Geochimica et Cosmochimica Acta, Vol. 69, No. 22, pp.

5233–5246, 2005
Copyright © 2005 Elsevier Ltd
Printed in the USA. All rights reserved
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doi:10.1016/j.gca.2005.06.022

Cu isotopic fractionation in the supergene environment with and without bacteria

RYAN MATHUR,1,* JOAQUIN RUIZ,1 SPENCER TITLEY,2 LAURA LIERMANN,3 HEATHER BUSS,3 and SUSAN BRANTLEY3
1
Department of Geology, Juniata College, Huntingdon, Pennsylvania 16652, USA
2
Department of Geosciences, University of Arizona, Tucson, Arizona 85721, USA
3
Department of Geosciences, Pennsylvania State University, State College, University Park, Pennsylvania 16802, USA

(Received February 28, 2005; accepted in revised form June 21, 2005)

Abstract—The isotopic composition of dissolved Cu and solid Cu-rich minerals [␦65Cu (‰) ⫽ (65Cu/
63
Cusample/65Cu/63Custd) - 1)*1000] were monitored in batch oxidative dissolution experiments with and
without Thiobacillus ferrooxidans. Aqueous copper in leach fluids released during abiotic oxidation of both
chalcocite and chalcopyrite was isotopically heavier (␦65Cu ⫽ 5.34‰ and ␦65Cu ⫽ 1.90‰, respectively,
[⫾0.16 at 2␴]) than the initial starting material (␦65Cu ⫽ 2.60 ⫾ 0.16‰ and ␦65Cu ⫽ 0.58 ⫾ 0.16‰,
respectively). Isotopic mass balance between the starting material, aqueous copper, and secondary minerals
precipitated in these experiments explains the heavier isotopic values of aqueous copper. In contrast, aqueous
copper from leached chalcocite and chalcopyrite inoculated with Thiobacillus ferrooxidans was isotopically
similar to the starting material. The lack of fractionation of the aqueous copper in the biotic experiments can
best be explained by assuming a sink for isotopically heavy copper present in the bacteria cells with ␦65Cu ⫽ 5.59
⫾ 0.16‰. Consistent with this inference, amorphous Cu-Fe oxide minerals are observed surrounding cell
membranes of Thiobacillus grown in the presence of dissolved Cu and Fe.
Extrapolating these experiments to natural supergene environments implies that release of isotopically
heavy aqueous Cu from oxidative leach caps, especially under abiotic conditions, should result in precipitates
in underlying enrichment blankets that are isotopically heavy. Where iron-oxidizing cells are involved,
isotopically heavy oxidized Cu entrained in cellular material may become associated with leach caps, causing
the released aqueous Cu to be less isotopically enriched in the heavy isotope than predicted for the abiotic
system. Rayleigh fractionation trends with fractionation factors calculated from our experiments for both
biotic and abiotic conditions are consistent with large numbers of individual abiotic or biotic leaching events,
explaining the supergene chalcocites in the Morenci and Silver Bell porphyry copper deposits. Copyright
© 2005 Elsevier Ltd

