metal biosorption by fungi (continued)

Application
Metal sorbed
Authors
contact
Sphaerotilus natans, biomass
Cd2+, Cu2+
pH 3, 4, 5 and 6.0
N/A
biomass concentration 0.5, 1.0 and 2
Esposito et al.
(2001)
g/L
Funalia trogii, immobilized
Hg2+, Cd2+ and Zn2+
biomass
pH range 3.07.0
Hg, Cd and Zn were 403.2,
15 and 45 oC
191.6, and 54.0 mg/g
Arca et al. (2004)
Initial concentration 30600 mg/L
Pleurotus pulmonarius and
Cu2+
pH 2.0 and 4.0
Schizophyllum commune,
30 and 50 oC, 12 h
dead fungal biomass
Initial concentration 4 and 100 mg/L
1.6-5 mg/g
Veit et al. (2005)
70 %; 23.62 mg/g
Mukhopadhyay et
biomass concentration 0.5 and 3. g /L

Aspergillus niger, biomass
Cu2+
pH 6.0
30 min - 24 hr
al. (2007)
Initial concentration 100, 50, 25, 10

mg/L
100 mg biomass
19
Table 2.1c. Heavy metal biosorption by fungi (continued)

Application
Metal sorbed
Authors
contact
Penicillium sp., biomass
Zn2+
pH 3-7
70 % in 5 min
30-70 C
Mishra and
Choudhuri (1996)
Initial concentration 4-20 mg/L ions

0.1-0.5 g biomass
Rhizopus cohnii,
Cr6+
28 oC
immobilized living and dead
10 min
biomass
Initial concentration 50 mg/L Cr6+
Rhizopus arrhizus, biomass
Cr6+ and Fe3+
25 oC,
150-300 mg/g
Li et al. (2008)
50-500 mg/L
Sag et al. (1998),
50 and 500 mg/ L stock solution, 1 g
Sag (2001)
cell wt /L,
Streptomyces rimosus, biomass
Cu2+, Zn2+ and Cr6+
pH 5,
30 mg/g Cu
25C,
27.4 mg/g Zn and 26.7
Initial concentration 0300 mg/L,
mg/g Cr
Chergui et al. (2007)
biomass dry weight 3 g/L
20
2.4
Leaching of metals by fungi
Several fungi have been identified for their abilities in metal leaching (Dave and Natarajan,
1981; Baglin et al., 1992; Sukla and Panchanadikar, 1993; Torma et al., 1993; Tzeferis and
Agatzini-Leonardou, 1994; Castro et al., 2000; Brandl et al., 2001; Valix and Loon, 2003; Le
et al., 2006; Santhiya and Ting, 2005; 2006; Thangavelu et al., 2006; Mohapatra et al., 2007;
Ren et al., 2009; Simate et al., 2010). The microorganisms may be utilized either in vivo
(whole cells) or in vitro (cell free extracts); with the former mostly used.
The metals leached and the conditions utilized are presented in Tables 2.2. It can be seen from
the table that the most utilized fungal genus is Aspergillus (about 70% of the time), with
Aspergillus niger being the most versatile species. A. niger has been investigated for leaching
of ores and metallurgical products, and is capable of secreting organic components such as
oxalic and citric acids that dissolve the metals at temperatures between 30 and 40 oC. The
metals dissolved include Cu, Sn, Al, Ni, Pb, Mn and Zn, and extraction efficiencies are very
high up to 99%. For about 90% of the time, shake culturing is employed.
21
Table 2.2a. Heavy metal leaching with fungi

Application
Metal leached
Authors
contact
Aspergillus and Penicillium,
Leaching of metals from
Shake at pH 7.5 at 30 oC for 4 hr to
whole cells and supernatants
siliceous ores
5 days for leaching of various heavy
Zn and Ni
Castro et al., (2000)
metals from calamine, garnierite

Aspergillus niger, whole cells
Leaching of the metals from
Shake at pH 6.7 at 28-35 oC for 5-
11-87.4 % recovery of
Torma et al (1993)
municipal solid wastes
25 days for leaching of various
Al, Fe, Pb, Cd, Mn,
Wu and Ting (2006)
(MSW) incineration fly ash
heavy metals from fly ash,
and Zn
Xu and Ting (2009)

Yang et al. (2009)
Aspergillus niger, extra-cellular
Clean up of soil contaminated
Shake at near-neutral pH 7.8 at 30
15-100 % of Cu, Cd, Pb Ren et al., (2009)
components
by passage of wastewater
and Zn
C for 15 days for leaching of
various heavy metals from

contaminated soil
Aspergillus sp., whole cell
Nickeliferous ore leaching
Shake at pH 6.5 at 37 oC,
0.1-92 % of Ni, Fe, Co,
Sukla and
Nickeliferous lateritic ore
Mn, Mg
Panchanadikar,
(1993)
22
Table 2.2b. Heavy metal leaching with fungi (continued)

Application
Findings
Authors
contact
Aspergillus niger,
Adaptation of fungal strains
Penicillium simplicissimum,
to nickel toxicity in nickel
Aspergillus foetidus and
laterite leaching, Tolerance to
Aspergillus carbonarius strains,
Ni, Co, Fe, Mg and Mn
25-32 oC for 9-18 days
Up to 2000 ppm of Ni,
Valix et al. (2001)
Co, Fe, Mg and Mn
Valix and Loon

(2003)
Whole cell
Penicillia and
In situ leaching of
Shake at pH 3.3-4 at 25 C-30 C
Aspergilli, whole cell
nickeliferous laterite ores
for 48 days
Aspergillus foetidus
Salt tolerance development
N/A
whole cell
Aspergillus foetidus
Weathered saprolite ore
whole cell
25-60 % of Ni, Co, Fe
Alibhai et al., (1993)
Up to 2000 ppm of salt
Thangavelu et al.
concentration
(2006)
Shake at pH 3.85.7 at 30 C, for 12
Le et al. (2006)
-14 days
Aspergillus niger, CMI 31821
Batch and semi-continuous
Shake at pH 1.8-3.7 at 30 oC for 30-
9-64 % Ni, Fe, Al, V,
King et al. (1987)
whole cell and cell-free
leaching of nepheline
60 days
Mo, and Sb
Aung and Ting
bioleaching from fluid
(2005)
catalytic cracking catalyst
Santhiya and Ting

(2005)
23
Table 2.2c. Heavy metal leaching with fungi (continued)

Application
Findings
Authors
contact
Cladosporium oxysporum
In situ leaching of mainly low
Aspergillus flavus and
grade oxide uranium ore
50-71 % U dissolution
Mishra et al. (2009)
P dissolution
Xiao et al. (2008)
10-99 %
Baglin et al (1992)
65-95 % Cu, Sn, Al, Ni,
Brandl et al. (2001)
Curvularia clavata
Candida krissii, Penicillium
TCP, aluminium phosphate
Shake at pH 7 at 30-32 oC at 140-
expansum and Mucor
(AlPO4)
160 rpm for 7 days
Aspergillus niger
Manganese ore
4-57 weeks
Aspergillus niger, Penicillium
Electronic scrap
ramosissimus
simplicissimum
Penicillium sp and Aspergillus
Pb, and Zn
Nickeliferous laterites
30 C for 50 days
55-60 % of Ni
Tzeferis (1994)
Aspergillus niger and
Raw or roasted low-grade
30 C for 28 days
14-34 % extraction of
Mohapatra et al.
Aspergillus fumigates
chromite overburden
Ni
(2007)
sp
24
2.5
Biotransformation of sulfur compounds by fungi
Different fungi are involved in the oxidation or reduction of inorganic/organic sulfur

compounds, and biotransformation activities may involve the use of whole cell, cell-free
extracts and enzymes (Wainwright, 1978; Wainwright and Killham, 1980; Killham et al.,
1981; Schreiner et al., 1988; Faison et al. 1991; Ray et al, 1991; Van Hamme et al., 2003).
Various species of fungi have been employed in bio-desulfurization of coals with organic and
inorganic sulfur content of up to 6 %. Removal of up to 90 % of inorganic sulfur and 30 % of
organic sulfur has been reported (Faison et al., 1991; Acharya et al., 2005; Gonsalvesh et al.,
2008). The fungal-decomposition of sulfur has been attributed to the secretion of oxidative
enzymes (Schreiner et al., 1988; Aranda et al., 2009). Some fungi, for example Fusarium
oxysporium, are found to adapt to anaerobic conditions by replacing the energy-producing
mechanism of oxygen respiration with sulfur reduction, and in the process sulfur is converted
to hydrogen sulfide (Abe, 2007).
Table 2.3 presents fungal biotransformation of some sulfur compounds. It can be seen that
fungi are mainly utilized in the desulfurization of coal and degradation of sulfur in soils. In
coal, organic sulfur was the target while in soils, S 2O32- S4O62- and SO42- were tested with
varying degrees of success.
Table 2.3. Fungal transformation of sulfur compounds

Fungus name and
Application
Findings
Authors
mode of contact
Aureobasidium
pullulans, cell-free
Sulfur oxidation
in soil
extracts and enzyme

Rhizopus oryzae
whole cell
Sulfur oxidation
in soil
Oxidation of S, S2O32- and

2-
S4O6 at 25 C, for 24 h
Enzymatic oxidation; 141

mg/L of dissolved S2O3
S4O62-,
and
Oxidation of S, S2O32- and
52-290 mg/L of S2O32-
2-
S4O6 , S2O5 , CN at 28-32

o
2-
S4O6 and
Killham et al.
(1981)
SO42-
under shake culturing

2-
and
2-
SO42-
Ray et al.
(1991)
C at pH 6 for 7, 10 days,
under stationary culturing

Paecilomyces sp.
Desulfurization
Oxidation of
2,2-dihydroxybiphenyl
Faison et al.
whole cell
of coal-related
dibenzothiophene, ethyl
sulfones produced
(1991)
compounds
phenyl sulfide and diphenyl

sulfide
Phanerochaete
Desulfurization
Oxidation of organic sulfur
More water soluble
Schreiner et al.
chrysosporium BKM-
of coal and
thianthrene
thianthrene
(1988)
F-1767
petroleum.
monosulfoxide produced
Purified enzymes
Aspergillus sp.
Desulfurization
Incubation of 2 wt% of -74
Removal of 7080 % total
Acharya et al.
whole cell
of coal
m coal with 2.13 %
sulfur
(2005)
inorganic and 1.21 % organic

for 10 days
Agrocybe aegerita and
Desulfurization
Incubation of
oxidized
Aranda et al.
Coprinellus radians,
of fossil fuels
dibenzothiophene in vivo and
dibenzothiophene (DBT)
(2009)
whole cells and
in vitro at pH 7 at 100 rpm at
(110 M) by 60-100 %
purified extracellular
24 C for up to 16 days under
into DBT sulfoxide, DBT
peroxygenases
shake culturing
sulfone
30-37 C
reduction of 13 % organic
Gonsalvesh et
sulfur and 79 % inorganic
al. (2008)
Trametes Versicolor
Desulfurization
ATCC No. 200801,
subbituminous
Phanerochaete
coal
Chrysosporium
ME446, Pleurotus
Sajor-Caju
whole cell
26
2.6
Biotransformation of organic carbon-containing materials
Several investigations have shown that there are some microorganisms involved in the
degradation and/or mineralization of organic carbon-containing materials such as coal, humic
acid, lignin, plastics and rubber. Others are automobile tires, diesel oil and sewage (Glazer
and Nikaido, 1995; Fakoussa and Hofrichter, 1999; Tsuchii and Tokiwa, 2002).
Mineralization converts the organic substances into inorganic substances and water (Wilson et
al., 1987; Pyne et al., 1987; Strandberg and Lewis, 1987; Quigley and Dugan, 1989). The
biotransformation of these compounds impact positively on metal extraction and/or
environmental clean-up (Kergomard and Renard, 1982; Bumpus et al., 1985; Bumpus and
Aust 1986; Sterflinger, 2000; Tunde and Tien, 2000).
2.6.1 Biotransformation of rubber and rubber products

Rubber and various kinds of rubber products have been used as substrates by fungi. Fusarium
solani and Spicaria violacea have been found to biotransform automobile tires and latex.
Biodegradation of rubber by the fungi generally leads to mineralization of the rubber but the
carbon black found in automobile tires may be left as residue as the components responsible
for degradation are not able to attack carbon black (Tsuchii and Tokiwa, 2002). This indicates
that these microorganisms fall short in complete degradation of the substrates. Other fungal
species that are known to biotransform natural rubber and rubber processing wastes include
Mucor racemous, Mucor sp. and Aspergilus niger (Atagana et al, 1999).
2.6.2 Biotransformation of coal
Many fungi species have been reported to possess the ability to solubilize and/or degrade coal.
Such fungi are either isolated from coal seams or tested in the laboratory on coal. The well
known species include Phanerochaete chrysosporium, Trametes versicolor, Penicillium
27
waksmanii, Penicillium sp., Aspergillus sp., Candida sp., Poria placenta, Mucor sp,
Paecilomyces sp, Fusarium oxysporum, Trichodemul atroviride, Nematoloma frowardii,
Clitocybula dusenii, Auricularia sp., Coprinus sclerotigenis and Punus tigrinus (Cohen and
Gabriele, 1982; Ward, 1985; 1993; Wilson et al., 1987; Stewart et al., 1990; Catcheside and
Mallet, 1991; Ho1ker et al., 1999). The prominent fungi involved in the transformation of
humic acids and different ranks of coal include the two white rot basidiomycetes,
Phanerochaete chrysosporium and Trametes versicolor (Cohen et al., 1987; 1990;
Fredrickson et al., 1990; Perez and Jeffries, 1992; Catcheside and Ralph, 1999; Ralph and
Catcheside, 1994; 1997; Fakoussa and Hofrichter, 1999; Afidenyo, 2008).
2.6.3 Microbial transformation of carbonaceous matter in gold ores
Based on their constituents, the formation of carbonaceous matter (CM) has been likened to
that of coals. For example, many of the organic compounds in CM associated with gold ores
such as poly aromatic hydrocarbons and humic acids are present in lignite, while the more
matured graphitic carbon is also present in bituminous and anthracite coals. In addition, CM
and coal, which is also known as carbonaceous rock (Faison, 1992; Anon, 2009c), have
similar carbon-to-hydrogen ratios (Sibrell et al., 1990, Stenebraten et al., 2000).
Consequently, most microorganisms that attack coal may be able to attack CM.
Several investigations on the microbial transformation of CM in gold ores, with the aim of
increasing gold extraction are available in the literature. Brierley and Kulpa (1992; 1993)
showed that a consortium of heterotrophic bacteria, consisting of Pseudomonas maltophila,
Pseudomonas oryzihabitans, Achromobacter sp. and Arthrobacter sp. could reduce gold
uptake by a carbonaceous ore leading to increase in gold recovery during cyanidation. Portier
(1991) used a mixture of heterotrophic fungal and bacterial species including Aspergillus
bruneio-uniseriatus and Penicillium citrinum fungi, followed by sulfide biooxidation using
Acidithiobacillus and Leptospirilum bacteria. The microbial action enhanced cyanidation gold
recovery from bituminous grade carbon-containing gold-bearing sulfur-containing ores.
28
Increase in gold extraction as a result of carbon degrading microbial pretreatment with

Streptomyces setonii, following sulfide oxidation of double refractory gold ores has also been
observed by other workers (Amankwah et al., 2005; Amankwah and Yen, 2006; Yen et al.,
2008).
Using S. setonii, Amankwah and Yen (2006) observed a more extensive biodegradation in
lignite and bituminous coal than in anthracite. In a solution of 5 mg/L Au, biotreatment
reduced the gold adsorption capacity of bituminous, lignite and anthracite coals respectively
from an average of about 19 % to 0.3 %, 22 % to 9 % and 45 % to 20 %. A white-rot fungus,
Trametes versicolor, has also been reported to reduce the preg-robbing effect of bituminous
coal from 40.3 % to 8.3 % and anthracite from 99.5 % to 27.7 % (Afidenyo, 2008). Most of
the treatments mentioned above, though effective with bituminous grade CM, could not
reduce the gold adsorption ability of anthracite-grade CM drastically. Anthracite-grade CM,
however, comprises about 50 % of the entire CM in refractory gold ores (Hausen and
Bucknam, 1984; Osseo-Asare et al., 1984; Sibrell et al., 1990; Pyke et al., 1999; Stenebraten
et al, 2000; Schmitz et al., 2001; Vaughan and Kyin, 2004). Thus, further investigations into
anthracite degrading microorganisms are required.
2.7 Biotransforming action of white rot fungi
White rot fungi are the main microorganisms involved in the biotransformation of organic
carbon-containing materials. Though many members make up the family of white rot fungi,
the two that have been most researched are Phanerochaete chrysosporium and Trametes
versicolor (Cohen et al., 1987; Tien and Kirk, 1988; Fredrickson et al., 1990; Perez and
Jeffries, 1992; Ralph and Catcheside, 1994; 1997; Afidenyo, 2008). Of the two,
Phanerochaete chrysosporium is the most studied, and the general characteristics of white rot
fungi are inferred from this fungus.
29
P. chrysosporium belongs to the basidiomycetous family; a family of fungi whose filaments

are characterized by an extensive network of cross-walls, and produce mushrooms as fruiting
bodies (Sachs et al., 1989; Tortora et al., 2004; Madigan and Martinko, 2006; Anon, 2009a;
2009b). The fungus secretes enzymes that digest wood and allow its filaments to penetrate
wood and degrade lignin (the most recalcitrant component of wood) with the aim of getting
access to cellulose and hemicellulose (Tien and Kirk, 1983; Sachs et al., 1989). This leaves
specks of white delignified areas amidst thin areas of firm wood, a phenomenon called white
rotting. A network of fungal hyphae of P. chrysosporium growing on aspen wood chips is
presented in Fig. 2.1 (Sachs et al., 1989). The authors reported the hyphae to be of 1-3 m in
diameter.
Fig. 2.1. Photomicrograph of Web-like hyphal network of Phanerochaete chrysosporium

(Sachs et al., 1989)
P. chrysosporium is a prominent lignin-degrading fungus, from which the first lignindegrading enzyme, lignin peroxidase, was isolated and characterized by Tien and Kirk (1983;
1984). P. chrysosporium has hence been studied in many laboratories as a model organism as
it grows quickly, degrades lignin rapidly, and has a relatively high temperature optimum (37-
30
39oC) and low pH optimum (3.5-5.0) though it can grow over a wider range of temperature
and pH (Tien et al., 1986; Andrawis et al., 1988).
Aside from lignin peroxidase (LiP), P. chrysosporium secretes manganese peroxidase (MnP)
(Glenn et al., 1983) and H2O2-generating enzyme, glyoxal oxidase (Kersten and Kirk, 1987)
under secondary metabolism in response to nutrient starvation (Kirk et al., 1986). The strong
oxidizing environment created by the extra-cellular components of the fungus catalyzes the
biodegradation of lignin and a variety of lignin-containing substrates such as low rank coals
(Tien and Kirk, 1984; Faison and Kirk, 1992; Bumpus and Aust, 1986; Hammel et al., 1986;
Kirk and Farrel, 1987; Ralph and Catcheside, 1997; Fakoussa and Hofrichter, 1999; Tunde
and Tien, 2000). The structure of lignin peroxidase produced by P. chrysosporium is
presented in Fig. 2.2. As shown in the figure, the enzymes of P. chrysosporium are hemecontaining glycoproteins possessing one ferric heme per molecule of enzyme (Tien and Kirk,
1988) and can be represented simply as P[Fe(III)].
Fig. 2.2. Structure of lignin peroxidase (A), and manganese peroxidase (B); enzymes
generated by P. chrysosporium (Banci et al., 1992)
31
When activated by hydrogen peroxide, lignin peroxidase (LiP) and manganese peroxidase
(MnP) catalyse one-electron oxidation of lignins aromatic substructure leading to formation
of cation free radicals and bond cleavage (Tien and Kirk, 1984, 1988; Glenn et al., 1983;
Hammel et al., 1986; Kersten et al., 1985; Kirk et al., 1986; Gold and Alic, 1993). The general
mechanism is shown in Equations 2.1 to 2.3 (modified after Andrawis et al, 1988; Banci et al.,
1992; Edwards et al., 1992; Gold and Alic, 1993; Fakoussa and Hofrichter., 1999). The
radicals may also react with the lignin polymer to depolymerize it, and the products may
undergo further modification to form carbon dioxide (Bumpus and Aust, 1986).
P[Fe(III)] (native enzyme)

P[O Fe(IV) ] (comp I) R
H 2 O2
P[O Fe(IV) ] (comp I) H2O
[2.1]
P[O Fe(IV)] (comp II) R
P[O Fe(IV)] (comp II) R 2H
[2.2]
H 2O
[2.3]
Compounds I and II are the two free radical intermediates involved in the catalysis of the
enzymes (Tien and Kirk, 1984; Kersten et al., 1985; Hammel et al., 1986; Kirk et al., 1986;
Fakoussa and Hofrichter, 1999). At the initial stage of the catalysis, the enzymes utilize H2O2
to get oxidized first to iron (IV) and then to a cation radical (Tien and Kirk, 1988; Kurek and
Kersten, 1995) called compound I (Equation 2.1), which is 2 oxidation jumps above the native
enzyme. Compound I subsequently reacts with a substrate molecule, R (Equation 2.2), to form
compound II, which is one-electron oxidized. LiP Compound II returns to the resting enzyme
state by reacting with the same or another molecule of substrate (Equation 2.3). The substrate
cation radical (R+) produced, can undergo a variety of non-enzymatic reactions and
spontaneous rearrangement leading to further degradation and formation of a wide range of
final products including oxygen-containing compounds as shown in Equation 2.4
(Paszczynski et al., 1985; Pease et al., 1989; Wariishi et al., 1991; Gold and Alic, 1993;
Catcheside and Mallett, 1991; Fakoussa and Hofrichter, 1999).
32
OH
OH
[2.4]
MnP Compound II, on the other hand, exhibits an absolute requirement for Mn2+ as the
reducing agent to get reduced to the resting enzyme state (Glenn et al., 1986; Pasczynski et
al., 1985; Wariishi et al., 1991; Gold and Alic, 1993). The Mn3+ ions, so produced, are
stabilized to high redox potentials by forming complexes with organic acids such as oxalate
((COO)22-),
malonate
(CH2(COO)22-),
malate
(CH3CHO(COO)22-),
tartrate
(CH3CHO2(COO)22-), and lactate (CH3CH(OH)COO-), all secreted by P. chrysosporium

(Catcheside and Ralph, 1999; Fakoussa and Hofrichter, 1999). The chelated Mn3+ diffuses
from the surface of the enzyme and acts as diffusible redox mediator that oxidizes insoluble
terminal substrates, cleaving chemical bonds such as non-phenolic and polycyclic aromatic
hydrocarbons that are normally not accessible to attack by MnP directly (Catcheside and
Ralph, 1999; Fakoussa and Hofrichter, 1999). By oxidizing the substrates, Mn3+ gets reduced
thus recycling Mn2+.
It can be deduced from the above mechanism that the oxidation of carbon by the fungus will
lead to reduction in carbon to oxygen ratios in the carbonaceous materials undergoing fungal
treatment (Equation 2.4). This can happen via complete loss of carbon through the formation
of carbon dioxide or the introduction of oxygen groups on the carbon which disrupts the
continuous nature of the graphitic planes.
As mentioned earlier, P. chrysosporium also produces hydrogen peroxide which is known to

solubilize
sulfides such as pyrite and arsenopyrite as shown in Equations 2.5 and 2.6
(McKibben and Barnes, 1986; Antonijevic, et al., 1997; Jennings et al., 2000; Aydogan,
2006).
33
2FeS2 15H2O2
2Fe3
FeAsS 7H2O2
Fe3
4SO4
AsO34
2H
SO4
14H2O
2H
6H 2O
[2.5]
[2.6]
The iron (III) and sulfuric acid produced constitute reagents that can be used for oxidation of
sulfides (Bennett and Tributsch, 1978; Brierley, 1978; 1995; Hackl, 1997; Keller and Murr,
1982; Sand et al., 2001; Rawlings et al., 2003; Marsden and House, 2006). The strong
oxidizing environment produced by the oxidative enzymes of P. chrysosporium is also known
to be responsible for the oxidative removal of sulfur from coal (Schreiner et al., 1988; Aranda
et al., 2009). Furthermore, P. chrysosporium has been used to degrade gold-bearing wood
chips, produced from underground timber support, to liberate gold for cyanide leaching
(Martin and Petersen, 2001). The authors observed a 10 % increase beyond the original 60 %
cyanidation gold recovery of untreated wood chips. It is therefore believed that P.
chrysosporium can catalyze the biotransformation of carbonaceous matter and sulfides present
in double refractory gold ores, and hence enhance gold extraction in the subsequent
cyanidation step.
2.8 Conclusions
Fungi are utilized extensively in the biotransformation of organic matter in paper and pulp
industries, breweries, coal solubilization, environmental clean-up, nutrient recycling in soils
and more recently in hydrometallurgy. Mycohydrometallurgy has been adopted to define
the application of fungi in hydrometallurgy. This review considered the literature on the
application of fungi in hydrometallurgy and expounded on the diversity, versatility and
hydrometallurgical applications such as biosorption, transformation of sulfur compounds,
leaching of metals and degradation of recalcitrant organic matter. The oxidative action of the
versatile fungus, P. chrysosporium, was discussed and the microorganism has the potential to
biodecompose metal sulfides and carbonaceous matter in refractory gold ores.
34
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42
Chapter 3
Mycohydrometallurgy: Coal model for potential reduction of preg-robbing capacity
of carbonaceous gold ores using the fungus, Phanerochaete chrysosporium
Abstract
During the cyanidation process for gold extraction, natural carbonaceous matter (CM) present in
refractory gold ores adsorbs dissolved aurocyanide complexes, thereby reducing gold extraction - a
phenomenon known as preg-robbing. The natural CM consists mainly of elemental carbon, humic
acids and hydrocarbons, and coal can be used to model the behavior of CM. In this study, samples of
lignite, subbituminous, bituminous and anthracite coals were used as surrogate materials to test the
capability of the fungus, Phanerochaete chrysosporium, to reduce the preg-robbing capacity of CM.
By utilizing several growth media including glucose, millet and wheat bran, it was established that
millet and wheat bran provided the best environment for fungal growth and biomodification of coal.
As-received, control and biotreated coal samples were subjected to gold adsorption tests and the results
indicate that P. chrysosporium can decrease the gold adsorption ability of coal by over 90% depending
on the growth media used. Reduction in gold adsorption was more pronounced in anthracite due to its
high ability to adsorb gold in the as-received form. The results demonstrate a potentially effective
alternative pretreatment process that utilizes the fungus, P. chrysosporium, in reducing preg-robbing
in gold extraction.
Keywords: Mycohydrometallurgy; Fungi; Phanerochaete chrysosporium; Biomodification; Culture

media; Controls; Coals; Carbonaceous matter; Preg-robbing; Gold adsorption; Cyanidation.
3.1 Introduction
3.1.1 Mycohydrometallurgy
Mycology is the study of the properties of fungi; their beneficial and adverse effects on
humans and the environment (Madigan and Martinko, 2006), whereas hydrometallurgy is a
branch of extractive metallurgy which focuses on the use of aqueous chemistry to recover
metals from materials such as ores, concentrates, scrap and residues (Gupta and Mukherjee,
1990; Osseo-Asare, 2009). Mycohydrometallurgy is therefore adopted to define the
connection between mycology and hydrometallurgy, thus the application of fungi in the field
of metal recovery (Chapter 2; Osseo-Asare et al., 2009).
3.1.2 Preg-robbing effect of carbonaceous matter on gold extraction