1. INTRODUCTION table fluctuations can lead to subsequent leaching of the en-

Recent studies have demonstrated that biologic and kinetic
processes cause measurable isotopic shifts for stable transition
metal isotopes (Shields et al., 1965; Halliday et al., 1998; Gale
et al., 1999; Maréchal et al., 1999; Anbar et al., 2000; Zhu et
al., 2000; Brantley et al., 2001; Bullen et al., 2001; Matthews et
al., 2001; Zhu et al., 2002; Ellis et al., 2002; Beard et al., 2003;
Anbar, 2004). In natural samples, copper exhibits the greatest
variation in isotopic composition (up to 9‰) of the transition
metals (Johnson et al., 2003; Larson et al., 2003).
An ideal natural location to observe the fractionation of
transition metal isotopes is the supergene enrichment environ-
ment. The supergene enrichment process involves leaching of
metal-rich igneous rocks that contain significant concentrations
of sulfide minerals. The interaction of pyrite and similar min-
erals with low-temperature aqueous fluids in the so-called
leached capping generates dilute sulfuric acid, which in turn
further dissolves the sulfide minerals releasing aqueous oxi-
dized metals. These metals subsequently precipitate in deeper
horizons after oxidants are consumed, and sulfides determine
the redox state (e.g., Brimhall, 1980; Guilbert and Park, 1986;
Titley, 1995). The process naturally concentrates metals in
what is termed the enriched supergene blanket below the water
table. As erosion progresses and/or climate changes, water
* Author to whom correspondence should be addressed
(mathur@juniata.edu).
5233
riched supergene blanket and reprecipitation in new enrichment
blankets at depth (Alpers and Brimhall, 1989). The dominant
Cu-containing sulfide that leaches in the oxidative vadose zone
to initiate the supergene leach cycle is chalcopyrite (CuFeS2),
whereas the dominant precipitate in enrichment blankets is
chalcocite (Cu2S).
The behavior and distribution of copper during enrichment is
complex. The oxidative dissolution process does not com-
pletely remove all of the Cu in the leach cap as evidenced by
Cu occurring in both primary chalcopyrite and secondary sul-
fate minerals such as chalcanthite, brochantite, and antlerite in
the leach cap. In contrast, chalcocite (Cu2S) and other related
copper sulfides dominate the mineralogy in enrichment blan-
kets; chalcocite probably forms by the progressive replacement
of chalcopyrite (CuFeS2) and pyrite (FeS2) (Brimhall et al.,
1985). Stokes (1970) demonstrated the importance of pyrite as
a template for precipitating chalcocite, although Sillitoe et al.
(1996) found bacteria forms in etched chalcocite from ancient
supergene systems and argued that nanobacteria promoted the
fixation of copper in the supergene environment.
To begin to understand the biologic, kinetic, and equilibrium
chemical behavior of copper during this process, we designed
several batch leach experiments to compare with samples taken
from ancient supergene environments. The experiments allow
us to compare the nature of both redox and precipitation reac-
tions working simultaneously during the biotic and abiotic
leaching of chalcopyrite and chalcocite.
5234 R. Mathur et al.
1.1. Chemistry of Leaching
In an effort to understand the biotic and abiotic mechanisms
that may cause Cu fractionation, it is worthwhile to compare
the process of industrial leaching of copper ores to the super-
gene environment (Dold and Fonbonte, 2001; Dold, 2003). The
industrial process involves placing large quantities of crushed
chalcocite ore (⬃2 wt% Cu) into piles called leach pads. A
network of plumbing distributes dilute sulfuric acid on top of
the leach pads. Chemically resistant pads underneath the piles
of ore collect the percolated copper-rich solution. Electrolytic
processes are then used to extract copper from the solution.
Oxidative leaching of chalcopyrite with ferric sulfuric acids
results in the release of Cu2⫹ to solution. Other reactions
occurring on the surface of the chalcopyrite involve the pre-
cipitation of native sulfur, polysulfides, and/or iron hydroxides
(Dutrizac et al. 1969; Hackel et al., 1995; Stott et al., 2000).
Mineral precipitates are thought to armor the surface of chal-
copyrite and cause the relatively slow kinetics associated with
leaching of chalcopyrite.
The extraction of Cu from chalcocite (Cu2S) by ferric ion-
containing sulfuric acid also causes the release of aqueous
Cu2⫹. As copper is lost, chalcocite is replaced sequentially by
digenite (Cu1.96S), anilite (Cu1.75S), geerite (Cu1.6S), spionko-
pite (Cu1.4S), yarrowite (Cu1.1S), and covellite (CuS) (Goble,
1985; Whiteside and Goble, 1986). The proportion of Cu2⫹
increases in the precipitated mineral phase until all of the Cu in
the final product, covellite, is thought to be Cu2⫹. However,
Brelle et al. (2000) analyzed nanoparticles of CuxS phases and
discovered that covellite precipitated from copper-rich solu-
tions contains as much as 33% Cu1⫹. Thus, the leaching
process generally does not completely oxidize all of the copper
in the system. A summary reaction describing formation of
covellite from chalcocite during leaching is (Lizama, 2001):
Cu2S ⫹ 2Fe(aq)
3⫹
? CuS ⫹ Cu(aq)
2⫹
⫹ 2Fe(aq)
2⫹
(1)
As described in this reaction, Fe3⫹ acts as the oxidant for the