The presence of carbonaceous matter (CM) in gold ores leads to two main deleterious effects:
(a) confinement of gold with attendant difficult release from the CM matrix and (b) loss of
dissolved metal via the ability of CM to adsorb gold from the pregnant solution (Osseo-Asare
et al., 1984; Hutchins et al., 1988; Portier, 1991; Brierley and Kulpa, 1992; 1993; Amankwah
et al., 2005). Adsorption of gold by CM leads to gold losses due to the fineness of the carbon
(0.002 2 m) (Afenya, 1991) which does not permit it to be screen-separated for gold
recovery, and this phenomenon is termed as preg-robbing (Osseo-Asare et al., 1984; Hausen
and Bucknam, 1985) The most serious cause of preg-robbing is the presence of CM, though
other minerals such as silicates and sulfides in gold ores have also been implicated
(McDougall and Hancock, 1981; Hausen and Bucknam, 1985; Afenya, 1991; Pyke et al.,
1999; Rees and van Deventer, 2000a; Schmitz et al., 2001; Vaughan and Kyin, 2004; Tan et
al., 2005). CM is composed mainly of hydrocarbons, humic acids, and graphitic/amorphous
elemental carbon. The elemental carbon is the component that behaves like activated carbon
and thus adsorbs gold, and the humic acid may form complexes with gold while the
hydrocarbon fraction has negligible interaction with gold (Radtke and Scheiner, 1970; Osseo44
Asare et al., 1984; Kohr, 1994; Adams and Burger, 1998; Stenebraten et al., 1999; Pyke et al,
1999; Schmitz et al., 2001).
The extent to which CM preg-robs gold generally increases with its maturity. The graphitic
elemental carbon has maturity similar to anthracitic grade coal, which is the most matured
form of coal, whereas the amorphous elemental carbon may have maturity similar to the lower
rank coals (Osseo-Asare et al., 1984; Hausen and Bucknam, 1985; Abotsi and Osseo-Asare,
1986; Sibrell et al., 1990; Pyke et al., 1999; Stenebraten et al., 2000; Van Vuuren et al., 2000;
Vaughan, 2004; Schobert, 2007). Due to the complexity of CM, coal has been used as
surrogate in its characterization as many of the components in CM such as kerogens and
humic acids are present in lignite (the youngest form of coal) while the more matured
graphitic carbon is also present in anthracite (Ibrado and Fuerstenau, 1992; Stenebraten et al.,
1999; Van Vuuren et al., 2000; Amankwah and Yen, 2006). Indeed, kerogen and graphite
respectively span the beginning and end of the coalification process (Van Krevelen, 1993;
Schobert, 2007).
3.1.3 Biomodification of carbonaceous matter

Carbonaceous matter (CM) has to be eliminated or passivated before cyanidation in order to
reduce preg-robbing, and some of the available techniques include roasting, chlorination and
treatment with carbon-blanking agents such as kerosene and other organic reagents (Arriagada
and Osseo-Asare, 1984; Abotsi and Osseo-Asare, 1987; Hutchins et al., 1988; Adams and
Burger, 1998). The use of activated carbon to simultaneously adsorb gold during cyanidation
has also been applied commercially to out-compete the natural CM in refractory gold ores in
the carbon-in-leach technology (Matson and Fisher, 1981; Hutchins et al., 1988; Rees and van
Deventer, 2000b; Marsden and House, 2006).
Currently there are studies into biomodification of carbon, considered more attractive for
economic and environmental reasons as they occur at relatively low temperatures (25-40oC)
45
and at atmospheric pressure, and the microorganisms can easily be regenerated (Portier, 1991;
Brierley and Kulpa, 1992; 1993; Amankwah et al., 2005; Yen et al., 2008). Portier (1991)
used a mixture of heterotrophic fungal and bacterial species including Aspergillus bruneiouniseriatus and Penicillium citrinum fungi, followed by sulfide bioxidation using
Acidithiobacillus and Leptospirillum bacteria. The microbial action progressively enhanced
cyanidation gold recovery from bituminous grade carbon-containing gold-bearing sulfurcontaining ores. Brierley and Kulpa (1992, 1993), who used a carbon de-activating microbial
consortium comprising of different combinations of species of Pseudomonas, Achromobacter,
Arthrobacter and Rhodococcus, observed an increase in overall gold extraction which they
attributed to partial deactivation as a result of blinding of active sites on carbon. Increase in
gold extraction as a result of microbial pretreatment of CM has also been observed by other
workers (Amankwah et al., 2005; Amankwah and Yen, 2006; Afidenyo, 2008; Yen et al.,
2008). Amankwah and Yen (2006) investigated the effect of bacterial-treatment on different
carbonaceous materials with varying degrees of maturity (lignite, bituminous and anthracite
coals). The authors observed degradation with some degree of carbon dioxide formation when
the coals were incubated with the actinomycete, Streptomyces setonii, and they reported a
more extensive biotransformation in lignite and bituminous than in anthracite. It has also
been reported that a white-rot fungus, Trametes versicolor, is able to passivate bituminous
coal but only passivates anthracite to a lesser extent (Afidenyo, 2008).
In this study, the white-rot basidiomycetous fungus, P. chrysosporium, was investigated for its
effectiveness in deactivating the active sites of CM for gold adsorption. The fungus was
selected based on its ability to degrade a wide range of organic carbonaceous materials (Tien
and Kirk, 1984; Faison and Kirk, 1985; Bumpus and Aust, 1986; Hammel et al., 1985; 1986;
Kirk et al., 1986; Kirk and Farrel, 1987; Ralph and Catcheside, 1994, 1997; Tunde and Tien,
2000). Various ranks of coal were used as surrogates to represent CM in gold ores so as to
monitor the changes in the preg-robbing behavior as a result of biomodification. The results
reported here indicate that P. chrysosporium could reduce the adsorption behavior of
anthracite by more than 90% depending on the growth media used.
46
3.2 Phanerochaete chrysosporium

P. chrysosporium is a white rot fungus which belongs to the basidiomycetous family; a family
of filamentous fungi that exhibits mushroom-like growth with extensive network of crosswalled filaments (Madigan and Martinko, 2006; Anon, 2009a; 2009b). The filaments are able
to penetrate wood and degrade lignin (the most recalcitrant component of wood) with the aim
of getting access to cellulose and hemicellulose (Tien and Kirk, 1983), thus leaving specks of
white delignified areas amidst thin areas of firm wood, a phenomenon referred to as white
rotting. P. chrysosporium has
been studied as a model organism as it grows quickly,
degrades lignin rapidly, and has a relatively medium temperature optimum (37-39 oC) and low
pH optimum (3.5-4.5), though it can grow over a wider range of temperature and pH (Tien et
al., 1986; Andrawis et al., 1988). The fungus secretes the enzymes, lignin peroxidase (Tien
and Kirk, 1983; 1984), manganese peroxidase (Glenn et al., 1983) and a hydrogen peroxide
generating enzyme, glyoxal oxidase (Kersten and Kirk, 1987). These oxidative enzymes are
known to catalyze the biodegradation of lignin and a variety of lignin-containing substrates
including some low rank coals and persistent environmental pollutants (Tien and Kirk, 1984;
Faison and Kirk, 1985; Bumpus and Aust, 1986; Hammel et al., 1985; 1986; Kirk et al., 1986;
Kirk and Farrel, 1987; Ralph and Catcheside, 1994, 1997; Fakoussa and Hofrichter, 1999;
Tunde and Tien, 2000). P. chrysosporium has also been used to degrade gold-bearing wood
chips, produced from underground timber support, to liberate gold for cyanide leaching
(Martin and Petersen, 2001). The authors observed a 10 % increase beyond the original 60 %
cyanidation gold recovery of untreated wood chips.
3.3 Experimental investigations
3.3.1 Materials
Coal (anthracite, bituminous, sub-bituminous and lignite) samples with particle size less than
850 m were provided by the Coal Bank of the EMS Energy Institute and these were crushed
47
and sieved to all passing 250 m before incubation. Fungal spores of P. chrysosporium
ME446, were kindly obtained from Prof. Ming Tien of the Department of Biochemistry and
Molecular Biology all of the Pennsylvania State University. Standard gold solution (50 g/mL
in 0.1 % sodium hydroxide and 0.05 % sodium cyanide) was supplied by High Purity
Standards, and all other chemicals used were of reagent grade.
3.3.2 Media preparation and incubation

Biotransformation was explored by incubating coal samples with the fungus, P.
chrysosporium, cultivated in different media at 37 oC for growth periods of up to 4 weeks, and
within pH of 4.0-7.0 depending on the growth medium. The coal samples were autoclavesterilized at 121 oC, and the different media were either liquid or solid substrates as described
in the subsections below.
Glucose broth and glucose agar media

Glucose broth and glucose agar media were prepared as described by Tien and Kirk (1988).
The media without thiamin were autoclaved for 30 minutes and cooled down before addition
of thiamin, which is heat-sensitive. Moist culturing with the fungus was done in agar plates
with the glucose agar whilst liquid-submerged culturing was done in Erlenmeyer flasks with
the glucose broth. The sterilized coal samples were added 7 days after inoculation of fungal
spores in the media. Oxygen was introduced into the liquid cultures every three days as
described by Tien and Kirk (1988).
Millet and wheat bran media

The use of solid substrates such as millet alone, and millet + wheat bran (MWB) obtained
from Natures Pantry, State College, was also investigated. The media were prepared by using
48
10 g of millet for millet alone medium, and 8 g millet plus 2 g wheat bran for MWB medium.
The media, in Erlenmeyer flasks, were moistened with water in the ratio of 1 g media: 1 mL
double-distilled (dd) H2O, and autoclaved at 121 oC for 30 minutes. On cooling, the media
were inoculated with 1 mL each of spore suspension of P. chrysosporium (made by
suspending 1 vial of spores in 25 mL of dd H2O). The optical density of the cell suspension,
used for inoculation, as measured with a Pharmacia LKB Ultraspec II UV-visible
spectrometer at 600 nm was 0.3 relative to water. After a growth period of one week, 10 g of
sterilized moist samples of the various coals were introduced into the cultures. After one week
of incubation, the fungus grew over all of the coal samples in the millet, and MWB, and
sandwiched the coals within two weeks. Control experiments were also conducted to establish
the exclusive effect of P. chrysosporium on preg-robbing capacity of coal. Fig. 3.1 shows the
autoclaved media (3.1a and 3.1b), after one week incubation with P. chrysosporium (3.1c and
3.1d), and after two weeks incubation with coal (3.1e and 3.1f). A closer look showing the
hyphal growth of P. chrysosporium on anthracite coal after 2-week incubation in MWB
medium is presented in Fig. 3.2
Fig. 3.1. Incubation of P. chrysosporium in millet and wheat bran. Autoclaved millet (A),
autoclaved MWB (B), 1-week culture of fungus and millet (C), 1-week culture of fungus and
MWB (D), 2-week incubated culture of P. chrysosporium with anthracite and millet (E), and
2-week incubated culture of P. chrysosporium with anthracite and MWB (F)
49
Fig. 3.2. Growth of P. chrysosporium on anthracite coal after 2-week incubation in MWB
medium
3.3.3 Incubation and harvesting of carbonaceous samples

All experiments with the fungus were done under aseptic conditions to avoid introduction of
other organisms. The coals were introduced into 1-week pre-grown cultures and incubated in
triplicates within the given contact time at 37 oC. At the end of the period the samples were
washed with water to get rid of the media and fungal biomass. Complete removal of the
fungal biomass in the case of glucose media was not possible, especially with the lower rank
coal samples, as the biomass adhered tenaciously to the carbon samples. The washed samples
were dried at 37 oC for 7 days. In general, visual observation indicated that growth of the
fungus was very impressive for the incubation conditions and the substrates investigated,
confirming the broad substrate utilization by P. chrysosporium (Tien and Kirk, 1988).
50
3.3.4 Gold adsorption experiments

Gold solution (50 g/mL = 254 M) was diluted with 1 g/L (25 mM) NaOH at pH 12 to
obtain a 5 g/mL (25.4 M) gold solution which was used for the adsorption tests. Triplicate
samples of biomodified, control and as-received coals of various weights (0.1 g - 1 g) were
contacted with 25 mL of 5 g/mL gold solution and agitated at 150 rpm on a New Brunswick
Series 25 incubator shaker for 24 hours. At the end of the contact time the residual solution
was filtered and gold in the filtrate determined using Perkin-Elmer Optima 5300 inductively
coupled plasma atomic emission spectrometer (ICP-AES). The difference between the
concentration of gold in solution before and after the adsorption test was computed with
respect to the amount of carbon-containing material used for the adsorption, as shown in
Equation 3.1, to obtain the preg-robbing effect of carbon (PEC) in mol/g.
PEC in
mol of gold
g of carbon
IC
25 mL x
FC
197 g
g
mL
mol
1
WC g
[3.1]
In Equation 3.1, IC and FC are the initial and final concentrations of gold in solution, WC is
the weight of carbon material used in the adsorption test, 25 is the volume of gold solution
used and 197 is the molar mass of gold. The difference in PEC values between as-received
and treated carbonaceous material gives an indication of the effect of fungal-biomodification
on preg-robbing of gold by the carbon-containing materials.
3.4 Results and Discussion

P. chrysosporium was incubated with various carbonaceous materials; lignite, subbituminous,
bituminous and anthracite coals in liquid and solid substrate media. The response to fungal
biomodification was determined by the reduction in the ability of carbon materials to adsorb
gold which can be translated into a reduction in preg-robbing ability of carbonaceous matter.
Equation 3.1 was used to compute the preg-robbing effect and the fungal-treatment variables
51
considered were coal ranks, contact time and growth media as demonstrated in the ensuing
figures. Control experiments were set up to monitor the direct contribution of the media
utilized. The results are presented and then discussed afterwards.
3.4.1 Effect of carbonaceous characteristics on biotransformation

For tests performed in submerged glucose media, Fig. 3.3 shows the effect of incubation time
on the adsorption of gold by the various coals. The gold adsorption capabilities of as-received
lignite, subbituminous and bituminous coals were very low, below 0.5 mol of gold per gram
of carbon while that of anthracite was very high, at 4.5 mol/g.
Fig. 3.3. Effect of fungal-treatment time on gold adsorption by lignite, subbituminous,

bituminous and anthracite coals. The growth medium was glucose broth
As indicated in Fig. 3.3, within four weeks of incubation in submerged glucose medium, the
adsorption capacities of lignite, subbituminous and bituminous coals did not change
significantly owing to the fact that the as-received did not adsorb much gold (less than 0.5
52
mol/g). However, the adsorption capacity of anthracite was greatly reduced from about 4.5
mol/g to 0.4 mol/g after incubation with P. chrysosporium for one week, and described a
plateau at that value as the contact time was extended beyond one week up to four weeks.
Figure 3.4 presents the effect of 2-week fungal-incubation in moist glucose agar medium on
the preg-robbing ability of various coals. The diagram depicts about the same level of gold
adsorption before and after fungal incubation for all the coal materials except for anthracite.
Figure 3.5 illustrates the effect of 2-week fungal-incubation in millet and MWB on the gold
adsorption ability of various coals. The trend in Fig. 3.5 confirms those observed in Figs. 3.3
and 3.4, with anthracite demonstrating significant adsorption in the as-received state, and
significant reduction in gold adsorption (over 90%) after fungal-treatment.

subbituminous, bituminous and anthracite coals. The growth medium was moist glucose agar
53

subbituminous, bituminous and anthracite coals. The media used were millet and MWB
Since the as-received samples of lignite, subbituminous and bituminous coals did not show
significant gold adsorption capabilities in the cyanide medium, it might not be appropriate to
measure the degree to which these coals have been biotransformed using aurocyanide
adsorption test. Gold adsorption test in cyanide medium, on the other hand, can be used to
predict the extent of biotransformation in anthracite by P. chrysosporium. The low gold
adsorption for both the as-received and biomodified lignite, sub-bituminous and bituminous
coals was observed throughout this work. Bio-treated samples of both lignite and
subbituminous coals showed slightly higher gold adsorption when treated in glucose agar
medium and this may be attributed to the entrained biomass, which was found in this research
to possess some minor degree of adsorption capacity.
54
In contrast, higher gold adsorption in as-received lignite and bituminous coals, and a more
intense bacterial-biodegradation in lignite and bituminous than anthracite coals was observed
by Amankwah and Yen (2006) using S. setonii. The literature contains varied reports on
adsorption of gold onto different coal materials from different origins and of different
maturity. These observations are due to the wide variations in coal deposits resulting, in part,
from different plant inputs and coal generating conditions (Faison, 1992; Fakoussa and
Hofrichter, 1999; Schobert, 2007), which precludes the direct transfer of the characteristics of
coals in one area to coals of other regions. The trend, however, is always very high gold
adsorption on anthracite relative to lignite, subbituminous and bituminous coals (Ibrado and
Fuerstenau, 1992; Van Vuuren et al., 2000; Amankwah and Yen, 2006).
The atomic ratio of carbon to oxygen in coal decreases with coal maturation or extent of
coalification from lignite to anthracite. Lignite, the geologically youngest coal, is formed from
the decomposition of kerogen, which occurs at the halfway point across the conversion of
organic matter to fossil fuel in the coalification process. The process beyond lignite is marked
initially by demethoxylation of lignin, decarboxylation of resins and coupling of phenolic
compounds liberated by lignin decomposition to form subbituminous coal. This is followed by
partial dealkylation of aromatic ring systems and resins, and aromatization of resin structures
to form bituminous coal. The final coalification jump to anthracite occurs through substantial
demethanation of aromatic systems, thermal breakdown of aliphatic structures to poly-enes,
and cyclization of poly-enes to aromatics, stacking and growth of aromatic ring system and
structural ordering (Faison, 1992; Van Krevelen, 1993; Schobert, 2007; Anon, 2009c).
Using data from the Coal Bank of the EMS Energy Institute, the Pennsylvania State
University, atomic ratios of carbon to oxygen in the coal samples were estimated to be
between 5 and 11 for lignite, subbituminous and bituminous coals, whereas it was 42 for
anthracite. This might explain the relatively higher aurocyanide adsorption on anthracite due
to higher degree of graphitization as against the other coal ranks (Fig. 3.6).
55
Fig. 3.6. Correlation between the atomic ratios of carbon to oxygen and gold adsorption by
lignite, subbituminous, bituminous and anthracite coals
The two most important properties of carbon for gold adsorption are large specific surface
area and highly developed graphitic structure (Jones et al., 1989; Ibrado and Fuerstenau;
1992). It has been reported that for low rank coals, micro-porosity is more important while for
high rank coals, the importance is more on active sites for adsorption (Van Vuuren et al.,
2000). Though bituminous is more matured than lignite and sub-bituminous, it is the least
porous among all the coal ranks. The porosity of anthracite is higher than that of bituminous
coal but lower than that of lignite. Nevertheless, anthracite has far more active sites for gold
adsorption than lignite owing to its higher degree of graphitization for the reason that
increased graphitization is known to boost gold adsorption than porosity (McDougall and
Hancock, 1981; Jones et al, 1989; Van Krevelen, 1993).
56
3.4.2 Effect of growth media

Visual inspection indicated that the fungus preferred millet and wheat bran over the glucose
media. This gives a technical advantage since, as compared to the glucose media, millet and
wheat bran are simpler and more cost effective in terms of components and expertise required
for preparation. Millet and wheat bran constitute complete food containing the ingredients
required by the fungus, including carbohydrates, sugar, proteins, iron, calcium, manganese,
magnesium, phosphorus, potassium, selenium, sodium, zinc, copper, boron, vitamins, fatty
acids and amino acids (Anon, 2009d; 2009e). The moist millet and wheat bran media possess
higher surface area for microbial attachment and have better aeration as a result of air pockets
created due to the interstices between the grains. Increased aeration has been shown to
enhance the lignin-degrading activity of P. chrysosporium (Tien and Kirk, 1988). In contrast,
the glucose media which is either liquid or continuous solid may be deficient of oxygen.
Figure 3.7 compares the effect of different media on 2-week biotransformation of anthracite.
It is evident from Fig. 3.7 that both millet and MWB media offered a better system for fungal
growth leading to a more pronounced reduction in preg-robbing (more than 95 %) whereas
glucose media reduced preg-robbing to about 90 %. On the basis of cost and nutritional
values, supplementing millet with wheat bran is advantageous since in general millet is more
expensive than wheat bran which is a discard from wheat processing. The use of wheat bran
alone is however not preferred due to its very low density which requires a large volume for
the process.
57
Fig. 3.7. Effect of different media utilized on gold adsorption behavior of anthracite; asreceived (AR), glucose broth (GB), glucose agar (GA), millet + wheat bran (MWB), millet
(M). The biotransformation period was 2 weeks.
The possible contribution of the MWB medium to the biotransformation action of coal by P.
chrysosporium was investigated by means of control experiments which were set up without
fungal addition. The gold adsorption behavior of coals used in the control experiments was
compared with the fungal-treated coals in Fig. 3.8. It is evident from the figure that MWB
medium can reduce the preg-robbing capacity of anthracite coal by about 25% within 14 days
of treatment as compared to over 95% reduction in gold adsorption by anthracite in the case of
MWB cultured with P. chrysosporium. The slight decline in preg-robbing due to media alone
may be attributed to fine suspended particulates that might have blinded some of the pores on
the carbonaceous material.
58
Fig. 3.8. A comparison of the gold adsorption behavior of controls and fungal-treated
anthracite.
3.5 Biotransforming action of P. chrysosporium

P. chrysosporium is known to produce oxidative enzymes that catalyze oxidation of lignins
aromatic substructure leading to formation of cation free radicals and bond cleavage (Tien and
Kirk, 1984, 1988; Glenn et al., 1986; Hammel et al., 1985; Kersten et al., 1985; Kirk et al.,
1986; Bumpus and Aust, 1986; Andrawis et al, 1988; Banci et al., 1992). The radicals may
also react with the lignin polymer to depolymerize it and the products may undergo further
modification to form carbon dioxide (Bumpus and Aust, 1986). The general mechanism was
presented in Chapter 2 and is represented below in Equations 2.1-2.3 (modified after
Andrawis et al, 1988; Banci et al., 1992; Edwards et al., 1992).
59

P[O Fe(IV) ] (comp I) R
H 2 O2
P[O Fe(IV) ] (comp I) H2O
[2.1]
P[O Fe(IV)] (comp II) R
P[O Fe(IV)] (comp II) R 2H
[2.2]
H 2O
[2.3]
The equations present enzymes and free radical intermediates referred to as compounds I and
II, which are involved in the catalysis (Tien and Kirk, 1984; Kersten et al., 1985; Hammel et
al., 1985; Kirk et al., 1986; Fakoussa and Hofrichter, 1999). During the initial stages of the
catalysis, H2O2 generated by the fungus is utilized by the enzymes (Kurek and Kersten, 1995),
to get oxidized first to iron (IV) and then to a cation radical (Tien and Kirk, 1988) called
compound I (Equation 2.1), which is 2 oxidation levels above the native enzyme (Gold and
Alic, 1993). Compound I subsequently reacts (Equation 2.2) with a substrate molecule, R, to
form Compound II, which is one-electron oxidized, and Compound II returns to the resting
enzyme state by reacting (Equation 2.3) with another molecule of substrate (Andrawis et al.,
1988; Banci et al., 1992; Gold and Alic, 1993). The substrate cation radical (R+) produced in
Equation 2.3, can undergo a variety of non-enzymatic reactions and spontaneous
rearrangement leading to further degradation and formation of a wide range of final products
(Pasczynski et al., 1985; Glenn et al., 1986; Pease et al., 1989; Wariishi et al., 1991; Gold and
Alic, 1993; Catcheside and Mallett, 1991; Fakoussa and Hofrichter, 1999).
From the above mechanism, it can be inferred that oxidation of carbon by the fungus may lead
to an increase in oxygen to carbon ratios through formation of carbon dioxide or introduction
of oxygen groups on the carbon which disrupts the continuous nature of the graphitic planes
required for gold adsorption (Jones et al., 1989; Klauber, 1991; Ibrado and Feurstenau, 1992;
Van Krevelen, 1993). Infrared analysis of bacterial treated coal samples by Amankwah and
Yen (2006) revealed an increase in oxygen groups on the coal and a subsequent reduction in
gold adsorption ability. It is therefore possible that biotransformation of anthracite leading to a
reduction in gold adsorption was as a result of introduction of oxygen groups and/or loss of
carbon following oxidation. Another possibility is the blocking of carbon pores by the spores,
60
hyphae and/or biofilms generated by P. chrysosporium which obviously will lead to

passivation and a decrease in active sites for adsorption.
The findings suggest that P. chrysosporium can potentially reduce the preg-robbing ability of
carbonaceous matter in gold ores. Other aspects of this on-going research focus on
establishing the optimum parameters for incubation and the effect of fungal treatment on
refractory gold ores among others. Preliminary results are promising though the process is far
slower compared to the existing high temperature and chemical methods used in deactivating
carbonaceous matter. Nevertheless, biological processes are normally slower than chemical
processes but once tested to work within engineering design capabilities, biological processes
such as biooxidation of sulfides are accepted as alternatives on the basis of cost and
environmental issues (Hutchins et al., 1988; Brierley, 1995). In addition, in a batch culture
such as that used in this study, microbial growth and metabolic activity increase to a
maximum and then reduce with further increase in processing time. Conversely, in a
continuous system, growth phase is sustained indefinitely (Shuler and Kargi, 1992). Thus it is
expected that in a continuous system processing time will be shorter without sacrificing
biotransformation of the carbonaceous matter. The initial findings reported here make this
process a potential alternative to other processes that can modify carbonaceous matter, and
thus merits further investigations into the engineering possibilities.
3.6 Conclusions
The ability of P. chrysosporium to interact with, and reduce the gold adsorption capabilities of
carbonaceous matter (CM) has been investigated and reported for the first time by this work.
Various ranks of coal were used as surrogate materials to predict the response of CM to
biotransformation by P. chrysosporium. Reduction in gold adsorption capabilities of the
surrogates was used to estimate declining preg-robbing ability. Various culture media used
under different incubation conditions affirmed that P. chrysosporium can grow on all the coal
ranks. The results, however, show that of the coal samples tested, only as-received anthracite
61
coal exhibited significant gold-cyanide adsorption ability, which was used as a means of
analyzing the extent of biotransformation. After contact with the fungus, preg-robbing ability
of anthracite reduced by between 90 and 97 % depending on the media.
By comparing submerged culturing using glucose broth medium and moist culturing using
glucose agar, solid millet and wheat bran, it was established that millet and wheat bran
constitute the best media for incubation, resulting in more than 95% reduction in pregrobbing. The findings demonstrate a potentially effective alternative pretreatment process for
reducing preg-robbing and require further investigations into possible commercial application.
62
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67
Chapter 4
Fungal biotransformation of anthracite-grade carbonaceous matter: Effect on
gold cyanide uptake
Abstract
Refractory carbonaceous gold ores contain carbonaceous matter that adsorbs gold during cyanidation
leading to reduction in gold recovery. In a related investigation (Chapter 3), the white rot fungus,
Phanerochaete chrysosporium was shown to reduce the gold adsorption ability of anthracite coal by
more than 90 % in a mixture of millet and wheat bran medium. This study focused on establishing the
incubation parameters that led to maximum reduction in gold adsorption by anthracite. The results
indicate that P. chrysosporium deactivates anthracite within a wide range of time, pulp density,
temperature, pH, and level of agitation. A processing time of 5-7 days, temperature and pH of 37 oC
and 4 respectively were the best conditions. The best pulp densities for stationary and shake culturing
respectively were 60 % and 25 %.
Analysis of the nature of anthracite using Raman, FTIR and XANES spectroscopies indicated that
fungal-treatment led to a reduction in carbon content and a boost of oxygen-containing groups such as
carboxyls and carbonyls. In addition, BET surface area and micro pore volume reduced by 76 % and
80 % respectively whereas average pore diameter increased by 65 %. The results suggest that P.
chrysosporium biotransforms anthracite by surface oxidation, which leads to a decrease in C/O ratio
and disruption of the continuous graphitic structure, thus decreasing the active sites necessary for
adsorption. Another factor is reduction in surface area via plugging of pores possibly by slimy
substance formed through fungal interaction with the growth medium, and hence decreasing
accessibility of gold to the adsorption sites. Fungal action also led to pore enlargement, thus rendering
the pores unsuitable for adsorption of the aurocyanide ion.
Keywords: Phanerochaete chrysosporium; Surface modification; Biotransformation; Preg-robbing,

pH; Pulp density; Temperature; Anthracite; Porosity; Aurocyanide; Adsorption; Characterization.
4.1 Introduction
Refractory carbonaceous gold ores are those that contain carbonaceous components, which
adsorb dissolved (or preg-rob) gold during cyanidation, resulting in reduced gold extraction.
Carbonaceous matter (CM) presents a serious and challenging problem in the extraction of
gold from refractory carbonaceous gold ores, and several techniques have been investigated to
deactivate CM and prevent preg-robbing. Among these processes are roasting, chemical
treatment using organic reagents and microbial treatment (Arriagada and Osseo-Asare, 1984;
Abotsi and Osseo-Asare, 1987; Hutchins et al., 1988; Afenya, 1991; Portier, 1991; Brierley
and Kulpa, 1993; Kohr, 1994; Adams and Burger, 1998; Rees and Van Deventer, 2000;
Amankwah and Yen, 2006).
Microbial pretreatment processes are gaining importance as potential alternatives on the basis
of cost and environmental issues, due to the relatively lower operating temperatures and the
fact that microorganisms can rejuvenate themselves to recycle the reagents needed for the
reaction. A number of bacteria and fungi have been tested for deactivation of carbonaceous
matter having various degrees of maturity, and there are reports of increase in overall gold
extraction. Among the microorganisms used are the bacteria Streptomyces setonii, species of
Pseudomonas, Achromobacter, Arthrobacter and Rhodococcus, and fungi Trametes
versicolor, Aspergillus bruneio-uniseriatus and Penicillium citrinum (Portier, 1991; Brierley
and Kulpa, 1993; Amankwah et al., 2005; Afidenyo, 2008; Yen et al., 2008).
In an investigation of biodegradation of lignite, bituminous and anthracite coals using S.

setonii, Amankwah and Yen (2006) observed a more extensive biodegradation in lignite and
bituminous coal than in anthracite. In a solution of 5 mg/L Au, biotreatment reduced the gold
adsorption capacity of bituminous, lignite and anthracite coals respectively from an average of
about 19 % to 0.3 %, 22 % to 9 % and 45 % to 20 %. A white-rot fungus, Trametes
69
versicolor, has also been reported to reduce the preg-robbing effect of bituminous coal from
40.3 % to 8.3 % and anthracite from 99.5 % to 27.7 % (Afidenyo, 2008).
Anthracite grade CM comprises more than fifty percent of the components of CM in

refractory gold ores (Hausen and Bucknam, 1984; Osseo-Asare et al., 1984; Sibrell et al.,
1990; Pyke et al., 1999; Stenebraten et al, 2000; Schmitz et al., 2001; Vaughan and Kyin,
2004). In addition, anthracite grade CM has a far higher capacity to adsorb gold than
bituminous and lignite coals due to the maturity and thus well-developed graphitic structure of
anthracite (Jones et al, 1989; Klauber, 1991; Ibrado and Feurstenau, 1992; Van Vuuren et al.,
2000; Amankwah and Yen, 2006; Ofori-Sarpong et al., 2010). Consequently, identifying a
microorganism that can deactivate anthracite and reduce its gold adsorption significantly will
be of immense benefit in the treatment of carbonaceous gold ores.
In Chapter 3 and Ofori-Sarpong et al. (2010), it was shown that anthracite interacted the
strongest with dissolved gold and also gold adsorption reduced the most after fungal
treatment. In this chapter, the interaction of P. chrysosporium with anthracite and its effect on
aurocyanide adsorption is further explored to ascertain the response to changes in pH, pulp
density, temperature and agitation.
4.2.1 Materials, medium preparation and incubation

Anthracite coal samples, the fungus P. chrysosporium and the MWB medium were obtained
and incubated as presented in Chapter 3, Section 3.3 and Ofori-Sarpong et al. (2010). The coal
was incubated under different conditions including processing time, pulp density, temperature,
pH, agitation and sterile versus non-sterile culturing. The pH was buffered to 4 and 6
respectively with succinic acid and potassium phosphate. Reagent grade potassium phosphate,
succinic acid and sodium hydroxide were obtained from VWR.
70
4.2.2 Characterization of anthracite

In order to better understand the adsorption results, characterization was performed on both
as-received and 14-day fungal treated anthracite using various techniques. The changes in
surface characteristics of anthracite were observed by measuring surface area, pore volume
and size by BET analysis, and the presence of oxygen groups and aliphatics by Fourier
Transform Infrared (FT-IR) and X-ray absorption near edge structure (XANES)
spectroscopies.
BET and pore size analyses were conducted using a Micromeritics ASAP 2000 Multi-Point
BET analyzer and samples were analyzed for pore size by high pressure nitrogen absorption.
FT-IR analysis was conducted by diluting milled samples of both as-received and biotreated
anthracite with spectroscopic grade KBr in the ratio of 1:100 (Miller and Stace, 1972). Each
mixture was ground in an agate mortar to homogenize the two components and reduce the
scattering effect of the relatively larger particles. Analysis was carried out in a Bruker IFS
66/S FT-IR Spectrometer. For XANES analysis, the samples were dried and ground with a
mortar and pestle to achieve homogeneous thickness (Szulczewski et al., 2001; Seiter et al.,
2008). The sample was then mounted on adhesive tape attached to the sample holder and
analyzed with the National Synchrotron Light Source in Brookhaven National Laboratory,
NY.
Raman scattering is a spectroscopic technique based on inelastic scattering of a laser source

monochromatic light, used to diagnose the internal structure of molecules and crystals
(Potgieter-Vermaak, 2000). In this chapter, Raman spectroscopy was used for semiquantitative analysis of the changes in graphitic nature of carbonaceous samples following
fungal-treatment by P. chrysosporium. Samples were mounted on adhesive sample holder and
the spectra were collected via the Confocal WITec XY Raman spectrometer, with a polarized
laser light of 488 nm wavelength using a 40X objective lens. The spectra were collected at 5
min interval with 1 s integration time.
71
4.2.3 Photomicrographs of P. chrysosporium

P. chrysosporium maintained on millet and wheat bran medium for 2 weeks was observed
with a Nicon Elipse 80i Microscope and the photomicrograph taken with the attached camera.
The NIS-Element AR 3.0 software was used to process the image. The photomicrographs
were taken at 100, 400 and 1000 magnification.
4.2.4 Analysis of data

The preg-robbing effect of carbon (PEC) which gives an indication of the effect of fungalbiomodification on adsorption of gold by carbon was computed as described in Chapter 3,
Section 3.4. Comparing to the amount (mol/g) adsorbed by the as-received anthracite
(AAR), percentage reduction in preg-robbing (PRP) as a result of fungal-treatment was
computed as in Equation 4.1. All experiments were carried out in triplicates and the data
reported in the figures are mean values of the 3 samples, with the error bars representing the
standard error (SE) of the means. In Equation 4.2, SD is the sample standard deviation
(Equation 4.3) where n is the sample size, xi and x-bar are the ith value and the mean
respectively.
PRP(%)
SE x
PEC
x100%
AAR
SD
n
[4.2]
3
SD x
[4.1]
xi
n 1
[4.3]
72
4.3 Results and discussions
4.3.1 Reduction in gold adsorption following fungal-treatment

The reduction in gold adsorption (preg-robbing) ability of anthracite following fungal
treatment is presented in Fig. 4.1. Fungal-treatment was conducted at 60 % solids for 14 days
at 37 oC and pH 6.5. From an initial adsorption value of 4.5 mol/g for the as-received
sample, gold adsorption reduced to 0.25 mol/g after 14 days of treatment representing a
Gold cyanide adsorption (mol/g)
reduction of about 95 %.
5
4
3
2
1
0
As-Received
Fungal-treated
Carbon material
Fig. 4.1. Reduction in gold adsorption by anthracite after fungal treatment at 60 % solids
density for 14 days at 37 oC and pH 6.5. For adsorption tests, 0.1 g of anthracite was contacted
with 25 mL of 5 g/mL gold solution and agitated at 150 rpm for 24 hours
73
4.3.2 Effect of pulp density

The effect of pulp density on biotranformation of anthracite by P. chrysosporium is shown in
Fig. 4.2. The incubation conditions used were 14 days, 37 oC and pH 6.5. After stationary
incubation for 7 days at a pulp density of 25 % solid, reduction in preg-robbing was 88 %
from a solution of concentration 5 mg/L Au. By using the same initial fungal dosage for
stationary incubation with pulp densities of 25 %, 40 %, 60 %, and 75 %, the average
percentage reduction of gold adsorption were 88 %, 92.5 %, 95.5 % and 95 % respectively.
Fig. 4.2. Effect of pulp density on biotransformation of anthracite by P. chrysosporium in

stationary culturing (37 oC, pH 6.5; 14 days)
From Fig. 4.2, pulp densities of 60 % and 75 % led to the highest reduction in preg-robbing of
treated anthracite. The higher the pulp density the greater the mass available for fungal
growth, hence the higher biotransformation in the 60 % and 75 % pulp densities as against the
25 % and 40 % pulp densities. At solid densities of 60 % and 75 %, the media and sample
were moistened but did not form slurry. The discontinuous nature of the moist substrate
74
allowed for the formation of air pockets which provided the oxygen necessary for growth and
activity of P. chrysosporium which is an aerobe (Tien and Kirk, 1988).
For shake cultures, as shown in Fig. 4.3, lower pulp densities were utilized, and there was 95
% reduction in average gold adsorption for materials incubated at 15 % and 25 % solids while
that of the sample treated at 40 % solids reduced by 91 %. From the figure, pulp densities of
15 % and 25 % for shake culturing led to higher reduction in preg-robbing of treated
anthracite than 40 % pulp density. At the lower pulp densities there is improved diffusion of
oxygen into the slurry as the sample is disturbed by agitation (Marsden and House, 2006),
providing the dissolved oxygen required for fungal-growth and activity. In addition, the ratio
of oxidants to material being treated is higher with the lower pulp densities. However, 25 %
pulp density was chosen over 15 % due to the mass of material that can be treated per unit
time.
Fig. 4.3. Effects of pulp density on biotransformation of anthracite by P. chrysosporium in

shake culturing (37 oC, pH 6.5; 14 days)
75
Since the reduction in gold adsorption achieved at pulp densities of 60 % and 75 % after 7
days of fungal treatment were equally good, further investigations were carried out to monitor
the effect of fungal-treatment time (Fig. 4.4) on reduction in preg-robbing. After an incubation
period of 1 day, the gold adsorption abilities reduced by 78 % for the sample cultured at 60 %
solids and 68 % for that cultured at 75 % solids. After the 5th day, preg-robbing by both
samples reduced by more than 90 %, and over 95 % by the 7 th day. It can be deduced from
Fig. 4.4 that a processing time of 5-7 days is suitable for the process. Due to the reduced
presence of water in the 75 % solids material, the fungus grew and formed a matrix that
covered the anthracite making separation between biomass and sample more difficult.
Consequently, a pulp density of 60 % may be preferred on the basis of reduced material losses
during post incubation harvesting of treated materials.
Fig. 4.4. Effect fungal-treatment time on biotransformation of anthracite at pulp densities of

60 % and 75 % solids under stationary culturing (37 oC, pH 6.5)
76
The choice between stationary and shake culturing will depend on a balance between volume
of material available for treatment and the time and technique available for harvesting treated
material. Whereas per unit time, much more material can be treated via stationary than shake
culturing, washing of treated material from shake culturing is less cumbersome and can be
accomplished at a relatively shorter time with much less material losses than in stationary
culturing where fungal biomass and ore particles become entangled making separation
difficult.
4.3.3 Effect of temperature

The effect of incubation temperature as a function of time for stationary culturing at 60 %
solids and pH of 6.5 is demonstrated in Fig. 4.5.
Fig. 4.5. The effects of incubation temperature as a function of time in stationary culturing (60
% solid, pH 6.5)
77
At processing temperature of 37 oC, gold adsorption ability reduced by 80 % after the 1 st day
and further to about 96 % after the 7th day. Beyond this day, the gold adsorption capabilities
remained the same with increasing incubation time to the 21 st day. The sample incubated at
25 oC followed a similar trend though the reduction was much slower as after an incubation
period of 1 day, the gold adsorption ability was reduced by 66 %. Sorption reduced further to
a final value of 96 % after 21 days.
Shake culturing was conducted at 25 % solids, pH 6.5, and at temperatures of 25 oC and 37 oC
over a period of 21 days. At 37 oC, the gold adsorbed reduced by 78 % after the 1 st day, and
followed a plateau at 95 % reduction after the 7th day as shown in Fig. 4.6. The trend is similar
to that observed for stationary culturing (Fig. 4.5). For incubation at 25 oC, gold adsorption
was halved after the 1st day and reduced by 92 % after 21 days.
Fig. 4.6. The effects of incubation temperature as a function of time in shake culturing (25 %
solid, pH 6.5)
78
It is clear from Fig. 4.5 and 4.6 that incubation at 37 oC gives a higher reduction in pregrobbing ability of anthracite than at 25 oC for both stationary and shake culturing. A
processing time of 5 to 7 days is also more favorable.
Though the temperature for optimum growth of P. chrysosporium is 37 oC (Tien and Kirk,
1988) the study at 25 oC was done to investigate changes in preg-robbing behavior at ambient
temperature. Work done by Kirk and Farrel (1987) shows that 37 oC provides the
microorganism the right temperature for growth and production of oxidative enzymes. This is
thus responsible for the higher performance at 37 oC compared to 25 oC. The challenge would
be settling for lower preg-robbing reduction under ambient temperature as against extra
heating cost for a higher preg-robbing reduction. Nevertheless, the catalysis of P.
chrysosporium leads to the generation of heat which can sustain reactions above ambient
temperature.
4.3.4 Effect of pH
The effect of pH on biotransformation of anthracite by P. chrysosporium was investigated at
pH 4 and 6.5. Incubation was conducted at 25 % solids and 37 oC for 7 days under shake
culturing. A pH of 4 was obtained by buffering the pulp with succinic acid and using NaOH to
adjust the pH, whereas pH of 6.5 was obtained with potassium phosphate buffer. Fig. 4.7
reveals that after one day of incubation about 85 % reduction was realized at pH of 4 as
against 78 % reduction at near neutral pH. By the 7 th day, there was near parity in sorption
reduction after fungal-treatment for the two pH values at 95 % and 97 %.
79
Fig. 4.7. Effect of pH on biotransformation of anthracite as a function of time in shake

culturing (25 % solid, 37 oC)
Investigations by Tien and Kirk (1984) indicate that the growth and ability of the fungus to
secret oxidative enzymes peak around a pH of 4, and this may be responsible for the higher
reduction in gold adsorption at that pH compared to the near neutral condition. The pH of the
fresh culture media was around 6.5 and for tests where pH was not controlled, it was observed
that pH reduced over time to between 4 and 5.5. This is because P. chrysosporium has the
tendency to buffer itself by producing organic acids such as oxalic acid ((COOH)2) malonic
acid (CH2(COOH)2) and tartaric acid (CH3CHO2(COO)2) that enables it to function around its
optimum pH (Tien and Kirk, 1984; Catcheside and Ralph, 1999; Fakoussa and Hofrichter,
1999). At both pH values, a residence time of 5-7 days was found to be suitable.
80
4.3.5 Effect of sterilization

The effect of sterilizing the medium and material before fungal inoculation is shown in Fig.
4.8. When incubation was conducted for 3 days, adsorption onto the non-sterile sample
decreased by 90 % as against the sterile system that recorded 92 %.
Fig. 4.8. Effect of sterilizing on the time dependence of the biotransformation action of P.
chrysosporium (37 oC, pH 6.5; 60 % solids; stationary)
Sorption reduction for the 5th and 7th days were 97 % for the non-sterile while for a similar
period the sterile averaged 96 %. Judging from the average values, the non-sterile appeared to
offer a slight advantage over the sterile. The main aim of sterilization is to get rid of resident
microorganisms that are naturally present in the media and/or substrate to be treated. Thus, in
the unsterilized culture, there will be other microorganisms aside the one specifically used for
inoculation. Consequently, the seemingly slight advantage might be due to positive
81
contribution of native microorganisms in the medium and/or the anthracite to the

biotransformation action of P. chrysosporium. It might also be the result of excess production
of oxidative enzymes by P. chrysosporium in response to competitive and antagonistic
survival against other possible native microorganisms (Waksman, 1941; Mille-Lindblom and
Tranvik, 2003; Madigan and Martinko, 2006; Mille-Lindblom, 2006).
4.3.6 Effect of growth medium

Figure 4.9 depicts the contribution of the growth medium (MWB) to the biotransformation
action of P. chrysosporium. The lower portions of the bars represent percentages of the total
biotransformation attributed to the medium alone whereas the upper portions show the
additional reduction in preg-robbing capacity of anthracite when the medium is cultured with
P. chrysosporium.
Reduction in preg-robbing (%)
100
Treated
Control
75
50
25
0
14
Fungal-treatment time (days)

Fig. 4.9. Combined effect of MWB and fungus in the biotransformation of anthracite (37 oC,
pH 6.5; 60 % solids; stationary; up to 14 days)
82
In the presence of the fungus, there was 4-8 fold decrease in gold adsorption in comparison to
the media alone. The slight decline in preg-robbing due to the medium alone may be
attributed to fine suspended particulates that might have blinded some of the pores on the
carbonaceous material (Chapter 3; Ofori-Sarpong et al., 2010).
4.4 Investigations of surface biomodification of anthracite

From the above section, it is clear that P. chrysosporium possesses the ability to biotransform
anthracite-grade carbonaceous matter under various incubation conditions and reduce its gold
adsorption activity substantially. The following section investigated the nature of surface
biomodification imparted onto anthracite by the fungus.
4.4.1 Surface area and pore characterization of anthracite

The adsorption sites for gold are located in the pores of the adsorbent and hence it was
necessary to know how fungal transformation changes the nature of these pores. The surface
area and porosity measurements are presented in Fig. 4.10. BET surface area of the asreceived anthracite was found to be 4.08 m2/g, a number similar to that of anthracite samples
studied by Miller and Sibrell (1991) who obtained 4.0 m2/g. After fungal treatment, the BET
surface area reduced by 76 % to 0.98 m2/g.
The micro pore volume and average pore diameter of the as-received sample were 15.09 x
10-4 cm3/g and 5.7 nm respectively and after treatment, the micro pore volume reduced to 2.82
x 10-4 cm3/g while the average pore diameter increased to 9.5 nm respectively. These translate
into micro pore volume reduction of 80 % and increase of 65 % in average pore diameter (Fig.
4.10). The micropore surface area on the other hand reduced by about 80% following fungal
treatment to 0.61 m2/g.
83
Percentage value of parameter (%)
120
As-received
Treated
100
80
60
40
20
0
BET surface
area
Micropore
area
Micropore
volume
Average pore
diameter
Parameter
Fig. 4.10. A comparison of surface area and pore characteristics of as-received and biotreated
anthracite. Anthracite was treated for 14 days in MWB mixture at 60 % solids, 37 oC and
pH 4.
All these changes potentially reduce gold adsorption onto carbon. A reduction in both pore
volume and surface area led to subsequent decrease in adsorption as both parameters influence
adsorption of the aurocyanide complex (McDougall and Hancock, 1981).
4.4.2 Photomicrograph of P. chrysosporium

The photomicrograph of P. chrysosporium maintained on MWB medium for 2 weeks was
taken with Nicon Elipse 80i Microscopic camera, and is presented in Fig. 4.11. The picture
shows hyphae and vegetative spores of the fungus, and diameter of the hyphae was 1-3 m
84
which is similar to that obtained by Sachs et al. (1989) when P. chrysosporium was cultured
on wood within a week. This number is within the range (0.5-10 m) reported by other
researchers, and the size is known to depend on the substrate and the culturing time (Kirk et
al., 1986; Sachs et al., 1989; Friese and Allen, 1991; Lyford, 1966; Steinberg and Rillig,
2003). The spores were estimated to be 0.4-2 m in diameter. Though the sizes are bigger
than the micropores and mesopores (< 0.2 m) of carbon, the tiny spores may be able to
access the over 0.2 m sized macropores which are the admission pores to the smaller pores
where gold adsorption usually takes place (McDougall and Hancock, 1981). It is important to
investigate the nature of the material that causes blockage of the pores and accounts for the
sharp reduction in the surface area and volume of the smaller pores (Fig. 4.10). However,
TEM analysis did not reveal information in that direction.
Hyphae
Spores
20 m
Fig. 4.11. Photomicrograph of P. chrysosporium. The fungus was maintained on MWB

medium for 2 weeks.
85
4.4.3 Modification of graphitic structure
Raman Spectroscopy
The degree of graphitization of carbonaceous materials such as anthracite can be studied by
identifying the G and D lines under Raman spectroscopy (Sadezky et al., 2005; PotgieterVermaak et al., 2010). The G-line indicates the graphitic or ordered nature of the structure
whereas the D-line, disordered nature of the structure due to edge defects or lattice disorder.
There are various peaks that have been assigned to graphitic and disordered structures in
various ways by different researchers but the two most distinct ones are the peaks around
wavelengths of 1580 cm-1 known as the G line, and 1370 cm-1, the D line (Sadezky et al.,
2005; Potgieter-Vermaak et al., 2010). Figure 4.12 presents the Raman spectrograms of as-
Raman Intensity
received and treated anthracite.
As-Received
600
Treated
400
1000
2000
3000
4000
Wavenumber, cm-1
Fig. 4.12. Raman spectra of anthracite; as received and 14-day fungal treated at 37 oC and
pH 4.
86
Changes in intensities of the peaks signal a change in degree of orderliness or disorderliness