Cu mineral. Aqueous ferric ions can also oxidize the S in
sulfide minerals, as summarized in this composite reaction for
pyrite oxidation (e.g., Singer and Stumm, 1968; Amend and
Shock, 2001):
FeS2 ⫹ 14Fe(aq)
3⫹
⫹ 8H2O ? 2⫹
15Fe(aq) ⫺
⫹ 2SO4(aq) ⫹
⫹ 16H(aq)
(2)
If similar sulfur-oxidizing reactions are written for copper
sulfide oxidation, mineral precipitates with Cu2⫹ can include
such minerals as chalcanthite (CuSO4·5H2O), brochantite
(Cu4SO4(OH)6), and antlerite (Cu3SO4(OH4)). The chemistry
of precipitates and aqueous leach fluids during supergene
leaching thus depends upon the chemistry and extent of leach-
ing and transport.
Ample evidence suggests that acidiphilic organisms exist
and thrive in leach pads and in natural supergene-leaching
environments. Enders (2000) and Hong et al. (2000) discovered
Thiobacillus ferrooxidans in the fluids of currently active su-
pergene zones of a porphyry copper deposit and Carlin gold
deposit, respectively. This species catalyzes the oxidation of
sulfur and/or metal, which in turn leads to the solubilization of
copper (Colmer and Hinkle, 1947). Several studies confirm that
the rates and mechanisms of the oxidation in the presence of T.
ferrooxidans (e.g., Breed and Hansford, 1999; Hansford and
Vargas, 2001; Crundell, 2003) affect the release of copper in
industrial leach pad environments and enhance the copper
yields. Singer and Stumm (1968) suggests that the rate-deter-
mining step of pyrite oxidation is oxidation of ferrous to ferric
iron in solution under low pH conditions. T. ferrooxidans is
thought to catalyze this reaction.
This brief overview of the controls of copper sulfide leaching
provides fundamental information of the multiple and inter-
twined biologic and kinetic mechanisms that may influence
copper isotope fractionation.
2. EXPERIMENTAL DESIGN
The experimental design is composed of two 30-day batch experi-
ments, one containing copper sulfides to monitor leaching of minerals
and one with bacteria and copper-rich medium to monitor copper
uptake into the cells without sulfide minerals present. A 24-h dissolu-
tion experiment containing copper sulfide minerals was also run to
investigate the effect of partial dissolution. Other measurements of
copper isotopes from completely dissolved copper-rich minerals asso-
ciated with the hypogene and supergene mineralization of porphyry
copper deposits sampled from many localities around the world were
conducted.
2.1. Leach Experiments
In the 30-day leach experiments, copper was leached from chalcopy-
rite from the El Teniente porphyry copper mine (e.g., Skewes and
Stern, 1994; Maksaev et al., 2004) and from chalcocite purified from
several ores in the Morenci porphyry deposit (described in Enders
[2000]). The samples, exhibiting grain sizes varying from 1- to 2-mm
granules to 1.5- to 2-mm very coarse sand, were homogenized before
use. As shown by X-ray diffraction (XRD), the chalcopyrite contained
only chalcopyrite, whereas the chalcocite contained two observable
phases, chalcocite and quartz. However, the amount of quartz is min-
imal (ⱕ10%) as indicated by XRD and optical petrography.
Chalcopyrite and chalcocite were leached in 250 mL flasks contain-
ing dilute sulfuric acid (pH 2.3) medium chosen to enhance the growth
of T. ferrooxidans (13598 from The American Type Culture Collection
[ATCC] Manassas, VA). The medium contained 0.8 g l-1 (NH4)2SO4,
2.0 g l-1 MgSO4 · 7H2O, 0.4 g l-1 K2HPO4, 20.0 g l-1 FeSO4 · 7H2O,
and 5.0 mL l-1 Wolfe’s mineral solution (Wolin et al., 1963). The pH
of the solution was adjusted to 2.3 with sulfuric acid, and the medium
was filter-sterilized (4.5 micron). A 1.56 g sample of either chalcopyrite
or chalcocite was autoclaved in each of 12 flasks (six of each mineral),
to which 200 mL of the sterile medium was aseptically transferred. The
experimental conditions were (1) chalcocite ⫹ medium (n ⫽ 3), (2)
chalcocite ⫹ medium ⫹ bacteria (n ⫽ 3), (3) chalcopyrite ⫹ medium
(n ⫽ 3), and (4) chalcopyrite ⫹ medium ⫹ bacteria (n ⫽ 3); where n
⫽ number of replicates of each condition (Table 1). Where indicated,
flasks were inoculated with 100 ␮L of T. ferrooxidans in late-log to
stationary phase of growth. The inoculated experiments are termed
biotic and the experiments which were not inoculated are termed
abiotic.
The two sets of six flasks were capped with sterilized plastic caps
and set in a shaker-incubator (New Brunswick Scientific) at a constant
temperature of 25°C. Approximately every 7 to 10 days, one inoculated
and one noninoculated flask was removed from each set. The solutions
from these flasks were filtered through a 0.2 ␮m filter and the remnant
mineral grains were separated and rinsed ultrasonically three times in
acetone. During the experiment, yellow-orange minerals encrusted the
mineral surfaces. The fine-grained coatings could not be separated from
the copper mineral residues.
The dried solid mineral grains were separated into two aliquots. A
0.1 g aliquot was separated for copper isotope analysis; this portion was
placed in a 5 mL Teflon beaker and dissolved in concentrated nitric
acid (8 N). Approximately 0.2 g of the other aliquot was powdered for
XRD analysis on a Scintag Pad V X-ray powder diffractometer. XRD
 Cu isotopes in the supergene environment 5235