(Leventhal an Hofstra, 1990; Bustin et al., 1995). It can be seen from the figure that all the
peak intensities reduced following fungal-treatment. Considering the two major peaks (1580
cm-1 and 1370 cm-1), reduction was more apparent in the disordered line (1370 cm-1) than the
graphitic line (1580 cm-1). This is because, like many processes in nature, microbial attack
initiates at points of weakness and thus the less structured zones (Wackett and Ellis, 1999).
Altogether, reduction in carbon content possibly due to complete oxidation to carbon dioxide
will result in lower carbon-to-oxygen ratio, known to be unfavorable to gold adsorption
(Ibrado and Fuerstenau, 1992; Ofori-Sarpong et al., 2010).
4.4.4 Modification of surface functional groups
Infrared analysis
Infrared spectroscopy measures the ability of matter to absorb, transmit or reflect infrared
radiation and relates it to the chemical composition of that substance (Miller and Stace, 1972).
Crystals and molecules absorb infrared radiation depending on the inter-atomic vibrations.
Certain vibrations are related to specific groups and these are termed characteristic
frequencies. The absorption/transmission of radiation plotted as a function of the wavelength
gives information about the structure and functional groups present on materials (Dyke et al,
1971). For a given substance, changes in the functional groups due to a pretreatment process
may be registered by a change in the spectral configuration. Figure 4.13 shows the infrared
spectra of as-received anthracite and a 14-day biotreated sample. The peak assignments on the
samples were compared with earlier work by Rao (1963), Silverstein et al. (1981) and Painter
(1983). The OH peak between 3200 and 3000 cm-1 on both the as-received and biotreated
anthracite samples is generally attributed to the presence of water while the peak between
2400 and 2200 cm-1 is due to carbon dioxide. The C=C band at 1582 cm-1 is considered to be
characteristic of the hexagonal ring structure of graphite (Painter et al., 1983).
87
By comparing the two spectra, it is realized that the major changes occurred at 2929 cm -1,
2856 cm-1, and 1715 cm-1. The peaks at 2929 cm-1 and 2856 cm-1 are due to the presence of
aliphatic hydrocarbons while carbonyl (C=O) groups are responsible for the peak at 1715 cm-1
(Rao, 1963; Silverstein et al., 1981; Painter, 1983). The C=O group which is present in the
treated sample indicates that there was formation of more oxygen containing groups on the
surface of anthracite. With the increase in the aliphatic groups and the carbonyl groups on
anthracite, it can be inferred that the degree of aromaticity was reduced due to fungal
treatment. These changes will result in reduced adsorption as aurocyanide adsorption, which
requires large continuous areas of graphitic plates, not interrupted by edges terminating with
1.1
C-H or C-O groups (Jones et al., 1989; Ibrado and Fuerstenau, 1992; Jia et al., 1998).
As-received anthracite
0.9
0.8
As-received vs treated anthracite
0.7
Absorbance Units
1.0
Biotreated anthracite
3200
3000
2800
2600
2400
2200
2000
1800
1600
Wavenumber cm-1
Fig. 4.13. Infrared spectra of both as-received and biotreated anthracite. Anthracite was
treated for 14 days at 60 % solids, 37 oC and pH 4.
88
XANES Spectroscopy
In this study, x-ray absorption near edge structure (XANES) spectroscopy was utilized in
observing changes in the surface functional groups on anthracite. Figure 4.14 compares the asreceived and 14-day biotreated anthracite, and the spectra shows an increase in intensity of the
oxygen-containing groups. Compared with studies by Braun et al (2005), the peaks between
285 and 286 eV are associated with carbonyls and phenols while the hump at 287 eV is for
aliphatics and the peak between 288 to 289 eV signifies the presence of carboxylic acids.
Though these peaks were on the as-received sample, they became more pronounced following
fungal treatment. This observation confirms that of the infrared analysis which also showed an
increase in oxygen-containing groups.
0.4
As-received
anthracite
Carbon nanopowders
0.2
units
Absorbance
x (E)
0.6
Biotreated
anthracite
280
285
290
295
E (ev)
300
305
Energy, eV
Fig. 4.14. A comparison of the as-received and biotreated anthracite spectra from oxygen
XANES. Anthracite was treated for 14 days, at 60 % solids density, 37 oC and pH 4.
89
4.5 Proposed mechanism responsible for reduction in gold adsorption after

biotransformation of anthracite by P. chrysosporium
The current theory of gold adsorption from alkaline cyanide solution favors adsorption of the
gold dicyano complex on the surface of graphitic planes of carbon via donation of delocalized
pi electrons from the graphitic planes to the empty 6s shell of gold ion (McDougall et al.,
1980). Though the surface of carbon is negatively charged due to sp2 hybridization, this
interaction outweighs the electrostatic repulsion between the negatively charged surface of
carbon and the Au(CN)2- complex (Jones et al, 1989; Klauber, 1991; Ibrado and Feurstenau,
1992; 1995; Jia et al., 1998).
In a review by Osseo-Asare et al. (1984), it was noted that modification of the surface of
carbon by organic additives such as kerosene and heavy oils, rather than complete oxidation to
carbon dioxide, can substantially reduce gold uptake by carbon. Similar results have been
reported by several other investigators (Abotsi and Osseo-Asare, 1987; Adams and Burger,
1998; Pyke et al., 1999). The reduction in gold adsorption has been interpreted to be the
consequence of preferential attachment of the organic compounds to the hydrophobic
graphitic planes (Jia et al., 1998). Destruction of graphitic planes, complete and/or partial
oxidation of carbon and thus increase in oxygen-to-carbon ratios are also known to decrease
the gold adsorption capacity of carbon (Osseo-Asare et al, 1984; Sibrell et al, 1990; Ibrado
and Feurstenau, 1992; Amankwah and Yen, 2006; Ofori-Sarpong et al., 2010).
Based on information provided by Klauber (1991) and Poinen and Thurgate (2003) as shown
in Fig. 4.15, the radius of the gold cyanide ion, Au(CN)2-, can be estimated as 0.733 nm, and it
can lie approximately on 3 adjacent graphitic planes (3 x 0.2456 = 0.7368 nm). The
continuous nature of the graphene layers is thus required for the adsorption of aurocyanide
complex from alkaline solutions (Jones et al., 1989; Klauber, 1991; Ibrado and Feurstenau,
1992, 1995).
90
0.2456 nm
C-Au(CN)-2
Au-C 0.212 nm
C-N
0.117 nm
Fig. 4.15. Interaction between the gold cyanide complex, Au(CN) 2 -, and the graphitic structure
of carbon (after Jones et al. 1989; Klauber, 1991; Ibrado and Fuerstenau; 1992; 1995; Poinen
and Thurgate, 2003)
In this study, Raman, infrared and XANES spectra indicated that the biotransformation of
anthracite by P. chrysosporium led to interruptions in the graphitic layers and subsequent
introduction of oxygen/hydrogen groups which can partly explain the reduction in gold
adsorption following fungal biotransformation.
The hyphae of P. chrysosporium, which are known to penetrate wood and soil, is estimated to
be between 0.5-10 m in diameter (Kirk et al., 1986; Sachs et al., 1989; Friese and Allen,
1991; Lyford, 1966; Steinberg and Rillig, 2003) depending on the substrate and the culturing
time. In this study, the diameters of the hyphae and spores produced by P. chrysosporium
taken under high magnification were 1-3 m and 0.4-2 m respectively. These sizes are
bigger than micropores and mesopores in anthracite but may be able to access macropores
(>0.2 m) (Marsden and House, 2006) and reduce gold adsorption by decreasing the ease of
access of aurocyanide ion to the adsorption sites (McDougall et al., 1980; Jia et al., 1998; Van
Vuuren et al., 2000; Marsden and House, 2006).
Surface area and pore size measurements as shown in Fig. 4.10 signify a drastic reduction in
surface area and micro pore volume with increase in average pore size diameter following
91
fungal-treatment. Microbial transformation of solids begins with the formation of a slime

layer around the area of attack. This layer is composed of polysaccharides (Glazer and
Nikaido, 1995; Madigan and Martinko, 2006) and it is possible that these organic substances
penetrated and coated more of the smaller pores. As discussed by Osseo-Asare et al. (1984),
coating of the surface by organic matter will lead to reduction in gold adsorption. Since gold
adsorption is reported to occur mostly in the micropores (McDougall and Hancock, 1981) a
reduction in available micropores and hence increase in available pore diameter contributed to
the reduction in gold adsorption. It is important to investigate the nature of the material that
caused blockage of the pores and accounted for the sharp reduction in surface area and
volume of the smaller pores. Surface examination using TEM has so far not provided answers
in this direction.
4.6 Conclusions
The white rot basediomycetous fungus, Phanerochaete chrysosporium was utilized in
reducing gold adsorption by anthracite coal which served as surrogate for carbonaceous matter
in refractory gold ores. Fungal treatment resulted in reduction of the gold adsorption ability of
anthracite by more than 90 % in a mixture of millet and wheat bran medium.
Fungal modification with P. chrysosporium was found to take place in a wide range of time,
pulp density, temperature, pH and level of agitation. After contact with the fungus, gold
adsorption ability of anthracite reduced by about 90-97 %, depending on the incubation
conditions. A pH of 4 and temperature of 37 oC were found to enhance biotransformation.
Stationary culturing permitted the use of higher pulp densities and the best for stationary and
shake culturing respectively were 60 % and 25 % solids. Altogether, strict choices of the
parameters will depend on materials required to be treated, techniques available for treatment
and post-incubation harvesting. Other considerations are a balance between capacity/tonnage,
extra cost on reagents and processing time. Firm decisions can be taken when cost analyses
are conducted.
92
Raman, infrared and XANES analyses showed that fungal treatment led to a reduction in
carbon content and introduction of oxygen containing groups such as carbonyls and an
increase in aliphatic groups. Further investigations showed that BET surface area and micro
pore volume decreased by 76 % and 80 % respectively whereas average pore diameter
increased from 5.7 nm to 9.5 nm. The combined characterization results from Raman,
XANES, IR and BET suggest that P. chrysosporium biotransforms anthracite in two main
ways. In one way there is loss of carbon through complete oxidation to carbon dioxide and
surface oxidation by introduction of oxygen groups which disrupts the continuous graphitic
structure thus decreasing the active sites necessary for adsorption. The second route is
reduction in available surface area via plugging of pores possibly by slimy substances formed
by interaction of the fungus with the medium, hence decreasing accessibility of gold to the
adsorption sites.
93
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97
Chapter 5
Gold adsorption on carbonaceous matter following fungal treatment: Comparison of
cyanide and non-cyanide gold complexes
Abstract
Though cyanide has been the universal reagent for gold leaching on counts of higher stability, lower
cost and better understood chemistry, its high toxicity and slower leaching kinetics have called for
research into alternative leaching reagents, of which thiosulfate and thiourea appear to be promising.
Unlike the cyanide system, interaction of thiourea and thiosulfate systems with carbonaceous matter
(CM) has not received much research attention. As non-cyanide reagents become important
alternatives for gold leaching, it is imperative to assess the degree of gold adsorption (preg-robbing) in
these systems and find ways of reducing its effect. As part of on-going research, the effectiveness of
the fungus, Phanerochaete chrysosporium, to biotransform anthracite-grade CM and reduce pregrobbing in cyanide solution has been established.
This chapter presents the results of a study on reduction in gold uptake by CM in non-cyanide systems
due to fungal-treatment. Comparison is made between cyanide and non-cyanide systems on the bases
of changes in gold adsorption by surrogate CM and CM in refractory gold ores. The results indicate
that gold adsorption by as-received surrogates is in the order of cyanide > thiourea > thiosulfate,
whereas after fungal-transformation, reduction in gold adsorption is more pronounced in cyanide than
in thiourea and thiosulfate solutions. The average reduction in gold adsorption on the CM is about 7097 % in cyanide, 60-92 % in thiourea and 50-91 % in thiosulfate. In general, the results exhibit a
potential technique for reducing preg-robbing in the processing of carbonaceous gold ores with
thiourea and thiosulfate solutions.
Keywords: Phanerochaete chrysosporium; Biotransformation; Carbonaceous matter; Preg-robbing;

Gold adsorption; Cyanide; Thiourea; Thiosulfate.
5.1 Introduction
5.1.1 Lixiviants in gold extraction

Cyanide has remained the preferred complexing agent in the leaching of gold for over a
century though many other complexing agents exist. The major reasons for the acceptance of
cyanide over the other lixiviants include higher chemical stability and lower cost
(Groenewald, 1976; Osseo-Asare et al., 1984; Nicol et al., 1987; Hiskey and Atluri, 1988;
Smith and Martell, 1989; Meng and Han, 1993; Wan et al., 1993; La Brooy et al., 1994;
Ritchie et al., 2001; Muir and Aylmore, 2004; Senanayake, 2004). In addition, adsorption and
desorption of gold cyanide complex onto and from the most widely used adsorbent, activated
carbon, has been studied the most with great success (McDougall et al, 1980; Adams and
Flemings, 1989; Jones et al, 1989; Ibrado and Fuerstenau, 1995; Rees and van Deventer,
2000).
In spite of the good attributes, the high toxicity of cyanide, the need for faster leaching
kinetics and hence shorter processing times, and an environmentally friendly recovery process
for gold ores have necessitated research into alternative leaching reagents. The reagents
include thiourea, thiosulfate, iodine, bromine and thiocyanate (Wan et al., 1994; Sparrow and
Woodcock, 1995; Yen et al., 1998; Schmitz et al., 2001; Fleming et al., 2002; 2003; Navorro
et al., 2002; 2006). Of the non-cyanide reagents, those that have the potential to replace
cyanide, and have been researched most in the past 30 years include thiourea and thiosulfate
(Groenewald, 1976; 1977; Hiskey., 1981; 1984; Mensah-Biney et al., 1994; Grosse et al.,
2003; Muir and Aylmore, 2004; Gonen et al., 2007; Osseo-Asare, 1988).
5.1.2 Adsorption of gold complexes on carbon

The dissimilarities in complexing reagents present significant differences in their interactions
with carbon. Gold thiourea is cationic with a +1 charge while cyanide and thiosulfate are
99
anionic but with charges of -1 and -3 respectively (McDougall and Hancock, 1981; OsseoAsare et al., 1988; Muir and Aylmore, 2004; Marsden and House, 2006). The ability of
activated carbon to adsorb these three gold complexes is in the order of Au(CN) 2 - >
Au(CS(NH2)2)2+ > Au(S2O3)23- (Gallagher et al.,1989; Brown and Deschenes, 1993; Arriagada
and Gardia,1997; Grosse et al., 2003; Muir and Aylmore , 2004; Marsden and House, 2006).
The mechanism of adsorption of these complexes has been discussed extensively in the
literature (McDougal et al., 1980; Jones et al., 1989; Adams and Fleming, 1989; Gallagher et
aI.,1989; Kongolo et al., 1990; Brown and Deschenes, 1993; Yen et al., 1994; Arriagada and
Gardia, 1997; Zhang et al, 2004; Muir and Aylmore, 2004; Navarro et al., 2006). Several
theories have been proposed for gold cyanide adsorption but currently there seems to be a
consensus that adsorption of aurocyanide ion (Au(CN) 2-) onto carbon occurs without chemical
change and that the graphitic structure of carbon is of great importance. There is disparity
though as to where the adsorbed gold resides, and the two major theories are predominant
adsorption on the planes of graphitic layers, and adsorption at the edges of graphitic layers
where there are oxygen functional groups (Jones et al., 1989; Klauber, 1991; Sibrell and
Miller, 1992; Ibrado and Fuerstenau, 1995; Poinen and Thurgate, 2003).
Gold thiourea complex, Au(CS(NH2)2)2+ has been reported to adsorb onto activated carbon
without undergoing any chemical change (Fleming, 1987; Schmidt et al, 1988). Using
activated carbons of different precursors, Arriagada and Gardia (1997) noted that the carbons
with oxygenated surface functional groups; carboxylic-like and phenolic or quinonic-like, had
higher affinity for gold thiourea complex than gold cyanide complex. In a study by Zhang et
al. (2004), the authors proposed that gold thiourea is generally adsorbed onto activated carbon
in the form of Au(I)thiourea complex and partial decomposition into metallic gold may occur
at locally concentrated high gold loadings.
Gold thiosulfate complex, Au(S2O3)23- has been shown by many investigators to have
significantly less affinity for carbon compared to gold cyanide and thiourea complexes
(Gallagher et al.,1989; Aylmore and Muir, 2001; Kononova et al., 2001; Schmitz et al.,
100
2001; Navarro et al., 2006; 2002). Gold thiosulfate complex has a high anionic charge
resulting from the interaction between gold cation and large thiosulfate anions. The low
affinity of activated carbon for Au(S2O3)23- complex is thus ascribed to the long pathway
between the central gold atom in the complex and the adsorbent surface, as created by the
large thiosulfate ions (Grosse et al., 2003; Aylmore and Muir, 2001; Muir and Aylmore,
2004).
5.1.3 Application of P. chrysosporium in reducing adsorption of gold complexes on

carbon
Loss of gold due to preg-robbing is a major setback in the cyanide system and has been of
great interest to researchers for many years in finding ways to mitigate the problem
(McDougal et al., 1980; McDougall and Hancock, 1981; Osseo-Asare, 1988; Miller and
Sibrell, 1991; Sibrell and Miller, 1992; Klauber, 1991; Ibrado and Fuerstenau, 1995).
Considering the potential of thiourea and thiosulfate to replace cyanide or be used as
alternatives, it is essential to assess the status of preg-robbing in these systems and to find
possible ways of reducing it.
The application of the fungus, Phanerochaete chrysosporium, in the biotransformation of

carbonaceous matter (CM) in gold ores has been discussed by Chapter 4 and Ofori-Sarpong et
al. (2010) of this dissertation. In the study, biotransformation led to a reduction in aurocyanide
uptake by CM and hence preg-robbing in cyanide solutions. The present study therefore
focused on assessing the ability of P. chrysosporium to alter the gold adsorption ability of CM
in thiourea and thiosulfate solutions.
101

Anthracite coal samples, P. chrysosporium and the millet and wheat bran (MWB) medium
were obtained and incubated as presented in Chapter 3, Section 3.3 and Ofori-Sarpong et al.
(2010). The gold-containing samples utilized were flotation concentrate (FC) and bacterial
oxidized FC (BFC). Samples FC and BFC, which had been milled to all passing 75 m, were
obtained from the sulfide treatment plant at Bogoso Mine of Golden Star Prestea-Bogoso
Resources, Ghana. Standard gold cyanide solution (50 g/ml in 0.1 % sodium hydroxide and
0.05 % sodium cyanide) was supplied by High Purity Standards. Powdered gold (I) sodium
thiosulfate (Na3Au(S2O3)2.xH2O hydrate, 99.96 % metal basis), gold flakes (APS 1.5-3.0 m,
99.96 % metal basis), thiourea, sodium thiosulfate, sodium hydroxide and sulfuric acid were
supplied by Alfa Aesar. Biotransformation was explored by incubating coal and gold ore
samples with P. chrysosporium, cultivated in MWB medium at 37 oC for different growth
periods of up to 3 weeks, and at pH of 6.5. At the end of the incubation period the samples
were washed with water in the ratio of about 1 g sample to 200 ml H2O to get rid of the media
and fungal biomass, and then dried at 37 oC for 7 days.
5.2.2 Gold adsorption experiments

Gold-cyanide solution of concentration 50 g/mL Au was diluted with 1 g/L NaOH at pH
12.8 to obtain 5 and 10 g/mL gold solutions which were used for the adsorption analysis.
Gold thiosulfate solution was prepared by dissolving 130 g of gold (I) sodium thiosulfate in
500 ml of 0.1 M thiosulfate solution at pH 9-11 to obtain 50 g/mL Au. To obtain 50 g/mL
Au in thiourea solution, 50 g of gold flakes was dissolved in 500 mL of 0.001 M thiourea
solution at pH of 1-2 at 40 oC. Both thiosulfate and thiourea gold stock solutions were diluted
appropriately to obtain 5 and 10 g/mL solutions, which were used for the adsorption test.
Triplicate samples of biomodified, control and as-received coals of various weights (0.1 g 1
102
g) and gold ores of 2 g - 5 g each were contacted with 25 mL of 5 g/mL and/or 10 g/mL
gold solutions and agitated at 150 rpm for 24 hours. At the end of the contact time the residual
solution was filtered and gold in the filtrate determined using Perkin-Elmer Optima 5300
inductively coupled plasma atomic emission spectrometer (ICP-AES).
5.2.3 Petrographic analysis of gold concentrate

Petrographic evaluation and thermal maturity analysis on the organic carbon in the gold
concentrate was conducted using Zeiss Universal research microscope. The gold concentrate
was mounted in epoxy, ground and polished with 600-grit emery paper before analysis.
5.3 Results and Discussion
5.3.1 Analysis of gold concentrates

Results of analysis of the as-received gold concentrates are presented in Table 5.1. The grade
of gold in both samples is very high at above 30 g/t with total sulfur and sulfide sulfur in FC
at 19.3 % and 14.9 % respectively. BFC has sulfide sulfur content of 3.0 % which is due to
unoxidized sulfide minerals after biooxidation. Other constituents are as shown in Table 5.1.
103
Table 5.1. Partial chemical analysis of as-received gold concentrates

Constituent
Grade, %
FC
BFC
Gold
30.2 g/t
39.3 g/t
Silver
2.2 g/t
Organic carbon
Constituent
Grade, %
FC
BFC
Iron
23.3
8.7
3.1 g/t
Arsenic
3.3
0.5
3.6
4.0
Copper
0.12
0.04
Total carbon
6.1
4.0
Zinc
0.034
0.007
Sulfide sulfur
14.9
3.0
Lead
0.029
trace
Sulfate sulfur
4.6
4.5
Nickel
0.023
0.005
Total sulfur
19.3
7.5
5.3.2 Effect of carbonaceous characteristics on gold adsorption from cyanide, thiourea

and thiosulfate solutions onto surrogate CM
The gold adsorption behavior of anthracite, bituminous, sub-bituminous and lignite coals from
cyanide, thiourea and thiosulfate solutions before and after biotransformation by P.
chrysosporium are presented in Figs. 5.1, 5.2 and 5.3 respectively. The maturity of coal, and
thus, the extent of graphitization decreases in the order of anthracite > bituminous > subbituminous > lignite. Conversely, the percentage of oxygen and hydrogen and thus, the ratio
of oxygen to carbon, and hydrogen to carbon decrease in the opposite direction (Van Kreveren
et al., 1993). It has been shown that gold cyanide adsorbs preferentially on anthracite relative
to the other coals due to better developed graphitic structure in anthracite (Ibrado and
Feurstenau, 1992; Amankwah and Yen, 2006). Correlating the carbon-to-oxygen ratios with
gold adsorption, it was inferred in Chapter 3 and Ofori-Sarpong et al. (2010) that the
adsorption of gold cyanide on anthracite is due to its high carbon-to-oxygen ratio of 42
compared to 5-11 for bituminous, sub-bituminous and lignite.
104
Fig. 5.1. Effect of one-week fungal-treatment on the gold adsorption ability of lignite, subbituminous, bituminous and anthracite coals in cyanide (CN) solution (37 oC; pH 6.5; 60 %
solids)
It can be seen from Figs. 5.2 and 5.3 that gold adsorption from thiourea and thiosulfate
solutions follow similar trends as that observed in the cyanide solution (Fig. 5.1) with
anthracite adsorbing about 5-20 times as much as the other coals. Comparing the gold
complexes though, it is clear that there is very low adsorption from thiosulfate solution on all
the coals, and this trend is in agreement with what is reported in literature (Gallagher et al.,
1989; Grosse et al., 2003; Schmitz et al., 2001; Marsden and House, 2006). The low affinity
of activated carbon for Au(S2O3)23- complex is ascribed to the long pathway between the
central gold atom in the complex and the adsorbent surface, as created by the large thiosulfate
ions. In addition, the negatively charged carbon surface exerts a stronger electrostatic
repulsion with the highly negative thiosulfate complex compared to the other complexes
(Kononova et al., 2001; Grosse et al., 2003; Aylmore and Muir, 2001; Muir and Aylmore,
2004).
105
Fig. 5.2. Effect of one-week fungal-treatment on the gold adsorption ability of lignite, subbituminous, bituminous and anthracite coals in thiourea (TU) solution. (37 oC; pH 6.5; 60 %
solids)
Whereas anthracite adsorbed much more in the cyanide solution than the thiourea solution, the
opposite was true for lignite, sub-bituminous and bituminous coals. The positively-charged
thiourea gold complex is reported to have better interaction with oxygen groups than
aurocyanide complex, and this may explain the higher adsorption of gold thiourea by the
lower coal ranks which contain more oxygen groups (Arriagada and Gardia, 1997;
Nakbanpote et al., 2000). As mentioned earlier, adsorption of gold thiosulfate by all coal
ranks was relatively very low.
106
Fig. 5.3. Effect of one-week fungal-treatment on the gold adsorption ability of lignite, subbituminous, bituminous and anthracite coals in thiosulfate (TS) solution. (37 oC; pH 6.5; 60 %
solids)
Figure 5.4 compares the percentage reduction in gold adsorption due to fungal-treatment of
the coals in all the gold solutions. After treatment of the coals with P. chrysosporium, the
adsorption capacity for the gold complexes generally decreased. For all the solutions tested,
the average reduction in gold adsorption was much more significant on anthracite than the
other coals. Average reduction in gold adsorption for all coals was highest in cyanide solution
as depicted in Fig. 5.4.
107
Fig. 5.4. Reduction in cyanide (CN), thiourea (TU) and thiosulfate (TS) gold adsorption on
lignite, subbituminous, bituminous and anthracite due to surface biotransformation by P
chrysosporium (37 oC; pH 6.5; 60 % solids; 7 days)
The comparison of gold adsorption on anthracite from cyanide, thiourea and thiosulfate
solutions is presented in Fig. 5.5, whereas Fig. 5.6 shows the effect of the incubation medium
on gold adsorption on anthracite. Gold adsorption onto anthracite was highest in cyanide
solution at 4.5 mol/g followed by thiourea at about 2.2 mol/g and thiosulfate at 0.4 mol/g
as shown in Fig. 5.5. Fungal treatment reduced sorption in all solutions to below 0.3 mol/g.
It is clear from Fig. 5.6 that gold adsorption for the controls were very close to that of the asreceived samples, and that the reduction in gold adsorption is essentially due to
biotransformation by P. chrysosporium.
108
Fig. 5.5. Comparison of gold adsorption on anthracite from cyanide, thiourea and thiosulfate
solutions as a function of fungal-treatment time. (37 oC; pH 6.5; 60 % solids; up to 21 days)
Fig. 5.6. Gold adsorption on as-received, control and treated anthracite from cyanide, thiourea
and thiosulfate solutions (37 oC; pH 6.5; 60 % solids; 7 days).
109
5.3.3 Effect of fungal biotransformation on the adsorption of gold complexes onto gold
concentrates
Having established the effect of fungal biotransformation of the surrogate carbonaceous
materials on the adsorption of the gold complexes, the flotation concentrate (FC), and FC
biooxidized with iron and sulfur oxidizing bacteria (BFC) were also tested to ascertain the
extent to which their preg-robbing abilities change following fungal-transformation. These
results are presented in Figs. 5.7 and 5.8. Within the boundaries established by the error bars
the levels of gold adsorption for the as-received FC was slightly lower than BFC for each of
the complexing solutions utilized. The above statement is also true for adsorption after fungal
treatment. This observation is based on the fact that the bacteria treatment of FC to produce
BFC does not inhibit the ability of CM to preg-rob gold, and the reduction in mass of
concentrate due to the treatment process boosts up the effective concentration of CM making
it a higher preg-robber.
Fig. 5.7. Comparison of gold adsorption on flotation concentrate (FC) from cyanide, thiourea
and thiosulfate solutions before and after fungal-treatment. (37 oC; pH 6.5; 60 % solids; 7
days)
110
Fig. 5.8. Comparison of gold adsorption on bacterial-oxidized flotation concentrate (BFC)

from cyanide, thiourea and thiosulfate solutions before and after fungal-treatment. (37 oC; pH
6.5; 60 % solids; 7 days)
Petrographic evaluation and thermal maturity analysis on the organic carbon in the gold
concentrate using Zeiss Universal research microscope as shown in Fig 5.9, revealed that the
organic carbon is fine with particle size in the range of 10-40 m. The mean maximum
reflectance was 3.7 %, which puts it in the anthracite-range maturity (Van Krevelen, 1993).
These carbon particles are coarser than those in similar refractory materials that were analyzed
by Afenya (1991) in which he obtained sizes of minus 2 m. The figure also shows sulfide
particles in the double refractory gold ore.
Though the CM was characterized to have anthracite grade maturity it is clear from both Figs.
5.7 and 5.8 that the level of reduction in preg-robbing does not compare well with that of
anthracite, which was used as surrogate material. This observation may be due to the complex
nature of the refractory gold ores with 3-4 % organic carbon and many other minerals
composing about 96-97 % (Table 5.1) in contrast to anthracite which has more than 90%
111
organic carbon. It is not clear at this point whether there was concurrent gold leaching during
the gold adsorption test and/or if fungal-treatment activated some other components in the ore
to sorb gold. Nevertheless, preg-robbing reduction of 50-70 % in refractory gold ores is
promising.
Sulfides
CM
Fig. 5.9. Petrographic analysis of double refractory flotation gold concentrate
5.4 Fungal biotransforming action on the adsorption of gold thiourea and thiosulfate on
carbonaceous materials
In contrast to the cyanide system, extensive research in gold adsorption by carbonaceous
matter within the thiourea and thiosulfate systems is yet to be realized. This study is therefore
important as it reports for the first time, the extent of reduction in gold up-take in these
systems as a result of fungal-biotransformation of carbonaceous matter. The adsorption of
gold thiosulfate complex by activated carbon is low and not considered a practical way of
recovering gold from thiosulfate solutions, and thus not much research has been done
regarding the mechanisms of gold adsorption (Hu and Gong, 1989; Marsden and House,
2006).
112
The proposed mechanisms by which P. chrysosporium biotransforms the surface of carbon are
through surface oxidation and plugging or blockage of adsorption pores. The above was
confirmed through characterization using such techniques as Raman, Fourier Transform
Infrared (FT-IR) and X-ray absorption near edge structure (XANES) spectroscopies, and BET
pore volume and size analysis (Chapter 4). Figs. 5.1-5.3 demonstrate that, in like manner to
the cyanide system, the graphitic structure of carbon is important for gold adsorption from
thiourea and thiosulfate systems (McDougal et al., 1980; Jones et al., 1989; Miller and Sibrell,
1991; Ibrado and Fuerstenau, 1992). The reduction in gold adsorption from the thiourea and
thiosulfate media subsequent to fungal treatment can thus be explained on one hand by the
destruction in the graphitic structure. On the other hand, reduction in gold adsorption is due to
cutback in surface area and pore volume of carbon due to blockage possibly by fungal hyphae
or jelly-like substances generated by fungal-interaction with the medium. Despite the fact that
free growing fungal hyphae are bigger (>100 nm) than the micropore size, it can be postulated
that like all other organisms when constrained, the hyphae may grow and penetrate smaller
openings (<100). This requires further investigations.
Though adsorption on as-received carbon is highest in cyanide solution, it can be seen in Fig.
5.4 that the percentage reduction in adsorption after fungal treatment is also higher in cyanide
than thiosulfate and thiourea. This observation could be due to the larger ionic sizes of the
thiosulfate and thiourea which make it possible for them to adsorb in relatively bigger pores
available after the fungal treatment (Nakbanpote et al., 2000; Syna and Valix, 2003; Miur and
Allymore, 2004). Another possibility in the case of thiourea, in preference to cyanide, might
be the ability to interact with oxygen groups (Arriagada and Gardia, 1997; Nakbanpote et al.,
2000).
113
5.5 Conclusions
This chapter reports, for the first time, preg-robbing of gold thiourea and gold thiosulfate
complexes by different grades of carbonaceous matter (CM), and the extent of reduction in
preg-robbing in these systems as a result of biotransformation of CM by the fungus,
Phanerochaete chrysosporium. The adsorption of gold on the as-received CM occurred in the
order of cyanide > thiourea > thiosulfate, agreeing with the trend reported in literature. After
fungal biotransformation, reduction in gold adsorption was more evident in cyanide (70-97 %)
than in thiourea (60-92 %) and thiosulfate (50-91 %) solutions depending on the type of CM.
Biotransformation of CM led to reduction in micropore volume and increase in oxygen
content all of which militated against uptake of gold cyanide by CM. Comparing the gold
concentrates (FC and BFC) to the surrogate anthracite coal, gold uptake and reduction in gold
uptake by anthracite were far more than FC and BFC. This observation may be due to the
complex nature of the refractory gold ores having 3-4 % organic carbon as against 90 %
organic carbon in anthracite. The above notwithstanding, preg-robbing reduction of 50-70 %
in refractory gold ores is promising.
114
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van Krevelen, D.W., 1993. Coal: Typology-Physics-Chemistry-Constitution, 3rd ed., Elsevier, Amsterdam, 771
pp.
Wan, R. Y., Le Vier, M. and Clayton, R. B., 1994. Hydrometallurgical process for the recovery of precious metal
values from precious metal ores with thiosulfate lixiviant, U. S. Patent 5,354,359.
Wan, R. Y., Le Vier, M., Miller, J. D., 1993. Research and development activities for the recovery of gold from
non-cyanide solutions. In Hiskey, J. B., Warren, G.W., (Eds.) Hydrometallurgy Fundamentals, Technology and
Innovations SME, Littleton, Colorado, 415-436.
Yen, W. T., Aghamirian, M., Deschenes, G., Theben, S., 1998. Gold extraction from mild refractory ore using
ammonium thiosulfate, Proceedings of the International Symposium on Gold Recovery, Montreal, May 3-6,
CIM.
Yen, W.T., Jin, S., Deschenes G., McMullen, J., 1994. Comparison of elution methods for carbon loaded with
gold thiourea complex. Minerals Engineering 7(7), 851864.
Zhang, H., Ritchie, I. M., La Brooy, S. R., 2004. The adsorption of gold thiourea complex onto activated carbon,
117
Chapter 6
Biotransformation of sulfides using the fungus, Phanerochaete chrysosporium: A
potential pretreatment process for refractory sulfidic gold ores
Abstract
This study assessed the capability of the fungus, Phanerochaete chrysosporium, to decompose pyrite,
arsenopyrite and a sulfide-containing flotation concentrate in an effort to develop a microbial process
for pretreating refractory gold ores. The extent of biotransformation was monitored by analyzing for
iron, sulfur and arsenic in incubation solutions, and for sulfide sulfur in the residual solids. For
arsenopyrite, 1.5 wt%, 7.2 wt% and 10.3 wt% of iron, arsenic and sulfur respectively were present in
the incubation solution after 21 days of fungal treatment, whereas for pyrite, there was 1.2 wt% iron
and 6.0 wt% sulfur. For the same processing period in the case of the flotation concentrate, 1.8 wt%,
6.1 wt% and 10.7 wt% respectively of iron, arsenic and sulfur remained in solution. Overall, the
decomposition of sulfide sulfur in the samples was 15 wt%, 35 wt% and 57 wt% respectively for
pyrite, arsenopyrite and the flotation concentrate. Changes in sulfide sulfur concentration and the
formation of oxide phases during fungal treatment were confirmed using Raman spectroscopy and Xray diffraction analysis. These results suggest that P. chrysosporium is a promising microorganism for
oxidative decomposition of metal sulfides associated with refractory gold ores.
Keywords: Phanerochaete chrysosporium; Biotransformation; Sulfides; Pyrite; Arsenopyrite;

Flotation concentrate, Refractory gold ores
6.1 Introduction
6.1.1 Refractory sulfidic gold ores and bacteria oxidation

In sulfidic refractory gold ores, tiny gold particles typically < 1 m (Marsden and House,
2006) may be highly disseminated and locked up within the grain boundaries or fractures of
sulfide minerals such as pyrite and arsenopyrite. Thus, decomposition of the sulfides is
required to liberate the gold (Baako, 1972; Boyle, 1979; Cabri et al., 1989; Komnitsas and
Pooley, 1989; Benzaazoua et al, 2007). Commercially, roasting, pressure oxidation and
bacteria oxidation are among the processes employed in sulfide oxidation (Bennett and
Tributsch, 1978; Brierley, 1978; Arriagada and Osseo-Asare, 1984; Hutchins, 1988;
Berezowsky et al., 1988; Yannopoulos, 1991; Nyavor and Egiebor, 1992; Brierley, 1995;
Rawlings et al., 2003).
Biooxidation makes use of iron and sulfur chemolithoautotrophic bacteria, mainly

Leptospirillum ferrooxidans, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans
to catalyze the oxidation of sulfides, liberating gold for subsequent cyanidation (Lundgren and
Silver, 1980; Livesey-Goldblatt et al, 1983; Brierley, 1997; Hackl, 1997; Kelly and Wood,
2000). The bacteria gain energy by oxidizing ferrous iron and elemental sulfur which generate
in situ, ferric ions and sulfuric acid, the lixiviants responsible for leaching the sulfides (Keller
and Murr, 1982; Sand et al., 1995; Rawlings et al., 1999; Rawlings, 2002; Madigan and
Martinko, 2006). The main reactions involved in pyrite oxidation can be summarized in
Equations 6.1 to 6.5 (Bennett and Tributsch, 1978; Lundgren and Tano, 1978; Sand et al.,
2001; Rohwerder et al., 2003; Holmes, and Bonnefoy, 2006; Marsden and House, 2006).
2Fe2
2FeS2 7O2 2H2O

4Fe2
O2
4H
4Fe3
FeS2 14Fe3
8H2O
FeS2 2Fe3
3Fe2
2H2 O
15Fe2
2S0
4SO24- 4H
16H
[6.1]
[6.2]
2SO24
[6.3]
[6.4]
119
2 S0 3 O2
2 H2O
4H
2 SO24
[6.5]
Equation 6.1 is reported to occur much faster in an environment of suitable bacteria whereas
6.2 and 6.5 do not proceed to any appreciable limit under atmospheric conditions and are said
to rely entirely on bacterial catalysis (Holmes, and Bonnefoy, 2006; Marsden and House,
2006). Though Equations 6.3 and 6.4 can proceed without microbial involvement, their
products, Fe2+ and S0, have to be oxidized by the bacteria to regenerate the lixiviants for
ongoing oxidation of pyrite (Bennett and Tributsch, 1978; Lundgren and Tano, 1978; Murr,
1980; Rawlings et al., 2003; Sand et al., 2001; Holmes, and Bonnefoy, 2006; Marsden and
House, 2006).
At the end of biooxidation, gold may be totally liberated or associated with relatively more
porous iron oxides and thus amenable to cyanidation (Marsden and House, 2006).
Unfortunately, because the bacteria are autotrophs they synthesize carbon from carbon dioxide
(Silver, 1970; Glazer and Nikaido, 1995; Madigan and Martinko, 2006) and do not use
organic carbon. Hence, carbonaceous matter (CM) is not degraded in the case of double
refractory gold ores (DRGO). The CM is consequently routed into downstream cyanidation
circuits where it adsorbs (preg-robs) dissolved gold. It is therefore imperative to deal with this
second part of double refractoriness even after the biooxidation process. Current research
efforts have been focused on two-stage processes to oxidize sulfides and deactivate CM
(Brierley and Kulpa, 1993; Amankwah et al., 2005; Afidenyo, 2008; Yen et al., 2008).
However, using a one-pot process that can simultaneously oxidize sulfides and deactivate
CM will be of immense benefit in the treatment of DRGO as it will shorten process time, and
thus cut down cost.
120
6.1.2 Fungal-biotransformation of sulfides and carbonaceous matter

The fungus, Phanerochaete chrysosporium, secretes the oxidative enzymes, lignin peroxidase
and manganese peroxidase (Glenn et al., 1983; Tien and Kirk, 1983; 1988), which are capable
of degrading aromatic carbonaceous materials. Ofori-Sarpong et al. (2010a) used the fungus
to deactivate anthracite-grade carbonaceous matter and reduce its gold adsorption ability by
more than 90%. P. chrysosporium also secretes a H2O2-generating enzyme, glyoxal oxidase
(Kersten and Kirk, 1987), and hydrogen peroxide is known to solubilize
pyrite and
arsenopyrite as shown in Equations 6.6 and 6.7 (McKibben and Barnes, 1986; Antonijevic, et
al., 1997; Jennings et al., 2000; Antonijevic, et al., 2004; Aydogan, 2006; Aydogan et al.,
2007).
2FeS2 15H2O2
2Fe3
FeAsS 7H2O2
Fe3
4SO24- 2H
14H2O
AsO34- SO24- 2H
6H2O
[6.6]
[6.7]
The iron (III) and sulfuric acid produced constitute the reagents created by the
chemolithotrophic bacteria for the indirect biooxidation of sulfides, as discussed in section
6.1.1 above (Brierley, 1978; Hackl, 1997; Keller and Murr, 1982; Sand et al., 1995; 2001;
Holmes and Bonnefoy, 2006).
Various species of bacteria and fungi have been employed in bio-desulfurization of coals with
sulfur (organic and inorganic) content of up to 6 %. Removal of up to 90 % of inorganic sulfur
and 30 % of organic sulfur has been reported (Faison et al., 1991; Acharya et al., 2005;
Gonsalvesh et al., 2008). The fungal-decomposition of sulfur has been attributed to secretion
of oxidative enzymes (Schreiner et al., 1988; Van Hamme et al., 2003; Aranda et al., 2009),
such as those produced by P. chrysosporium. This study therefore aims at investigating the
ability of the CM-deactivating fungus, P. chrysosporium, to also solubilize/oxidize sulfides
found in refractory gold ores in order to develop a single-stage process for the pretreatment of
double refractory gold ores.
121
6.2.1 Materials
Fungal spores of P. chrysosporium ME446, were obtained from the Ming Tien lab of the
Department of Biochemistry and Molecular Biology, Penn State University. Pyrite and
arsenopyrite samples were supplied by VWR and Wards Natural Science Establishment
respectively. The sulfide-containing flotation concentrate (FC) already milled to all passing 75
m was obtained from the sulfide treatment plant at Bogoso Mine of Golden Star PresteaBogoso Resources, Ghana. The growth media for the fungus, millet and wheat bran (MWB)
were obtained from Natures Pantry, State College, PA, whereas reagent grades succinic acid
and sodium hydroxide were obtained from Alfa Aesar.
6.2.2 Medium preparation, incubation and harvesting of treated material

The millet and wheat bran (MWB) medium was prepared by using 8 g millet plus 2 g wheat
bran. Double-distilled (dd) H2O used for the incubation was buffered with succinic acid and
the pH adjusted to 4 using sodium hydroxide. The medium, in Erlenmeyer flasks, was
moistened with water in the ratio of 1 g medium: 1 mL of dd H 2O, and autoclaved at 121oC
for 30 min. On cooling, the medium was inoculated with 1 mL of spore suspension of P.
chrysosporium (made by suspending 1 vial of spores in 25 mL of dd H2O). The optical density
of the cell suspension, used for inoculation, as measured with a Pharmacia LKB Ultraspec II
UV-visible spectrometer at 600 nm was 0.3 relative to water.
Pyrite and arsenopyrite samples were ground to all passing 75 m, and 10 g each was
moistened with 5 mL of water and autoclave-sterilized for 30 min. Sterilized dd H2O was
added to each sample to achieve 30% solids, and the samples were incubated in triplicates
with the culture medium at 37 oC, for up 21 days on a New Brunswick Series 25 Incubator
shaker at 150 rpm. The potential was monitored throughout the experiments with a VWR
122
Scientific meter model 3000. Control experiments were also set up for 14 days under similar
conditions as described above, except that there was no addition of fungus. This was
necessary in order to establish the exclusive effect of the fungus on biotransformation of
sulfides. At the end of the incubation period the samples were washed with water in the ratio
of about 1 g sample to 200 ml H2O to get rid of the media and fungal biomass, and then dried
at 37 oC for 7 days.
6.2.3 Determination of dissolved components and sulfide sulfur analysis of fungaltreated material
Inductively coupled plasma atomic emission spectroscopy (ICP-AES) is a technique used
routinely for the qualitative and quantitative determination of elements in solution. ICP-AES
has also proven to be a quick and invaluable technique for the determination of total dissolved
sulfur and has found tremendous application in many fields including soil science and
environmental engineering. Soluble sulfur constituents determined by this technique include
Na2SO4, FeSO4, CuSO4, S2O32-, SO32- and Na2S (Zhao et al., 1994; Sarudi et al., 1998; Wang
et al., 1999; McGuire et al., 2001; Dewil et al., 2006). The liquid samples obtained after
fungal-treatment were tested for total dissolved arsenic, iron and sulfur using ICP-AES.
Before determining the sulfide sulfur in both the as-received and processed solids, sulfate
sulfur and elemental sulfur, if any, had to be removed. For elemental sulfur (S 0), samples were
digested in the ratio of 2 g solid sample to 25 mL tetrachloroethylene for 60 min in a boiling
water bath, after which the sample was filtered. The dried filter cake was then contacted with
3 M HCl in the ratio of 1g solid to 50 mL solution (Ciminelli and Osseo-Asare, 1995;
Caldeira et al., 2003; Rait and Aruscavage, 2006). The mixture was heated at 80 oC for 90 min
and then filtered. The filter cake was washed with 400 ml of water and dried at 70 oC. Sulfide
sulfur in the filter cake was then determined by the volumetric combustion technique using
the LECO Sulfur determinater SC-4444DR.
123
6.2.4 Characterization of flotation concentrate

Both X-ray diffraction and Raman spectroscopy were used to evaluate phase changes in the
flotation concentrate before and after treatment with P. chrysosporium. Raman scattering is a
spectroscopic technique based on inelastic scattering of a laser source monochromatic light,
used to diagnose the internal structure of molecules and crystals. Raman spectra was collected
using the Confocal WITec XY Raman spectrometer, with a polarized laser light of 488 nm
wavelength using a 40X objective lens. The spectra were collected at 5 min interval with 1 s
integration time.
For X-ray diffraction (XRD) studies, the samples were ground to fine powder in an agate
mortar and then sprinkled on the surface of a quartz zero-background sample holder. The
analysis was carried out using PANalytical XPert Pro powder diffractometer with Xcelerator
detector. A Ni-filtered CuK radiation produced at 45 KV and 40 mA was used for the
analysis. The scan was run from 10 to 70 degrees for 2-theta at a scan speed of 2 deg/min.
Data acquisition was done using MDI Jade 9 software.

The extent of biotransformation of sulfides by P. chrysosporium was evaluated by analyzing
the incubation solution for total dissolved iron, sulfur and arsenic, and by determining sulfide
sulfur in the residual solids. Fungal dissolution of constituents (FDC) was estimated in wt% as
shown in Equation 6.8, where V is the volume of solution used in fungal incubation, C is the
concentration of dissolved constituent as measured by ICP-AES, Pm is the percentage of the
constituent in the sulfide material and W is the mass of sulfide material used. Equation 6.9
also depicts the percentage conversion of sulfide sulfur (CoS) in the residual solids, where SI
and SF are respective sulfide sulfur contents before and after fungal treatment.
124
V (mL) x C g
FDC (wt%)
CoS (wt%)
Pm
mL
x100%
% xW g
(SI SF) (g)

x100%
SI g
[6.8]
[6.9]
All experiments were carried out in triplicate, and the data reported in the figures are mean
values, with the error bars representing the standard error (SE) of the means. In Equation 6.10,
SD is the sample standard deviation (Equation 6.11) where n is the sample size, xi and x-bar
are the ith value and the mean respectively.
SE x
SD
[6.10]
n
3
SD
xi
n 1
[6.11]
6.3 Results and discussion
6.3.1 Transformation of pyrite by P. chrysosporium

To discuss the direct effect of P. chrysosporium on the biotransformation of sulfides it was
necessary to examine the effect of the growth media, and this was done by running control
experiments. The results obtained for pyrite samples incubated for two weeks both in the
absence and presence of P. chrysosporium are illustrated in Fig. 6.1. The figure shows that
though there is some amount of dissolution of pyrite in the absence of fungal biomass, the
presence of the fungus led to 3-6 fold increase in dissolution of iron and sulfur. The minor
solubilization in the control experiment might be due to natural oxidation of sulfides
125
according to the reaction shown in Equation 6.1. These are, however, slow reactions and
cannot lead to any appreciable level of dissolution under atmospheric conditions. In a strong
oxidizing environment containing hydrogen peroxide and oxidative enzymes, such as that
generated by P. chrysosporium, the conversion of ferrous to ferric ion as shown in Equation
6.12 is possible (Brierley, 1978; Keller and Murr, 1982; Sand et al., 1995; Hackl, 1997;
Rawlings, 2002; Rohwerder et al., 2003; Marsden and House, 2006).
Fe3
Constituents in solution (% of
initial weight)
Fe2
[6.12]
6
Iron
Sulfur
4
3
2
1
0
Control
Treated
Dissolved constituents
Fig. 6.1. Two-week biotransformation of pyrite in the absence and presence of P.

chrysosporium. The experiment was conducted at 37 oC for 14 days at pH 4 and 30 % solids
The fungal-dissolution of pyrite as a function of time is depicted in Fig. 6.2. After 21 days of
fungal treatment there was 1.2 wt% iron and 6.0 wt% sulfur in the incubation solution. It can
be seen from the figure that the rate of dissolution of pyrite is higher at the initial stages (up to
7 days) but decreases afterwards. In a batch culture such as the one utilized in this study,
126
microbial growth and hence activity increases exponentially in the initial stages of growth,
and retardation takes place afterwards due to exhaustion of growth media and generation of
waste products of metabolism (Shuler and Kargi, 1992). The reduction in microbial activity
and availability of oxidants may account for the trend observed.
Constituents in solution (% of initial weight)
7
Fe wt %
S wt %
6
5
4
3
2
1
0
10
15
20
25

Fig. 6.2. Accumulation of dissolved sulfur and iron during the incubation of pyrite with P.
chrysosporium. The experiment was conducted at 37 oC for up to 21 days at pH and 30 %
solids
The pH for optimum operation of P. chrysosporium is 4 (Tien and Kirk, 1988) and it is at this
pH 0.3 that the experiment was carried out. It is known that in oxidizing leaching of pyrite
above pH of 3 there is production of insoluble iron oxides/hydroxides/sulfates such as Fe 2O3,
FeOOH and FeSO4, which precipitate on the surface of unreacted material (Osseo-Asare et al.,
1984; Marsden and House, 2006). Other components that may be present in the system
127
include sulfate, sulfite, thiosulfate, and elemental sulfur (Ciminelli, 1987; Ciminelli and
Osseo-Asare, 1995; Wei and Osseo-Asare, 1997; Shahverdi et al., 2001; Bhakta and Arthur,
2002; Caldeira et al., 2003). Accumulation of elemental sulfur in the system has the potential
to passivate the unreacted surfaces and thus obstruct movement of reactants and products to
and from the reaction sites leading to reduction in the overall rate of reaction (Lindstrom et
al., 1992; Marsden and House, 2006; Mousavi et al., 2006). Stoichiometrically, the molar ratio
of sulfur to iron in pyrite is 2:1. However, this ratio does not tally with the measurements
made as the ratio is about 5 moles of sulfur to 1 mole of iron. The deficiency of iron in the
solution is likely due to precipitation as explained above. The progressive decomposition of
pyrite with time during treatment with the fungus is shown in Fig. 6.3.
16
Unconverted pyrite (g)
14
9.8
12
10
9.4
Unconverted pyrite
Converted pyrite
9.0
8.6
2
8.2
Converted pyrite (wt % of initial)
10.2
5
10
15
20
25
Fig. 6.3. Biotransformation of sulfide sulfur in pyrite as a function of fungal-treatment. The

experiment was conducted at 37 oC for up to 21 days at pH 4 and 30 % solids
The mass of unconverted pyrite was estimated from the percentage of sulfide sulfur as
measured by the LECO technique. From initial sulfide sulfur content of 52 %, there was a
128
reduction in sulfide sulfur to 43 % within 21 days of incubation, representing an oxidation of