Table 1. Results of 30 leach experiments with chalcocite and chalcopyrite.

Collection Cu Fe Phases identified by

Sample day ␦65Cumin‰* ␦65Cuaq‰* ppm ppm % Cu leached** XRD ␣aq-min**
Chalcocite 2.60 – – – – chalcocite ⫹ quartz –

Abiotic
7 1.90 5.34 1320 3620 21.4 digenite ⫹ quartz 1.0034
14 1.99 4.68 2200 3730 35.7 covellite ⫹ quartz 1.0027
30 1.67 4.54 2240 1870 36.4 covellite ⫹ quartz 1.0029
Biotic
7 2.34 2.96 1320 2820 21.5 digenite ⫹ quartz 1.0006
14 2.75 2.63 3310 92 53.8 covellite ⫹ quartz 0.9999
30 0.69 2.81 3490 1100 56.7 covellite ⫹ quartz 1.0021

Chalcopyrite 0.58 – – – – chalcopyrite –

Abiotic
7 0.37 1.90 301 4170 11.3 chalcopyrite 1.0015
14 0.27 1.90 420 3430 15.8 chalcopyrite 1.0016
30 0.59 1.51 634 2000 24.0 chalcopyrite 1.0009
Biotic
7 0.69 0.79 272 2120 10.2 chalcopyrite 1.0001
14 0.33 0.86 386 2050 14.5 chalcopyrite 1.0005
30 0.29 0.81 444 2130 16.7 chalcopyrite 1.0005