15 %. About 90 % of the conversion was achieved within the initial 10 days of fungaltreatment. The generally low level of oxidation of pure sulfides having initial sulfide sulfur
content of about 52 % is likely to translate into higher percentage oxidation with refractory
sulfidic gold concentrates with lower sulfide sulfur content.
6.3.2 Transformation of arsenopyrite by P. chrysosporium

The dissolution of the constituents of arsenopyrite in the absence and presence of the fungus,
P. chrysosporium is presented in Fig. 6.4. In the absence of the fungus, dissolution of arsenic,
Constituents in solution (% of initial

weight)
iron and sulfur was marginal at 0.9 wt%, 0.16 wt% and 2.6 wt% respectively.
10
Control
Treated
6
4
2
0
Arsenic
Iron
Sulfur
Fig. 6.4. Two-week biotransformation of arsenopyrite in the absence and presence of P.
129
When the sample was incubated with P. chrysosporium, dissolution increased to relatively
high values of 6.3 wt%, 1.3 wt% and 9.2 wt% for arsenic, iron and sulfur respectively.
The minor solubilization in the control experiment might be due to natural oxidation of
sulfides according to the reactions shown in Equation 6.13 (Marsden and House, 2006). In
a strong oxidizing environment the conversion of arsenite to arsenate is possible as shown
in Equation 6.14. The progressive increments of iron, sulfur and arsenic in incubation
solution during the fungal-treatment of arsenopyrite over different incubation periods are
presented in Fig. 6.5.
4FeAsS 11O2
6H 2O
2H3AsO3 O2
4Fe2
4SO24
12H
4AsO33
[6.13]
2H 2 AsO 4
[6.14]
12
As wt %
Fe wt %
S wt %
10
8
6
4
2
0
0
10
15
20
25

Fig. 6.5. Dissolution of iron, sulfur and arsenic from arsenopyrite as a function of incubation
time. The experiment was conducted at 37 oC for up to 21 days at pH 4 and 30 % solids
130
Over a period of 21 days, the concentrations of iron, sulfur and arsenic in solution increased
consistently ending at 1.5 wt%, 7.2 wt% and 10.3 wt% respectively. The trends in Fig. 6.5
indicate that the concentrations of the constituents in solution are yet to reach their respective
plateaus, and the processing time for maximum degradation extends beyond 21 days. The
stoichiometric ratio of iron, arsenic and sulfur in 1 mole of arsenopyrite is 1:1:1. This ratio
changed based on the amount of these constituents in incubation solution as the ratio in
solution was about 0.14 mol of Fe, 0.7 mol of As and 1 mol of sulfur after 21 days of
treatment. This implies a deficiency of 0.86 mol of Fe and 0.3 mol of As in solution, due to
precipitation.
According to the Eh-pH diagram for Fe-As-S-H2O system in Fig. 6.6, at pH 4 as was used in
this study, the formation of Fe2+ and FeOOH is possible above Eh of 0.3 V (SHE). Goethite
(FeOOH) exists as the most dominant solid species of iron (Osseo-Asare et al., 1984). In the
present study, measured potentials were in the range 0.4 to 0.5 V (SHE) indicating the
possibility of goethite precipitation which may partly explain the very low values of total iron
(0.9 wt%) in solution.
In addition, precipitation of ferric arsenate at pH above 2 has been observed, with concomitant
decrease in reaction rate (Escobar et al., 2000; Brierley, 2003; Lindstrom et al., 2003;
Marsden and House, 2006). Overall the conversion of sulfide sulfur in arsenopyrite as
determined by the LECO combustion technique was 35 wt%.
The higher overall decomposition of arsenopyrite as compared with 15% in the case of pyrite
is because pyrite is more noble than arsenopyrite and the As-S bond is weaker than the Fe-S
bond (Marsden and House, 2006). Microbial attack is naturally initiated from weaker zones in
a material (Wackett and Ellis, 1999).
131
Fe3+
1.0
FeOOH
Fe
2+
Eh
0.0
FeS2
Fe2+
Fe3O4
FeAsS
-1.0
Fe
Fe(OH)3Fe(OH)42-
{Fe} = 10-4
{As} = {S} = 10 -4
-2.0
0.0
4.0
8.0
12.0
pH
Fig. 6.6. Eh-pH diagram for Fe-As-S-H2O system (Osseo-Asare, 1984)
6.3.3 Transformation of flotation (sulfidic gold) concentrates by P. chrysosporium

Figure 6.7 presents the direct dissolution of iron, sulfur and arsenic from sulfidic gold
concentrates by P. chrysosporium after 14-day contact with MWB medium with and without
fungal biomass. As explained in section 6.3.2, the slight dissolution obtained in the control
experiment may be due to natural dissolution of sulfides in the presence of water and oxygen,
which may cease in a limited oxidizing environment. However, in the presence of the fungus
and the associated oxidizing environment generated, about 2-5 fold increase in dissolution
was observed. Figure 6.8 depicts the dissolution of sulfides from FC by P. chrysosporium as a
function of incubation time in weight percent (Equation 6.9).
132

weight)
12
Control
10
Treated
6
4
2
0
Arsenic
Iron
Sulfur
Fig. 6.7. Two-week biotransformation of flotation concentrate in the absence and presence of
P. chrysosporium. The experiment was conducted at 37 oC for 14 days at pH 4 and 30 %
solids
The dissolution of sulfide begins after the initiation of incubation and rises rapidly within the
first week. Though the rate of dissolution declines after a week of incubation, Fig. 6.8 shows a
consistently increasing dissolution as the incubation period increases. This signifies that given
more time for incubation, dissolution will continue.
133
12.0
As wt %
Fe wt %
S wt %
10.0
8.0
6.0
4.0
2.0
0.0
0
10
15
20
25

Fig. 6.8. Dissolution of sulfides in flotation concentrate by P. chrysosporium as a function of
time for 21 days. The experiment was conducted at 37 oC for up to 21 days at pH 4 and 30 %
solids
Figure 6.9 presents the transformation of sulfide sulfur (Equation 6.10) in the residual solid
materials after incubation. The left vertical axis represents the actual mass of sulfide sulfur in
the flotation concentrate, and the mass was estimated from sulfide sulfur determined via the
LECO combustion technique. From an initial mass of about 5 g of sulfide sulfur in the
flotation concentrate, there was 48 % reduction within the initial 10 days of processing.
Beyond this period, biotransformation stalled leading to a further 9% decrease bringing the
total reduction in sulfide sulfur to 57% after 21 days.
134
60
50
4
40
Unconverted sulfide sulfur
30
Converted sulfide sulfur
20
2
10
1
Sulfide sulfur converted (wt % of initial)
Unconverted sulfide sulfur in gold concentrate (g)
0
0
10
15
20
25
incubation time. The experiment was conducted at 37 oC for up to 21 days at pH 4 and 30 %
solids
6.3.4 Characterization of flotation concentrate

Many metal sulfides show Raman peaks between 300 and 400 wavelength (Ushioda, 1972;
Mernagh and Trudu, 1993; Sasaki et al., 1995; Edwards et al., 1999) and this was confirmed
in the current investigation. Raman spectroscopy of the as-received and treated flotation
concentrate indicated a reduction in the intensity of sulfide peaks after fungalbiotransformation as presented in Fig. 6.10. These observations authenticate the decrease in
sulfide sulfur as determined by the LECO combustion technique. Further analysis by XRD
shows other products of sulfide sulfur oxidation (Fig. 6.11).
135
As-Received
Intensity
Raman
CCD,
cts
1000
800
Treated
600
400
200
400
600
800
1000
1200
Rel. 1/cm
Wavenumber, cm-1
Fig. 6.10. Raman spectrogram of as-received and fungal-treated flotation concentrate.
Treatment was conducted for 14 days at 37 oC and pH 4
By comparing the as-received and fungal-treated samples in Fig. 6.11, it was observed that the
treated material had two new oxygen containing phases; hematite (Fe2O3) and ferric arsenate
(FeAsO4.H2O). In similar systems, ferric arsenate has been observed earlier by researchers
such as Escobar et al. (2000), Brierley (2003) and Lindstrom et al. (2003). Though according
to Fig. 6.6 goethite (FeOOH) is generally expected to be the dominant phase, it was not
detected in the present study. It is likely that other oxides were precipitated but due to lower
resolutions, the peaks might have been masked by the higher intensity peaks. Also, the
diffractometer used for the study, has a detection limit of 5 % and thus any phase with less
than 5 wt% composition may not show a peak on the diffractogram.
136
As-received FC
Intensity (counts)
300
Treated FC
1 quartz
2 pyrite
3 arsenopyrite
4 hematite
5 ferric arsenate
6 muscovite
200
1 2
5
100
2
6
5 1
2
3
3
6
1
0
10
20
30
40
50
60
70
2 Theta (deg)
Fig. 6.11. X-ray diffractogram of as-received and fungal-treated flotation concentrate.

Treatment was conducted for 14 days at 37 oC and pH 4
The presence of the oxidation products indicates clearly that P. chrysosporium has the ability
to transform sulfides in refractory gold ores. These results compare well with those obtained
by Brierley, (2003) in the biooxidation of refractory gold ores of about 2 % sulfide-sulfur
content at 20-60 oC for 10 days using mesophilic and moderate-thermophillic bacteria, and
hyper-thermophillic archea. The author achieved over 50 % increase in the subsequent gold
recovery by cyanidation. In a related study, gold recovery increased appreciably following
fungal treatment (Ofori-Sarpong et al., 2010b).
137
6.4 Conclusions
The fungus, Phanerochaete chrysosporium, which has proven its ability to reduce the pregrobbing capacity of anthracite-grade carbonaceous matter, was investigated for its ability to
also transform sulfides, in an effort to develop a microbial process for pretreating refractory
gold ores. The results show that P. chrysosporium is capable of solubilizing sulfides. For an
incubation period of 21 days, sulfide sulfur decreased by 35 % in the treated arsenopyrite
sample and 15 % in the pyrite sample. These materials had initial sulfide sulfur content of
19.6 and 52 % respectively. For refractory sulfidic gold concentrate with about 15 % initial
sulfide sulfur content, a higher oxidation level of 57 % was realized. Changes in sulfide sulfur
concentration and the formation of oxide phases during fungal treatment were confirmed
using Raman spectroscopy and X-ray diffraction analysis. For all the samples, there was a
strong correlation between the decomposition of sulfides and incubation period. These results
indicate that the fungus, P. chrysosporium, is a potential microorganism for oxidative
decomposition of metal sulfides associated with refractory gold ores.
138
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142
Chapter 7
Biotransformation of double refractory gold ores by the fungus, Phanerochaete
chrysosporium
Abstract
Double refractory gold ores (DRGO) contain organic carbonaceous matter (CM) and metal sulfides,
and thus require pretreatment before cyanidation for gold extraction. Though there have been several
studies into microbial pretreatment of DRGO, available literature indicates that microbial degradation
of both sulfides and CM in a single-stage process has not achieved much success. In an on-going
research, the fungus Phanerochaete chrysosporium, has been used to effectively reduce the gold
adsorption (preg-robbing) nature of CM on one hand, and decompose sulfides on the other.
The present research reports for the first time; the ability of P. chrysosporium to simultaneously
decompose sulfides in refractory gold ores, liberating locked-up gold, and deactivating carbonaceous
matter, thus reducing preg-robbing. Flotation concentrate (FC) of DRGO and bacterial-oxidized FC
(BFC) were utilized in this investigation. Sulfur speciation of the biotreated solids indicated
progressive decomposition of pyritic sulfur with time, and incubation solution showed increase in
dissolved iron, sulfur and arsenic. Cyanidation of fungal-treated concentrates yielded an increase in
recovery of 37 % and 13 % for FC and BFC respectively over the as-received concentrates. The results
demonstrate a potential alternative pretreatment process for DRGO.
Keywords: Phanerochaete chrysosporium; Double refractory gold ores; Carbonaceous matter;

Flotation concentrate; sulfidic minerals;
Bacteria; biooxidation; Fungi;
Biotransformation;
Cyanidation; Gold recovery.
143
7.1 Introduction
Gold ores are termed double refractory when they contain organic carbonaceous matter (CM)
and sulfide minerals (Nyavor and Egiebor, 1992; Amankwah et al., 2005). Sulfide minerals
encapsulate fine gold particles and prevent direct contact between gold particles and leaching
reagents, thus interfering with gold dissolution, whereas CM adsorbs dissolved aurocyanide
complexes, thereby reducing gold extraction - a phenomenon known as preg-robbing (OsseoAsare et al., 1984a; Hausen and Bucknam, 1985; Schmitz et al., 2001).
For gold locked up in double refractory ore (DRGO) to be amenable to cyanidation,

pretreatment is required to decompose the shielding sulfidic minerals and in consequence
liberate the gold. Processes such as roasting, pressure oxidation and bacterial oxidation have
been used commercially to pretreat refractory gold ores (Bennett and Tributsch, 1978;
Arriagada and Osseo-Asare, 1984; Hutchins, 1988; Weir and Berezowsky, 1986; Berezowsky
et al., 1988; Afenya, 1991; Brierley, 1995; Rawlings et al., 2003). Roasting involves the use
of high temperature (450-700 oC) to decompose sulfidic and carbonaceous materials in the
presence of oxygen/air. The combustion reaction leads to generation of harmful gases such as
sulfur dioxide and arsenic trioxide, which cause environmental problems (Komnitsas and
Pooley, 1989; Marsden and House, 2006). Pressure oxidation makes use of high temperature
and pressure, typically 180-225 oC and 1500-3200 kPa, to decompose metal sulfides in the
presence of water. Though this process eliminates the harmful gaseous products, it is
associated with operational difficulties such as safety due to high operating temperatures and
pressures, higher rates of material corrosion, and higher equipment cost (Weir and
Berezowsky, 1986; Marsden and House, 2006). In view of the high cost and environmental
concerns associated with the above processes, bacteria oxidation has become a method of
choice (Brierley, 1997; Marsden and House, 2006).
For the past two decades, the use of biooxidation, which can be seen as the biological
counterpart of pressure oxidation, has become more prominent in part to curtail high cost and
also to improve upon gold recovery and environmental/safety aspects of processing (Livesey144
Goldblatt et al, 1983; Komnitsas and Pooley, 1989; Yannopoulos, 1991; Hackl, 1997;
Rawlings, 1998). According to Brierley (1995), biooxidation can generally reduce capital
costs of roasting by 12-20 %, operating costs by 10 % in some cases, and construction time by
25 % (Brierley, 1997). Several bacteria are known to oxidize sulfides but the ones commonly
used in commercial biooxidation of refractory gold ores are Acidithiobacillus thiooxidans,
Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans (Lundgren and Silver, 1980;
Livesey-Goldblatt et al, 1983; Brierley, 1997; Hackl, 1997; Kelly and Wood, 2000; Holmes
and Bonnefoy, 2006). Unfortunately, because the bacteria are autotrophs, they synthesize
carbon from carbon dioxide and do not use organic carbon, and hence, the CM is not degraded
in the case of DRGO (Rohwerder et al., 2003; Madigan and Martinko, 2006).
Apart from roasting that aims at eliminating the carbon, passivation has been employed using
carbon-blanking agents such as kerosene and other organic reagents (Abotsi and Osseo-Asare,
1987; Hutchins et al., 1988; Adams and Burger, 1998). Though yet to be applied
commercially, biomodification of carbon appears more attractive from economical and
environmental points of view, as it occurs at relatively low temperatures (25-40 oC) and at
atmospheric pressure. In addition, lixiviants are regenerated in situ due to the presence of the
microorganisms. Microorganisms employed in this manner include the bacteria; Pseudomonas
sp., Achromobacter sp., Streptomyces setonii, and the fungi; Aspergillus bruneio, Penicillium
citrinum, Trametes versicolor and Phanerochaete chrysosporium (Portier, 1991; Brierley and
Kulpa, 1992; 1993; Amankwah and Yen, 2006; Afidenyo, 2008; Yen et al., 2008; OforiSarpong et al., 2010a).
Available literature indicates that microbial degradation of both sulfides and CM in a singlestage has not yet been realized with great success (Brierley and Kulpa, 1993; Amankwah and
Yen, 2006; Afidenyo, 2008; Portier, 1991; Yen et al., 2008). The limitation to single-stage
processes has been attributed to the inability of the heterotrophic carbon deactivating
microorganisms, which work at relatively higher pH values (4-8), to coexist and function
effectively at the optimum conditions within which the acidophilic (pH: 1-2) sulfide oxidizing
bacteria work (Tortora et al., 2004; Amankwah et al., 2005; Madigan and Martinko, 2006).
145
In on-going studies, the white rot lignin-degrading fungus, Phanerochaete chrysosporium,

which secretes oxidative enzymes, has been utilized to deactivate anthracite-grade
carbonaceous matter and reduce gold preg-robbing by more than 90 % (Ofori-Sarpong et al.,
2010a). In a related study using pyrite, arsenopyrite and flotation concentrate of DRGO, P.
chrysosporium was able to decompose the sulfides progressively over time. For a processing
time of 21 days, conversions of sulfides in pyrite, arsenopyrite and flotation concentrate
respectively were 15 wt%, 35 wt% and 57 wt% (Ofori-Sarpong et al., 2010b). P.
chrysosporium has also been used to degrade gold-bearing wood chips to liberate gold for
cyanide leaching, and the authors observed a 10 % increase beyond the original 60 %
cyanidation gold recovery of untreated wood chips (Martin and Petersen, 2001). This study
investigated the simultaneous carbon passivating and sulfur oxidizing ability of P.
chrysosporium in a single stage process using double-refractory gold ores.

Gold-containing samples, P. chrysosporium and the millet and wheat bran (MWB) medium
were obtained and incubated as presented in Chapter 6, Section 6.2 and Ofori-Sarpong et al.
(2010b). Analysis of the incubation solution for dissolved iron, arsenic and sulfur, and sulfide
sulfur determination in residual solids were conducted as detailed in the same chapter. Sigma
flux used for fire assaying of gold was obtained from Sigma Chemicals.
146
7.2.2 Cyanidation
Cyanidation was conducted at 25 oC on 25 g of as-received and fungal treated materials at 30
% pulp density and 10 g/L cyanide concentration. The pH was maintained between 11 and 12
by the addition of sodium hydroxide. The samples were agitated on a New Brunswick Series
25 incubator shaker at 150 rpm for 24 hours in partially covered Erlenmeyer flasks to allow
oxygen into the system as required by the fundamental equation for cyanidation (Equation
7.1).
4Au 8CN- 2H 2 O O2
4Au(CN) -2 4OH-
[7.1]
At the end of the leaching period the pulp was filtered, and the solution obtained was analyzed
for gold by ICP-AES whereas the residual solids (tails) were assayed for residual gold by fire
assaying.
7.2.3 Fire Assaying

The gold in solid samples was determined by fire assaying with atomic absorption
spectrometric finish. In this method, a known mass of gold-containing sample is mixed with
three times its mass of Sigma flux made up of lead oxide, sodium tetraborate, wheat flour and
sodium carbonate. The mixture was heated to 1100 oC in a fireclay crucible and kept at the
temperature for 45 min. During the process, lead oxide is reduced by the carbon-containing
flour and the lead produced collects the precious metal to form the lead button. The other
fluxes react with unwanted materials in the gold ore to form a slag that separates from the lead
on pouring, due to its lower density. The lead was hammered into a rough cube and cupelled
at 950 oC on a shallow dish made of magnesium oxide, known as a cupel. During cupellation
lead is absorbed by the cupel, leaving the precious metal bead, which is digested with aquaregia and the gold determined by ICP-AES.
147

After incubating the concentrates with and without P. chrysosporium, the solutions were
filtered for determination of arsenic, iron and sulfur. Fungal dissolution of constituents (FDC)
and percentage conversion of sulfide sulfur (CoS) were calculated as defined in Chapter 6,
section 6.2.
The cyanidation test was conducted to evaluate the extent of liberation of gold, and thus
cyanide amenability following fungal-transformation of FC and BFC. Equation 7.2 was used
in calculating the percentage gold recovery (PGR), where W is the mass of gold concentrate
and V is the volume of water used in the cyanidation experiment, C is the concentration of
gold in the leachate after cyanidation as determined by ICP-AES whereas HG is the head
grade of the gold concentrate before cyanidation as determined by conventional fire assaying.
PGR ( wt %)
1
( g 1 ) xV (mL) xC ( g )
mL
W
x100%
HG ( g )
t
[7.2]

The double refractory gold ores mined by GSPBR contain about 2 % sulfur and this is lower
than about 10 % sulfur required for biooxidation. For this reason, the ore is ground and froth
flotation is used to concentrate sulfides which serve as feed for biooxidation. The biooxidation
process does not destroy carbonaceous matter and it continues to adsorb gold in the
subsequent cyanidation step. Consequently, it was necessary to subject both FC and BFC to
fungal treatment to ascertain the ability of the fungus to decompose sulfides and deactivate
carbonaceous matter. BFC was selected for testing to also allow comparison between bacteriatreated and fungi-treated concentrates.
148
7.3.1 Fungal-biotransformation of sulfides in flotation concentrate (FC)

The concentration of dissolved iron, arsenic and sulfur in incubation solution was determined,
presented and discussed in Chapter 6 under Figs. 6.7 and 6.8. Changes in the concentration of
sulfide sulfur is an important parameter in the biotransformation of sulfide minerals, and the
transformation of pyritic sulfur in the residual solids as a result of fungal-treatment of FC is
60
50
4
40
Unconverted sulfide sulfur
30
Converted sulfide sulfur
20
2
10
1
Unconverted sulfide sulfur in gold concentrate (g)
depicted in Fig. 7.1.
0
0
10
15
20
25
incubation time. The experiment was conducted at 37 oC for up to 21 days at pH 4 and 30 %
solids
It can be seen from the figure that the sulfide sulfur content of the residual solids reduced
from an initial value of 15 % of FC to about 6 %, representing a percentage decomposition of
57 %. About 48 % decomposition occurred within the initial 10 days of fungal-treatment as
149
shown in Fig 7.1, after which the decomposition rate reduced leading to additional 9 %
decomposition within the next 11 days. As explained in Chapter 6, precipitation of products
such as goethite, ferrous sulfate, ferric arsenate and elemental sulfur on the surface of
unreacted material can slow down the reaction (Osseo-Asare et al., 1984b; Ciminelli and
Osseo-Asare, 1995; Wei and Osseo-Asare, 1997; Shahverdi et al., 2001; Bhakta and Arthur,
2002; Caldeira et al., 2003; Marsden and House, 2006).
7.3.2 Fungal-biotransformation of sulfides in bacterial-treated FC (BFC)

The effect of P. chrysosporium on the biotransformation of BFC is presented in Fig 7.2. The
assessment of the direct effect became possible by running control experiments with the
growth media alone, under similar conditions as those used in the fungal experiment. It is
clear from the figure that there was a sharp increase in decomposition of the major
constituents in BFC by 3-5 folds in the presence of the fungus. Analysis of the residual solids
revealed an overall oxidation of about 85 % sulfide sulfur within 14 days of fungal-treatment.
The results obtained after biotreating both FC and BFC demonstrate the potential of P.
chrysosporium to oxidize sulfides in DRGO and thus liberate gold for cyanidation. The
fungal-decomposition of sulfides in BFC allowed for extra liberation of gold beyond the
bacterial oxidation of FC, as discussed in Section 7.3.3.
150

weight)
50
Control
Treated
40
30
20
10
0
Arsenic
Iron
Sulfur
Fig. 7.2. Two-week fungal-treatment of BFC in the absence and presence of P.
7.3.3 Effect of fungal-treatment on cyanide amenability of FC and BFC

The recoveries of gold from FC and BFC are presented in Fig. 7.3 for 14-day control and
fungal-treated materials. The figure shows an increase in gold extraction as a result of fungaltreatment. The recoveries of gold from the respective control experiments of samples FC and
BFC were 47 % and 84 %, whereas after treatment with P. chrysosporium, gold recovery
increased to 75 % and 93.5 % correspondingly. Changes in cyanide-amenability of sample FC
as a function of fungal treatment time is presented in Fig. 7.4.
151
Gold recovery (%)
100
80
Control
Treated
60
40
20
0
FC
BFC
Gold concentrate
Fig. 7.3. Gold recovery from fungal-treated and control FC and BFC. Cyanidation was
conducted at 30 % pulp density for 24 h at pH 11 and cyanide strength of 10 g/L
Gold recovery (%)
80
70
60
50
40
0
10
15
20
25

Fig 7.4. The effect of fungal treatment time on gold extraction from Sample FC. Cyanidation
was conducted at 30 % pulp density for 24 h at pH 11 and cyanide strength of 10 g/L
152
After 24-h cyanidation, the percentage of gold extracted from the as-received flotation
concentrate was 40 %. This value increased to 67 % within 7 days of incubation, and there
was progressive improvement over time to 78 % at the 21st day of fungal-treatment. The
sequence in Fig. 7.4 shows that extraction has not yet reached a plateau and it is possible to
extract some more gold if fungal treatment time is extended. This is expressed in the
correlation between fungal-decomposition of sulfide sulfur in FC over time, and gold recovery
80
70
Gold recovery (%)
60
70
50
40
60
30
20
50
Gold recovery (%)
10
Sulfide converted (%)
40
0
0
10
15
20
by cyanidation (Fig. 7.5).
25

Fig. 7.5. Variation of sulfur transformation and gold extraction as a function of fungaltreatment time. Cyanidation was conducted at 30 % pulp density for 24 h at pH 11 and
cyanide strength of 10 g/L
The trends of both sulfide-sulfur conversion and gold extraction show increments, and
consequently, it is expected that with increase in fungal-treatment time more sulfides will be
decomposed, more gold will be liberated for cyanidation, and gold extraction will increase.
The limitation in the action of the fungus on the flotation concentrate to achieve higher gold
153
recovery values could be due to the amount of refractory materials present in excess of the
oxidizing enzymes secreted by the fungus, thus leading to incomplete oxidation (Tien and
Kirk, 1983; 1988). With the same amount of gold concentrate, growth media and fungus,
treatment of BFC (Fig. 7.2) realized over 3 times percentage decomposition of sulfide as
compared to FC (Fig. 6.7 in Chapter 6). This disparity is due to the difference in initial
sulfide content; 3 % in BFC as against 15 % in FC, making the available oxidizer to sulfide
ratio higher in the case of BFC. Changes in cyanide-amenability of sample BFC as a function
of fungal treatment time is presented in Fig. 7.6.
Gold recovery (%)
100
95
90
85
80
0
10
15
20
25

Fig 7.6. Gold extraction from BFC as a function of fungal incubation time. Cyanidation was
In a manner similar to gold extraction from the flotation concentrate, recovery from sample
BFC also increased after fungal treatment (Fig. 7.6). From an initial recovery of 81 %, gold
extraction increased to 94 % within 10 days of incubation, beyond which the extraction
stagnated up to 21 days of treatment. This is because sulfide decomposition peaked at 85 %
within 14 days of fungal-treatment of BFC. In addition, the high levels of dissolved gold
concentrations in solution (17 mg/L) achieved during cyanidation prevented more gold from
being dissolved from the solids (Marsden and House, 2006).
154
7.4 Assessment of fungal- and bacterial- treatment of gold concentrates: Single-stage

against two-stage processes
To evaluate the effectiveness of the fungal process as a potential single-stage pretreatment
method for double refractory gold ores, it was necessary to compare the extent of gold
extraction from as-received flotation concentrate (FC), fungal-treated FC (FFC), as-received
bacterial-treated FC (BFC) and fungal-treated BFC (FBFC). The comparison is visually
accessible in Fig. 7.7, which shows increase in gold extraction in the order of FC (40 %) <
FFC (78 %) < BFC (81 %) < FBFC (94 %).
100
Gold recovery (%)
80
60
40
20
0
FC
FFC
BFC
FBFC
Gold concentrates:
FC-flotation concentrate;
BFC-bacterial-treated FC;
FFC-fungal-treated FC;
FBFC-fungal-treated BFC
Fig. 7.7. Comparison of gold extraction from flotation concentrate (FC), fungal-treated FC
(FFC), bacterial-treated FC (BFC) and fungal-treated BFC (FBFC). Cyanidation was
BFC and FFC are from single stage processes while FBFC is from a two-stage process. The
gold recovery from FBFC was 13 % and 16 % above BFC and FFC respectively. The average
gold recoveries from FC after fungal-treatment alone (FFC), and after commercial bacterial155
treatment process alone (BFC), compare favorably with respective values of 78 % and 81 %
(Fig 7.7). The closeness of the two values highlights the potential of P. chrysosporium in the
biotransformation of refractory gold ores.
P. chrysosporium has an added advantage of reducing gold adsorption, and Fig. 7.8 shows the
decomposition of sulfides, reduction in gold adsorption following deactivation of
carbonaceous matter (CM), and associated increase in gold extraction after fungal treatment of
double refractory gold ores. From Fig 7.8, a reduction in sulfide sulfur and gold adsorption by
CM which were 57 % and 64 % respectively, translated into an increase in gold extraction of
37 %.
Percentage change (%)
120
As-Received
Fungal-treated
100
80
60
40
20
0
Sulfide sulfur
content of FC
Gold adsorption Cyanidation gold

on carbon in FC recovery from FC
Type of process/material
Fig. 7.8. Changes in sulfide sulfur content, gold adsorption by carbonaceous matter and gold
extraction from FC following 14-day fungal treatment. Cyanidation was conducted at 30 %
pulp density for 24 h at pH 11 and cyanide strength of 10 g/L.
In an independent but related study, Yen et al. (2008) utilized the carbon deactivating fungus,
Trametes versicola, bacterium, Streptomyces setonii, the sulfide oxidizing bacteria
Acidithiobacillus
ferrooxidans,
Acidithiobacillus
thiooxidans
and
Leptospirillum
ferrooxidans, and abiotic processes to treat different types of double refractory gold ores. The
156
authors used different combinations of the microorganisms mentioned to treat the ores in two
stages, and reported increase in overall gold recoveries. To compare with the findings being
reported in this study, their recoveries for FC, FFC, BFC and FBFC (as defined above) were
in the order of 15-22 % for FC; 54-65 % for FFC; 71-81 % for BFC and 87-92 % for FBFC. A
first-stage fungal process from the various two-stage combinations by Yen et al. (2008) can be
viewed as equivalent to the single-stage fungal process (FFC) utilized in the current study.
Comparing the two studies therefore, it can be concluded that for fungal treatment alone
(single-stage) this study achieved a higher overall recovery (78 %), which is much higher than
the (54-65 %) achieved by Yen et al. (2008), an overall increase of 20-44 % in favor of the
present study.
The ability of P. chrysosporium to reduce the gold adsorption activity of carbonaceous matter
(CM) in addition to decomposing sulfides suggests that the recovery of FFC should
technically outweigh that of BFC, since the bacteria treatment does not deactivate CM.
Notwithstanding the above, the fungal-process is at its infant stage of just about 3-year
laboratory batch research. On the other hand, the bacteria process has undergone over 4
decades of research spanning from laboratory batch/continuous through pilot to commercial
scale. It therefore stands to reason that the fungal-process has yet to overcome teething
problems, such as optimization of kinetics and quantification of biomodifiers and materials
balance. When the experiments are conducted in a continuous system instead of the batch
units used, the processes could be faster due to continuous feeding of reactants and removal of
products.
7.5 Conclusions
This research examined the capability of Phanerochaete chrysosporim to simultaneously
biotransform sulfides and carbonaceous matter in double refractory gold ores (DRGO). It is
comprehensible from the investigations that P. chrysosporium has the ability to solubilize and
oxidize sulfide minerals and deactivate carbonaceous matter in DRGO. The results show that
157
at pH 4, substantial sulfide dissolution takes place in the presence of the fungus leading to
relatively high solution concentrations of iron, sulfur and arsenic with overall sulfide sulfur
decomposition of 57 % and 75 % of FC and BFC after 21 days of fungal-treatment. Within
the same period, gold extraction increased appreciably from 40 % to 78 % for sample FC, and
81 % to 94 % for sample BFC, signifying increase in recovery of 38 % and 13 % over the asreceived FC and BFC respectively. For sample FC which has residual sulfide sulfur content of
about 6 % after 21-day fungal treatment, there is a great potential for more sulfides to dissolve
leading to further increase in gold extraction. The single stage fungal-treatment of FC which
led to an overall recovery of 78 % compares well with the recovery (81 %) of the bacterialtreated FC. Given the infant stage of the fungal process, the closeness of its gold recovery
value to that of the commercial bacterial process highlights the potential of P. chrysosporium
in the biotransformation of refractory gold ores.
158
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Perek, K. T. (Eds.), Proceedings of the 23rd International Mineral Processing Congress, Instanbul, 1445-1451.
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Bhakta, P., Arthur, B. 2002. Heap bio-oxidation and gold recovery at Newmont Mining: First-year results.
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Caldeira, C.L. , Ciminelli, V.S.T., Dias A., Osseo-Asare, K., 2003. Pyrite oxidation in alkaline solution: nature
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Ciminelli V. S. T., Osseo-Asare K. 1995. Kinetics of pyrite oxidation in sodium carbonate solutions. Metall.
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Ciminelli, V.S.T., 1987. Oxidation of pyrite in alkaline solutions and heterogeneous equilibria of sulfur- and
arsenic-containing minerals in cyanide solutions. PhD Thesis, The Pennsylvania State University, University
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Microbiol. 50, 511-516.
Komnitsas C. Pooley F.D, 1989. Mineralogical characteristics and treatment of refractory gold ores. Minerals
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(Eds.), 627-641.
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Ofori-Sarpong, Osseo-Asare, K., G., Tien, M., 2010b. Biotransformation of sulfides using the fungus,
Phanerochaete chrysosporium: A potential pretreatment process for refractory sulfidic gold ores. In:
Proceedings of the Precious Metals 10 Conference, Falmouth, UK.
Osseo-Asare, K. Xue, T., Ciminelli, V.S.T., 1984b. Solution chemistry of cyanide leaching systems. In: Kudryk,
Osseo-Asare, K., Afenya, P. M., Abotsi, G. M. K., 1984a. Carbonaceous Matter in Gold Ores; Isolation,
Portier, R. J., 1991. Biohydrometallurgical processing of ores, and microorganisms therefor. US Patent
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Rawlings, D. E., 1998. Industrial practice and the biology of leaching of metals from ores. Journal of Industrial
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fundamentals and mechanisms of bacterial metal sulfide oxidation. Appl. Microbiol Biotechnol, 63, 239-248.
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Shahverdi, A. R., Yazdi, M. T., Oliazadeh, M. Darebidi, M. H. 2001. Biooxidation of mouteh refractory goldbearing concentrate by an adapted Thiobacillus ferrooxidans. J. Sci. I. R. Iran, 12, 209-212.
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161
Chapter 8
In vitro transformation of carbonaceous matter and sulfides in refractory gold ores using
cell-free extracts of Phanerochaete chrysosporium
Abstract
This chapter reports the findings of preliminary investigations aimed at assessing the use of hydrogen
peroxide and cell-free extracts from the fungus, Phanerochaete chrysosporium, to effect
biotransformation of sulfides and carbonaceous matter (CM) in double refractory gold ores (DRGO) to
increase gold extraction. The cell-free components were extracted from already grown fungal culture
by filtering through cheesecloth and fiberglass. After 72 hrs of treatment with the cell-free extracts (in
vitro), total dissolved iron and sulfur from pyrite stood at 2.6 wt% and 3.7 wt% respectively. For the
same period, total dissolved arsenic, iron and sulfur in solution were up to 5.2 wt%, 0.9 wt% and 6.0
wt% respectively from flotation concentrate. Analysis for sulfide sulfur in the residual solids of the
gold concentrate indicated about 25 wt% oxidation within 24 hours of cell-free treatment. Gold
adsorption capacity of anthracite (used as surrogate for CM) reduced by 30 % after the cell-free
treatment for 24 hr. Using cyanide-leaching, gold recovery increased by 24 % after 24-hr treatment
with the cell-free extracts as against 15% for treatment with 2 mM hydrogen peroxide alone. The in
vitro treatment had a slight advantage over treatment with whole cells of the fungus (in vivo) with gold
recovery of 66 % as against 62 % after 72 hr-treatment in vivo. In general, cell-free decomposition of
the samples did not increase beyond 24 hours of contact time, possibly due to exhaustion of the
oxidative components of the extracts. These initial results indicate clearly that in vitro processing is a
promising alternative to in vivo processing of DRGO using P. chrysosporium.
Keywords: Phanerochaete chrysosporium; Refractory gold ores; Flotation concentrate; Pyrite;

Anthracite; Fungi; Cell-free extracts; Enzymes; Hydrogen peroxide; Biotransformation; Gold
adsorption; Cyanidation; Gold recovery; In vitro; In vivo.
8.1 Introduction
Double refractory gold ores (DRGO) contain sulfide minerals and carbonaceous matter (CM)
which require pretreatment before cyanidation to decompose sulfide minerals and liberate
encapsulated gold particles, and deactivate CM to prevent gold uptake during cyanidation
(Osseo-Asare et al., 1984a; Hausen and Bucknam, 1985; Hutchins, 1988; Weir and
Berezowsky, 1986; Afenya, 1991). Biological pretreatment processes are gaining acceptance
on the bases of cost, safety and environmental issues (Livesey-Goldblatt et al, 1983;
Komnitsas and Pooley, 1989; Brierley, 1997; Hackl, 1997; Rawlings et al., 2003). Several
bacteria and fungi have been studied to assess their ability to decompose sulfides and/or
deactivate carbonaceous matter in refractory gold ores (Lundgren and Silver, 1980; Portier,
1991; Brierley and Kulpa, 1992; Amankwah et al., 2005; Afidenyo, 2008; Yen et al., 2008).
By utilizing whole cells of P. chrysosporium, the white rot lignin-degrading fungus

demonstrated its ability to deactivate anthracite-grade coal used as a model for CM in
refractory gold ores. The fungal-treatment led to reduction in the gold adsorption capacity of
anthracite by more than 90 % (Chapter 3; Ofori-Sarpong et al., 2010a). When the fungus was
used in the treatment of refractory gold concentrates for up to 2 weeks, gold adsorption
reduced by 64-70 % (Chapter 5). In a related study using pyrite, arsenopyrite and flotation
concentrate (FC), P. chrysosporium was able to decompose the sulfides progressively over
time. Conversions of sulfides in pyrite, arsenopyrite and FC respectively were 15 wt%, 35
wt% and 57 wt% after 21 days of fungal treatment (Chapter 6; Ofori-Sarpong et al., 2010b).
Subsequent cyanidation of the treated material led to an increase in gold recovery of 38 %
over the untreated material, bringing the overall recovery to 78 % (Chapter 7).
Phanerochaete chrysosporium produces the enzymes lignin peroxidase (LiP), manganese

peroxidase (MnP), and hydrogen peroxide, which are known to be responsible for the
degradation of a host of materials including carbon- and sulfur-containing substrates (Glenn et
163
al., 1983; Tien and Kirk, 1983; Kersten and Kirk, 1987; Schreiner et al., 1988; Gold and Alic,
1993; Ralph and Catcheside, 1997; Martin and Petersen, 2001). As explained by Tien and
Kirk (1984; 1988), these cell-free components can be extracted and utilized in vitro (absence
of fungus) for biotransformation (Couto et al., 2006). It was therefore necessary to investigate
the possibility of using the fungus as a factory to produce the oxidative enzymes, and
extracting the enzymes for use in biotransformation, instead of the biomass. Also, as detailed
in Chapter 3, Fig 3.2 and Ofori-Sarpong et al. (2010), the biomass of the fungus is fluffy.
Because of this, it is a daunting task to separate the biomass from ore particles after
processing due to entanglement. Consequently, the use of cell-free extracts may offset this
separation difficulty and avoid the possibility of materials loss during washing. In addition,
the use of cell-free components has the potential to facilitate better understanding of the
process chemistry and kinetics.
In this study, crude enzymes were extracted from already grown P. chysosporium, and the
potency of the cell-free extracts in deactivating carbon and decomposing sulfides in DRGO
was examined. The degree of in vitro transformation of the materials was determined by
investigating the gold adsorption ability of CM, reduction in sulfide sulfur content of sulfidic
materials and cyanide amenability of the gold concentrates. A processing time comparison
between in vitro and in vivo treatments was also made.
8.2.1 Materials
Gold-containing samples, pyrite, anthracite grade coal, P. chrysosporium and the millet and
wheat bran (MWB) medium were obtained as presented in Chapters 3 and 6. Reagent grades
of dextrose powder, ammonium tartrate, succinic acid, thiamin, manganese sulfate, sodium
tartrate, hydrogen peroxide, hydrochloric acid, sodium hydroxide and sodium cyanide were
supplied by VWR. Cheesecloth and fiber glass filter were also supplied by VWR.
164
8.2.2 Preparation of cultures and extraction of cell-free components

Glucose broth (liquid) medium was prepared with dextrose powder (carbon source) and
ammonium tartrate (nitrogen source) as described by Tien and Kirk (1988). Succinic acid was
used to buffer the solution, and the pH adjusted to 4.0 with hydrochloric acid or sodium
hydroxide. The medium without thiamin (protein source) was autoclaved for 30 minutes and
cooled down before addition of thiamin, which is heat-sensitive. The medium was inoculated
with 1 vial of spore suspension of P. chrysosporium per 1000 mL of medium. The inoculated
medium was distributed as 10 mL solution into 125 mL Erlenmeyer flasks, and allowed to
incubate for 14 days under stationary culturing at 37 oC. Oxygen was introduced into the
cultures on the first day of incubation and every three days as described by Tien and Kirk
(1988). Development of a brown coloration on the mycelia signifies appearance of ligninase
activity (formation of enzyme) in the extracellular fluid (Tien and Kirk, 1988). By the 14 th day
about 75% of the incubation flasks had the brown coloration and so the extract was harvested
on day 14. At the end of the incubation period, the cell-free liquid (supernatant) was extracted
from the culture by filtration through quadruple-layered cheesecloth and fiber glass. The cellfree extract (crude enzymes) was buffered with sodium tartrate to pH of 4 and used
immediately.
8.2.3 Preparation of reagents for the treatment of materials

Aside from using the cell-free extracts, it was necessary to assess the use of hydrogen
peroxide alone in the treatment of the substrates. This is because P. chrysosporium produces
hydrogen peroxide, in addition to secreting oxidative enzymes. In the catalytic action of the
fungus, hydrogen peroxide is required to activate the enzymes, lignin peroxidase (LiP) and
manganese peroxidase (MnP) (Glenn et al., 1983; Tien and Kirk, 1984; Kersten and Kirk,
1987), and this is detailed in Chapter 3 and Ofori-Sarpong et al. (2010a). Hydrogen peroxide
is produced in the range of 2-30 M by the fungus (Tonon and Odier, 1988) depending on the
amount of enzyme secreted (Kelly and Reddy, 1986). In abiotic sulfide dissolution however,
165
higher concentrations of 1-5 M have been used (McKibben and Barnes, 1986; Antonijevic, et
al., 1997; Jennings et al., 2000; Aydogan, 2006). In this research, 2 mM of hydrogen peroxide
which is about a hundred times that secreted by the microbes, and far less than that utilized in
abiotic processes was used to assess the extent to which hydrogen peroxide can transform
sulfides and carbonaceous matter. The concentration utilized in this study, thus mimics a
microbial system with excessive hydrogen peroxide production. In addition, MnP requires
Mn(II) ions in its oxidative cycle as detailed in Andrawis et al. (1988), Banci et al. (1992) and
Chapter 2, and concentrations in the range of 0.1-20 mM have been used (Gold and Alic,
1993). Overall, concentration used in this chapter was 100 M prepared from manganese
sulfate solution. The solutions were buffered using 20 mM sodium tartrate at pH 4 to ensure
high enzymatic activity (Tien and Kirk, 1988). The substrates (anthracite, pyrite, gold
concentrate) were treated with the hydrogen peroxide and the cell-free fungal extracts at 15 %
solids and pH 4 for up to 72 hrs at an agitation speed of 150 rpm on a New Brunswick Series
25 Incubator shaker. After treatment, the treated material was filtered and the residue dried at
37 oC for 7 days.
8.2.4 Post-incubation experiment

Total dissolved arsenic, iron and sulfur in the incubation solution was determined using
inductively coupled plasma atomic emission spectroscopy (ICP-AES), whereas sulfide
sulfur in the residual solids was determined using LECO volumetric combustion technique as
described in Chapter 6 and Ofori-Sarpong et al. (2010b). Gold adsorption experiments were
also conducted as described in Chapter 3 and Ofori-Sarpong et al. (2010a), whilst cyanidation
and fire assaying of the residual solids were conducted as detailed in Chapter 7. Experiments
used for the treatment of the substrates were carried out in duplicates, and the data are
reported as mean values.
166
8.2.5 Surface area and porosity analysis

Surface area and pore analysis was conducted using Micromeritics Gas adsorption Analyzer
ASAP 2020 by high pressure nitrogen adsorption. In this method, nitrogen is admitted to a
solid material in controlled increments, and after each adsorptive dose the pressure is allowed
to equilibrate and the quantity of gas adsorbed calculated. The gas volume adsorbed at each
pressure (at constant temperature) defines an adsorption isotherm, from which the quantity of
gas required to form a monolayer over the surface of the solid and its pores is determined. The
area covered by each adsorbed gas molecule is used to estimate the surface area whereas the
isotherms of adsorption and desorption are used to estimate the pore size, pore volume and
area of the material (Van Krevelen, 1993).
8.2.6 Transmission electron microscopy (TEM)

The structural features of the as-received and treated anthracite were examined using the
JEOL 2010 LaB6 transmission electron microscope (TEM). The sample was dispersed on a
Holey carbon-coated TEM support grid and placed on a sample holder in the sample chamber
of the TEM. TEM is a technique employed by irradiating a thin specimen with a high-energy
electron beam typically 100-200 keV. It can assess the structural features of materials in the
nanometer range (Van Krevelen, 1993). The electron beam is focused by a number of
magnetic lenses situated before and after the position of the specimen being analysed. These
help in controlling illumination, magnification and focusing of the specimen and the image
formed. As the beam interacts with the specimen, an image is formed and recorded by a
digital CCD camera.
167
8.3.1 Effect of cell-free extracts processing on gold adsorption behavior and surface
modification of anthracite
The amount of gold adsorbed on anthracite treated variously, is depicted in Fig. 8.1. The
diagram shows reduced gold adsorption of 13 wt% and 30 wt% respectively for hydrogen
peroxide and the cell-free extracts. P. chrysosporium secrets both peroxide and oxidative
enzymes, and thus the higher level of biotransformation observed with the cell-free extracts
may be attributed to the combined effects of hydrogen peroxide and the enzymes. The 30%
reduction in gold uptake is very low, compared to 65-85% achieved when whole cells of the
fungus was used in treating anthracite for the same period (Chapter 4).
Gold adsorbed (mol/g)
5.0
4.5
4.0
3.5
3.0
2.5
2.0
As-Received
H2O2
Fungal extracts
Treatment method
Fig. 8.1. A comparison of the gold adsorption behavior of treated anthracite (15 % solids,
37 oC, 24 hr, pH 4)
168
BET surface area, micropore area and micropore volume of the as-received anthracite were
4.07, 2.84 and 1.51 x 10-3 cm3/g. After treatment with the cell-free extracts, these values
reduced by 82 %, 85 % and 86 % respectively as shown in Fig. 8.2. The reduction in pore
volume (86 %) and surface area (82 %) after enzyme treatment compares well with the whole
cell-treated carbon, which registered 81 % and 78 % respectively (Chapter 4). Though the
crude enzyme-treated carbon showed slightly higher reduction in pore volume and surface
area, these did not translate into higher reduction in gold up-take.
Percentage value of parameter (%)
120
As-received
Whole cell
Cell-free extracts
100
80
60
40
20
0
BET surface
area
Micropore
area
Parameter
Micropore
volume
Fig. 8.2. Surface area and porosity analysis of anthracite samples before and after treatment
using whole cell and cell-free extracts of P. chrysosporium (15 % solids, 37 oC, 24 hr, pH 4)
Further analysis of the structural nature of anthracite using TEM indicated a more amorphous
surface following both the whole cell and cell-free treatments as compared to the as-received
anthracite (Fig. 8.3). The figure shows varying shades of grey with the lighter sections
169
indicating less dense material and the darker sections, denser material. Figure 8.3a shows a
well-ordered structure of as-received anthracite and this structure is similar to that obtained by
Pappano and Schobert (2009) for studies on graphitizability of a Pennsylvania anthracite. The
amorphous nature shown in Figs 8.3b and 8.3c, coupled with the extensive reduction in
surface area (Fig. 8.2), is due to surface oxidation (Chapter 4) and surface coating with
organic fungal metabolites (Glazer and Nikaido, 1995), which leads to blinding of adsorption
sites (Osseo-Asare et al., 1984a; Abotsi and Osseo-Asare, 1987; Adams and Burger, 1998).
Gold cyanide adsorption is known to be more favorable on structured carbon due to
interaction of the gold central ion with pi electrons of the graphitic planes (Jones et al., 1989;
Klauber et al., 1991; Ibrado and Fuerstenau, 1992).
10 nm
Fig. 8.3a. TEM analysis of as-received anthracite samples showing a very structured surface
170
10 nm
Fig. 8.3b. TEM analysis of anthracite samples after treatment using whole cells of P.
chrysosporium showing relatively more amorphous surface (37 oC, pH 4, 60 % solids, 7 days)
10 nm
Fig. 8.3c. TEM analysis of anthracite samples after treatment using cell-free extracts of P.
chrysosporium showing very amorphous surface (37 oC, pH 4, 15 % solids, 3 days)
Similar adsorption levels of the aurocyanide ion was expected on both in vivo and in vitro
treated carbons as the carbon structures were similar and quantitative changes in pore volume
171
and surface area were equally high. However, the in vitro adsorbed more gold. The much
higher reduction in gold adsorption after in vivo treatment indicates that the presence of the
fungus contributes extra components that enhance deactivation of the active sites on carbon
(Chapter 4). Since the results of the cell-free (in vitro) treatment are only preliminary, further
investigations are required to confirm the trends observed.
8.3.2 Effect of cell-free extracts and hydrogen peroxide processing on biotransformation

of sulfides
The results obtained for pyrite constituents in solution after being treated for up to 72 hr in the
cell-free extracts is presented in Fig 8.4 whilst comparison of 24-hr treatment in hydrogen
peroxide alone, and in extracts is illustrated in Fig. 8.5. It is clear from Fig. 8.4 that the
concentration of constituents in solution increased beyond 4 hr of treatment until 24 hr, after
which there was no further dissolution. However, the reaction was not complete as analysis of
the residual solids for sulfide sulfur indicated only about 10 wt% oxidation. The observed
trends can thus be due to depletion of oxidative enzymes in the solution after 24 hr of
treatment. Compared with Fig. 8.1 on gold adsorption, Fig. 8.5 shows a similar trend of the
extracts giving better results than the hydrogen peroxide alone. Hydrogen peroxide produced
by the fungus is in the range of 2-30 M depending on the amount of enzyme secreted (Kelly
and Reddy, 1986; Tonon and Odier, 1988). In this research, a much higher concentration of 2
mM was used to establish the effect a 100-fold increase in hydrogen peroxide concentration.
Yet, even at that concentration, the presence of the enzymes gave a more pronounced effect.
This is because the extract contains the oxidative enzymes and hydrogen peroxide both of
which possess oxidizing ability.
172
initial weight)
4
3
4 hr
24 hr
72 hr
2
1
0
Iron
Sulfur
initial weight)
Fig. 8.4. In vitro dissolution of constituents during pyrite transformation as a function of time
(15 % solids, 37 oC, up to 72 hr)
H2O2
Extracts
3
2
1
0
Iron
Sulfur
Fig. 8.5. Comparison of constituents in solution after pyrite treatment with H 2O2 and fungal
extract (15 % solids, 37 oC for 24 hr)
173
Stoichiometrically, the molar ratio of sulfur to iron in pyrite is 2:1. However, this ratio does
not tally with the observed solution concentrations in this study as the ratio is about 3 mol of
sulfur to 1 mol of iron. The deficiency of iron in the treatment solution is due to production of
insoluble iron oxides/hydroxides/sulfates such as Fe2O3, FeOOH and FeSO4 which precipitate
above pH of 2 (Osseo-Asare et al., 1984b; Ciminelli and Osseo-Asare, 1995; Shahverdi et al.,
2001; Bhakta and Arthur, 2002; Caldeira et al., 2003; Marsden and House, 2006; Chapter 6).
8.3.3 Biotransformation of flotation concentrate by cell-free extracts

The extent of decomposition of flotation concentrate (FC) using cell-free extracts was
assessed partly by analyzing for constituents in solution, and for sulfide sulfur in the residual
solids after treatment. The corresponding results are presented in Figs. 8.6 and 8.7. Figure 8.6
shows an increase in dissolution of arsenic, iron and sulfur between the processing periods of
4 and 24 hr. Not much dissolution occurred after 24 hr and this is due to depletion of
oxidizing components in solution.
In agreement with the trends observed for the constituents in solution (Fig. 8.6), sulfide sulfur
analysis of the residual solids (Fig. 8.7) indicated an increase in decomposition from a value
of 17 wt% within 4 hours of treatment to 25 wt% in 24 hr. Only 2 % extra decomposition was
observed between 24 and 72 hr bringing the final value to 27 wt%. The two major possibilities
for reduction in decomposition rate are exhaustion of oxidizing reagents and formation of
precipitates at the pH of 4 utilized in the process. The precipitates account for the difference
between the higher value (27 %) of overall decomposition of sulfide sulfur as compared to the
lower value of only 6 wt% total sulfur present in the treatment solution (Fig. 8.6).
174

weight)
7
4 hr
24 hr
72 hr
5
4
3
2
1
0
Arsenic
Iron
Sulfur
Sulfide sulfur converted (wt % of

initial)
Fig. 8.6. In vitro dissolution of constituents during transformation of flotation concentrate as a

function of time (15 % solids, 37 oC, up to 72 hr)
30
25
20
15
10
5
0
4 hr
24 hr
72 hr
Treatment time
Fig. 8.7. Decomposition of sulfide sulfur in flotation concentrate with fungal extracts (15 %
solids, 37 oC, up to 72 hr)
175
8.3.4 Cyanidation of flotation concentrate

The aim of using P. chrysosporium to biotransform double refractory gold ores is to reduce
uptake of gold by carbonaceous matter and to decompose sulfides to liberate locked up gold
for dissolution. Ultimately therefore, gold recovery is expected to increase after the treatment.
In the previous chapter (Chapter 7), in vivo treatment using whole cells of the fungus led to an
increase in gold recovery of 38 % over that of the as-received flotation gold concentrate.
Comparison of cyanide leaching of as-received and FC treated with H2O2, and cell-free
Gold recovery (% of initial weight)
extracts of P. chrysosporium is depicted in Fig. 8.8.
70
60
50
40
30
20
As-received
H2O2
Fungal extracts
Treatment method
Fig. 8.8. Cyanide amenability of flotation concentrate (as-received) and after 24-hr treatment
with H2O2, and cell-free extracts of P. chrysosporium. Both samples were processed at 15 %
solids and 37 oC at pH 4
176
Following the common trend observed, the fungal extract gave better results (64 %) than
treatment with hydrogen peroxide alone (55 %) within 24 hrs. Though both hydrogen
peroxide and the enzymes generated by the fungus possess oxidizing ability, the combined
effect as exhibited in the performance of the cell-free extracts is better.
The oxidation of pyrite and arsenopyrite with hydrogen peroxide is well established and the
reactions are illustrated in Equations 8.1 and 8.2 (McKibben and Barnes, 1986; Antonijevic, et
al., 1997; Jennings et al., 2000; Aydogan, 2006).
2 FeS2 1 5 H2O2
2 Fe3
FeAsS 7 H2O2
Fe3
4 SO4
AsO34
2H
SO4
1 4 H2O
2H
6 H2O
[8.1]
[8.2]
The oxidative enzymes, LiP and MnP, secreted by P. chrysosporium are so called because
they require peroxide to get activated for the oxidative action on substrates. The fungus
therefore produces hydrogen peroxide for this process (Glenn et al., 1983; Tien and Kirk,
1983; Bumpus and Aust, 1986; Kersten and Kirk, 1987; Gold and Alic, 1993; Ralph and
Catcheside, 1997; Chapter 3). However, since hydrogen peroxide is a strong oxidant it will,
apart from activating the enzymes, complement the oxidative action. This in consequence
explains the higher levels of decomposition and gold recovery obtained with the experiments
in which the fungal extracts were used.
The comparison of FC treated with whole cell and cell-free extracts is presented in Fig. 8.9.
The diagram shows that after 72 hr of treating the flotation concentrate with whole cells and
cell-free extracts of P. chrysosporium, cyanidation led to gold recoveries of 62 % and 66 %
respectively, giving the cell-free solution a slight edge over the whole cells. For a better
understanding of the kinetics, systematic quantification and purification of the extracts are
required to obtain the pure enzyme which may then be used in estimating the actual amount of
177
enzyme required to decompose a given amount of sulfides and carbonaceous matter in gold
Gold recovery (% of initial weight)
ores.
70
60
50
40
30
20
As-received
Whole cell
Cell-free
Mode of fungal-treatment of FC
Fig. 8.9. Cyanide amenability of flotation concentrate (as-received) and after treatment with
whole cell and cell-free extracts of P. chrysosporium (15 % solids, 37 oC, 72 hr).
8.4 Proposed mechanism of biotransformation of sulfides by P. chrysosporium

The enzymes of P. chrysosporium are heme-containing glycoproteins possessing one ferric
heme per molecule of enzyme (Tien and Kirk, 1988) and can be represented as P[Fe(III)]. The
mechanism of lignin degradation using the enzyme, lignin peroxidase, is established and has
been used to predict the degradation of a host of other aromatic substrates, including coals and
carbonaceous matter (CM) in gold ores (Andrawis et al, 1988; Banci et al., 1992; Edwards et
al., 1993; Gold and Alic, 1993; Fakoussa and Hofrichter, 1999; Ofori-Sarpong et al., 2010a).
The mechanism of sulfide decomposition in the bacteria oxidation system has been discussed
extensively by several researchers (Bennett and Tributsch, 1978; Lundgren and Tano, 1978;
Sand et al., 2001; Rohwerder et al., 2003; Holmes, and Bonnefoy, 2006; Marsden and House,
178
2006; Ofori-Sarpong et al., 2010b). The ideas of fungal decomposition of CM and bacteria
oxidation of sulfides are adopted in predicting possible reactions in the fungal decomposition
of inorganic sulfides.
The initiation of enzymatic attack on substrates is triggered by activation of the enzyme via
reduction of hydrogen peroxide to water as shown in Equation 8.3. Having donated two
electrons, the enzyme forms a two-electron oxidized intermediate called compound I.
Reduction of Compound I back to the native enzyme state involves two steps of receiving one
electron at a time, with compound II as the one-electron oxidized intermediate (Equations 8.4
and 8.5), where SC represents sulfide or carbon used as substrate for degradation. The
oxidative degradation of pyrite is thus proposed as represented in Equations 8.6 and 8.7.
H2O2
P[O Fe(IV) ](Comp I) SC
2P[O Fe(IV)]
2P[O Fe(IV)]
FeS2 4H
H 2O
[8.3]
P[O Fe(IV)] (Comp II) SC
P[O Fe(IV)] (Comp II) SC 2H
2P[O Fe(IV) ] FeS2
P[O Fe(IV) ] (Comp I)
Fe2
2P[Fe(III)] Fe2
2S0
2S0 2H2O
[8.4]
SC
H2O
[8.5]
[8.6]
[8.7]
Being a catalyst, the enzyme is not consumed, and the overall reaction which is an addition of
Equations 8.3, 8.6 and 8.7 is given by Equation 8.8, and thus the enzyme is recycled.
2FeS2 2H2O2 4H
2Fe2
4S0 4H2O
[8.8]
Using ferrous ion in place of pyrite as the substrate, Equations 8.9-8.11 can be written in like
manner as Equations 8.6-8.8. Compound II may also react with elemental sulfur as shown in
Equation 8.12. The reactions in Equations 8.9-8.12 lead to the production of ferric ions and
sulfate, and these are responsible for indirect decomposition of pyrites in the biooxidation
system.
179
P[O Fe(IV) ] Fe2

Fe2
P[O Fe(IV)]
H2O2 2Fe2 2H
P[O Fe(IV)]
[8.9]
2H
P[Fe(III)] Fe3
2Fe3
2H2O
[8.11]
3P[Fe(III)] SO24- 2H
[8.12]
S0 H2O
3P[O Fe(IV)]
Fe3
H2O
[8.10]
The hydrogen peroxide reaction with pyrite also leads to the formation of Fe3+ and SO42(McKibben and Barnes, 1986; Antonijevic, et al., 1997; Jennings et al., 2000; Aydogan,
2006). Non-enzymatic reactions that can occur in the fungal degradation system may therefore
include Equations 8.13 8.16. All these would contribute to the oxidative decomposition of
pyrite, the dissolved constituents in solution and the solid products reported in this study.
Fe2
SO24
FeSO4
FeS2 2Fe3
3Fe2
2FeS2 7O2 2H2O

2Fe3
O2
[8.13]
2S0
2Fe2
FeOOH
[8.14]
4SO24- 4H
[8.15]
[8.16]
8.5 Conclusions
In this chapter, the cell-free extracts from the fungus, Phanerochaete chrysosporium, were
examined for their ability to biotransform sulfides and carbonaceous matter in double
refractory gold ores (DRGO) in vitro. The conditions of treatment included up to 72 hr of
contact time under agitation at 150 rpm, at 15 % solids, and pH of 4. Within 24 hr of
treatment, total dissolved iron and sulfur from pyrite stood at 2.6 wt% and 3.7 wt%
respectively, whereas arsenic, iron and sulfur in incubation solution were up to 5.2 wt%, 0.9
wt% and 6.0 wt% respectively from flotation concentrate. Analysis of sulfide sulfur in the
residual solids of the gold concentrate indicated about 25 wt% oxidation within 24 hr of
180
treatment. Within the same treatment period, gold recovery by cyanide-leaching was 64 %, an
increase of 24 % over the as-received material.
Comparing treatment by the cell-free extracts with that by hydrogen peroxide alone, gold
recovery was 9 % more for the extract-treated (64 %) as the hydrogen peroxide-treated
yielded 55 % within 24 hr of treatment. Comparison with whole cell treatment gives a slight
advantage in favor of the cell-free treatment, which recorded gold recovery of 66 % after 72
hr of treatment as against 62 % for the whole cell treatment. These preliminary findings
indicate clearly that in vitro processing is a promising alternative to in vivo or whole cell
processing of DRGO using P. chrysosporium.
181
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185
Chapter 9
General conclusions, recommendations and processing proposal
9.1 General Conclusions

Mycohydrometallurgy has been adopted in this research to define the application of fungi in
hydrometallurgy. The ability of the white rot basidiomycetous fungus, Phanerochaete
chrysosporium, to biotransform sulfides and carbonaceous matter has been studied.
Carbonaceous matter (CM) was tested for reduction in gold adsorption ability while sulfides
were evaluated to ascertain the oxidation capabilities of the fungus. The biotransforming
action of the fungus was assessed on various ranks of coal (lignite, subbutiminous, butiminous
and anthracite) that served as surrogates for CM, and also on pyrite and arsenopyrite, major
metal sulfides associated with gold ores. Actual gold concentrates; flotation concentrate (FC)
and bacteria-treated flotation concentrate (BFC) containing sulfides and CM were utilized to
confirm the fungal action on the surrogates.
For the various coals utilized, reduction in gold adsorption capabilities of the surrogates was
used to estimate declining preg-robbing ability. Various culture media used under different
incubation conditions affirmed that P. chrysosporium could grow on all the coal ranks. The
results, however, showed that of the coal samples tested; only anthracite coal exhibited
significant gold adsorption ability which was used as a means of analyzing the extent of
biotransformation. Fungal treatment resulted in reduction in gold adsorption ability of
anthracite by more than 95% in a mixture of millet and wheat bran medium.
Fungal treatment with P. chrysosporium was found to take place in a wide range of processing
time, pulp density, temperature, pH and level of agitation. After contact with the fungus, gold
adsorption ability of anthracite reduced by about 90-97 %, depending on the incubation
conditions. A pH of 4 and temperature of 37 oC were found to enhance biotransformation.

Stationary culturing permitted the use of higher pulp densities and the best for stationary and
shake culturing respectively were 60 % and 25 % solids.
When tested in different complexing systems, the adsorption of gold on the as-received CM
occurred in the order of cyanide > thiourea > thiosulfate. After fungal biotransformation,
average reduction in gold adsorption was more evident in cyanide (70-97 %) than in thiourea
(60-92 %) and thiosulfate (50-91 %) depending on the type of CM. In addition, there was a
reduction of 50-70 % in preg-robbing activity of the gold concentrates, FC and BFC.
Aside from deactivating the active sites on CM, P. chrysosporium showed the capability to
decompose sulfide minerals. For an incubation period of 21 days, sulfide sulfur decreased by
35 % in treated arsenopyrite and 15 % in pyrite. These materials had initial sufide sulfur
content of 19.6 % and 52 % respectively. For refractory sulfidic gold concentrate with about
15 % initial sulfide sulfur content, a higher oxidation level of 57 % was realized. For all the
samples, there was a strong correlation between the decomposition of sulfides and incubation
period.
When gold concentrates were contacted with the fungus, the investigations showed that P.
chrysosporium could simultaneously biotransform sulfides and CM in double refractory gold
ores (DRGO). The results showed that at pH 4, substantial sulfide dissolution took place in the
presence of the fungus leading to relatively high solution concentrations of iron, sulfur and
arsenic. Overall, sulfide sulfur decomposition of 57 % from FC and 75 % from BFC was
realized after 21 days of fungal-treatment. Within the same period, gold extraction increased
appreciably from 40 % to 78 % for sample FC, and from 81 % to 94 % for sample BFC. This
signifies increase in recovery of 38 % and 13 % over the as-received FC and BFC
respectively. The single stage fungal-treatment of FC which led to an overall recovery of 78
% compares very well with the recovery (81 %) of the bacterial-treated FC.
187
The investigations also revealed that both the whole cells of P. chrysosporium and cell-free
extracts were capable of transforming both surrogates and gold concentrates leading to
deactivation of CM, decomposition of sulfides and increase in gold extraction. The cell-free
extracts exhibited a slight advantage over the whole cell processing with 4 % increase in gold
recovery over a similar processing period.
During fungal degradation of sulfide minerals, changes in sulfide sulfur concentration and the
formation of oxide phases were confirmed using Raman spectroscopy and X-ray diffraction
analysis. For carbonaceous matter, Raman, infrared and XANES spectrocopies showed that
fungal treatment led to reduction in carbon, introduction of oxygen containing groups such as
carboxylic and carbonyls. Further investigations showed that BET surface area and micro pore
volume reduced by 76 % and 80 % respectively whereas average pore diameter increased
from 5.75 nm to 9.48 nm. TEM studies revealed an increase in the amorphous nature of the
surface of carbon after microbial action. The characterization results suggest that P.
chrysosporium biotransforms anthracite in two main ways. In one way, there is surface
oxidation by introduction of oxygen groups which disrupts the continuous graphitic structure
thus decreasing the active sites necessary for adsorption. The second route is reduction in
surface area via plugging of pores possibly by hyphae or biofilms produced through fungal
interaction with the substrate. These led to reduced accessibility of gold to the adsorption
sites.
9.2 Recommendations
The following recommendations are made based on experimental data and visual observations
made during the investigations. In the present study, the reduction in gold adsorption on CM
was due mainly to inaccessibility of the aurocyanide to the micropores. Further
characterization studies, probably using XRD mapping, should be conducted to verify the
cause of this blockage.
188
The fungus P. chrysosporium has the potential to grow and form a matrix that entangles ore
particles making separation difficult. It is recommended that a counter-current decantation
technique be investigated to aid washing and complete separation of fungal biomass from ore
particles. The entanglement caused by fungal biomass may be avoided when in vitro
biotransformation is adopted. Thus the study on in vitro treatment should be pursued further
so cell-free extracts instead of whole cells are employed. Quantification and purification of the
extracts to obtain the pure enzyme will help in estimating the actual amount of enzyme
required to decompose a given amount of sulfides and carbonaceous matter in gold ores.
The data obtained for sulfide processing indicated that further dissolution of sulfur is possible
when processing time is extended beyond 21 days. Further dissolution will promote release of
encapsulated gold and increase metal recovery. It is recommended that further experiments be
conducted to allow complete oxidation of sulfides and also at a faster rate.
P. chrysosporium is known to function best at a relatively low acidic pH of 4. This pH poses a

challenge regarding the ability of elemental sulfur and ferric arsenate to precipitate on the
unreacted sulfide particles leading to reduction in reaction and problems with downstream
gold recovery processes. To address this problem, it is suggested that P. chrysosporium be
adapted to function well at lower pH. If this becomes possible the precipitation reactions may
be reduced. In addition, P. chrysosporium may be tested in a consortium with the acidophilic
sulfide oxidizing bacteria to investigate a possible synergistic degradation within a shorter
processing time.
In the cyanidation experiments using BFC, recoveries did not rise beyond 94% due in part to
the very high solution gold concentration which prevented more gold from being dissolved
from the solids. For such high values, a carbon-in-leach (CIL) system, where activated carbon
is introduced into the system for simultaneous leaching and gold adsorption may be better.
Further studies should be conducted using the CIL system to increase gold extraction.
189
All the experiments using P. chrysosporium were conducted under batch conditions. It is
recommended that these reactions be conducted under continuous/semi-continuous conditions
to ascertain the implications on processing time. By using the continuous system, a scale-up
operation may be conducted to monitor how a pilot plant would perform.
9.3 Processing proposal

The results of the biodegradation of both sulfides and carbonaceous matter (CM) in a single
stage by the fungus P. chrysosporium constitute a novel technical process for microbial
pretreatment of refractory gold ores. In its application, flotation concentrate (FC) containing
sulfides and CM may be subjected to fungal treatment in stirred tanks and also in bioheaps as
the fungus has proven to work well under both stationary and shake conditions.
In bioheap application, the FC may be loaded onto support rocks of size between 10 and 20
mm in an agglomerator using fungal culture as wetting agent. The coated support rocks may
be stacked onto already prepared impervious pads where fungal culture is sprinkled for the
microorganisms to deactivate CM and decompose sulfides (Fig 9.1). The support rocks will
allow easy aeration which will enhance the process as the fungus is an aerobe. At the end of
the processing period, the biooxidized concentrate may be removed by a hydraulic system
after which washing and solid-liquid separation is performed in counter-current decantation
thickeners. The thickened slurry may then be conditioned, and gold in the solid residue
leached using the well-known process of cyanidation.
In its application in a continuously stirred tank reactor (CSTR), slurry of FC may be

conditioned with growth media and mixed with P. chrysosporium. With agitation and
sparging of air, the microorganism will biotransform sulfides and CM, and release gold for
subsequent cyanidation. After fungal pretreatment, solid-liquid separation may be performed
in counter-current decantation thickeners and the thickened slurry conditioned for cyanidation
as shown in Fig 9.2. In the investigations, in vitro studies showed comparable results to those
190
of whole cell treatment. Hence in its application, the fungus may be grown, the oxidative
enzymes extracted and fed into the CSTRs to be utilized in biotransformation.
Flotation concentrate containing gold,

sulfides and CM
Coating of support rocks with

flotation concentrate
Heaping, sprinkling and biotreatment
Removal with water jets

Washing of product in counter current
decantation thickeners
Washed and thickened

slurry
Conditioning
Wash water
Precipitation of arsenic with lime,

neutralizaton of residual acid and
disposal
Cyanidation and gold recovery
Fig. 9.1. Proposed flow sheet for bioheap leaching of flotation concentrate using P.
chrysosporium
191
Flotation concentrate containing gold,

sulfides and CM
Conditioning of flotation concentrate
Fungal treatment of flotation

concentrate in CSTR
Washing of product in counter current

decantation thickeners
Washed and thickened

slurry
Conditioning
Wash water
Precipitation of arsenic with lime,

neutralizaton of residual acid and
disposal
Cyanidation and gold recovery
Fig 9.2. Proposed flowsheet for fungal biotreatment of flotation concentrate using a
continuously stirred tank reactor (CSTR)
The operating pH was deduced from this study to be 4.0. To reduce energy consumption,
temperature may be maintained at ambient conditions. The processing time may, however, be
confirmed from pilot studies. The technical advantages of this proposal compared with the
current biooxidation process are that the pretreatment process leads to reduction in pregrobbing behavior of the ore and an increase in gold recovery during cyanidation. P.
chrysosporium has a biosafety of one and is therefore neither pathogenic nor harmful to
humans, which makes the process environmentally compatible.
192
VITA
Grace Ofori-Sarpong
Grace Ofori-Sarpong was born in Ghana and was educated at the Kwame Nkrumah University
of Science and Technology (KNUST), Kumasi, Ghana where she obtained a BSc in
Metallurgical Engineering and MSc in Environmental Resources Management in 1998 and
2002 respectively. In 2002, she was appointed a lecturer in Mineral Engineering in the
University of Mines and Technology (UMaT), Tarkwa, Ghana and became the first female
lecturer in the Department. In 2006, she won a scholarship from the Government of Ghana to
pursue PhD in Energy and Mineral Engineering at the Pennsylvania State University, USA.
Her areas of research interest include water quality monitoring, mine waste processing,
microwaves in extractive metallurgy, biotransformation of refractory gold ores and
environmental issues of small-scale gold mining. She is a member of the Ghana Institution of
Engineers (GhIE), Society for Mining, Metallurgical and Exploration Engineers (SME),
Women in Science and Engineering (WISE) and a fellow of the Schlumberger Faculty for the
Future (FFTF).

Simultaneous Biotransformation of Carbonaceous Matter and Sulfides in Double Refractory Gold Ores Usin The Fungus

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The Pennsylvania State University

The Graduate School

SIMULTANEOUS BIOTRANSFORMATION OF CARBONACEOUS MATTER AND

2010 Grace Ofori-Sarpong

Submitted in Partial Fulfillment

Robert Larry Grayson

*Signatures are on file in the Graduate School

3.3 Experimental investigations .........................................................................................47

5.2.1 Materials, medium preparation and incubation...................................................102

7.3.3 Effect of fungal-treatment on cyanide amenability of FC and BFC ....................151

1.1.1 Gold ores

1.1.2 Relevance of microbial pretreatment of double refractory gold ores (DRGO)

Most of the investigations have recorded substantial degradation for bituminous-grade CM

1.1.3 Phanerochaete chrysosporium

Specific research objectives

This study sought to investigate the simultaneous fungal-degradation of carbonaceous matter

Approach to accomplish experimental objectives

Table 1.1. Summary of the methodology required to accomplish the objectives

Coal type (lignite, sub-bituminous,

Gold adsorption test of

bituminous and anthracite)

Media (glucose, millet and wheat bran)

Time (1-21 days)

Pulp density (15-75 % solids)

Temperature (25 oC and 37 oC)

Sulfides (pyrite and arsenopyrite)

Time (1-21 days),

Pulp density (15-60 %)

Sulfur speciation and

surface oxidation products

Gold concentrate (flotation concentrate

(FC) and bacterial-oxidized FC)

Time (1-21 days)

Pulp density (15-60 %)

Sulfur speciation and

surface oxidation products

ICP-AES, fire assay

Table 1.2. Brief description of the dissertation write-up

Problem statement, motivation, hypothesis and

Mycohydrometallurgy: application of fungi in

Literature review of the diversity and application

of fungi biosorption, bioleaching and degradation

Mycohydrometallurgy: Coal model for potential

Experiments establishing the potential of P.

reduction of preg-robbing capacity of

chrysosporium to reduce gold adsorption (preg-

carbonaceous gold ores using the fungus,

robbing) capacity of carbonaceous matter in

refractory gold ores

Fungal biotransformation of anthracite-grade

Establishment of a set of incubation parameters

carbonaceous matter: effect on gold cyanide

that leads to maximum reduction in preg-robbing

of anthracite when using P. chrysosporium

Gold adsorption on carbonaceous matter

Ability of carbonaceous matter of different

following fungal treatment: comparison of

maturity to adsorb different gold complexes (i.e.,

cyanide and non-cyanide gold complexes

cyanide, thiourea, and thiosulfate), and reduction

Biotransformation of sulfides using the fungus,

Experiments establishing the ability of P.

Phanerochaete chrysosporium: A potential