* ␦65Cu reported in per mill with respect to the 976NIST standard, as demonstrated in Eqn. 1, with 2␴ ⫽ 0.16‰.
** % of Cu in starting material leached.
***␣aq-min⫽(␦65Cuaq ⫹ 1000)/(␦65Cumin ⫹ 1000), where aq ⫽ aqueous and min ⫽ mineral remaining at end of experiment.

scans were completed in slow, step-scan mode for precision analysis.
Some minerals might be present but not detected if they occur at less
than 5 vol% of the material analyzed or are amorphous.
In the inoculated experiments, evidence of cell growth was deter-
mined by a characteristic color change in the medium (from a yellow-
brown to a reddish-brown, which was not observed to occur in the
absence of cells), and by occasional cell count estimates under a light
microscope.
2.2. Bacterial Uptake Experiments
ensure that the medium was completely rinsed from the pellet. The
bacteria pellets were dried, weighed, and acid-digested for copper
analysis.
Cells were also pelleted and washed as described above from the
flask set up for TEM and EDS analysis. Single drops of the pellet were
placed on Formvar carbon-coated glow-discharged nickel TEM grids
and incubated for 3 min; after wicking away excess solution, 2%
aqueous uranyl acetate was added, then the samples were incubated for
30 s in the dark, wicked again, and dried overnight before TEM
viewing.

The bacterial uptake experiments were designed to monitor the Cu
content and Cu isotopic composition of the T. ferrooxidans. This
experiment was conducted because a Percol density gradient separation
of T. ferrooxidans from the mineral grains in the preceding experiment
was not successful. This organism has been renamed to Acidiphious
ferrooxidans (Kelly and Wood, 2002).
To run the bacterial uptake experiments, an aliquot from a dissolved,
pure (99.99%) copper nugget (2.45 g, from the Phelps Dodge’s
Morenci processing plant) in 10 mL of concentrated nitric acid (8 N)
was mixed into the Thiobacillus medium to a final concentration of 50
mM copper. Nielsen and Beck (1972), Leduc et al. (1997), Boyer et al.
(1998), Gericke and Pinches (1999) demonstrated that Thiobacillus can
exist in solutions with as much as 400 mM copper.
Six flasks were set up with 150 mL of this copper-rich medium,
inoculated with Thiobacillus as described above, and set on a rotating
shaker for 30 days. Three flasks were used to determine the copper
isotopic composition of the bacteria. Two flasks were used to determine
the concentration of copper in the bacteria, and one flask was used for
imaging by transmission electron microscopy (TEM) and scanning
electron microscopy (SEM). Bacteria were also analyzed using energy-
dispersive X-ray spectroscopy (EDS). The Cu concentration of the
medium for this experiment remained the same after 30 days of
bacterial growth (initial [Cu] ⫽ 50 mM, final [Cu] ⫽ 50 mM). This
concentration represented the approximate midway point between the
two final aqueous copper concentrations of the chalcopyrite and chal-
cocite biotic leach experiments after 30 days ([Cu] ⫽ 110 mM and 14
mM, respectively).
Two of the three flasks were combined for copper isotope analysis to
increase the cell mass and entrained total copper. The cells were
pelleted by centrifugation and washed with 18 ⍀ water three times to
2.3. Partial Dissolution Experiments
In the 24-h partial dissolution experiments, copper was leached
incompletely from chalcopyrite and bornite (Cu5FeS4). The chalcopy-
rite sample was a mineral concentrate from the Ertsberg District, Irian
Jaya (described in Mathur et al. [2000]). The bornite sample was from
Collahuasi, Chile (described in Masterman et al. [2004]). The samples
have a grain size similar to the El Teniente and Morenci samples
discussed above, and no other phases were observed in the XRD
patterns of the samples.
Instead of using sulfuric acid to leach chalcocite and chalcopyrite
(which is known to produce phase changes during partial leaching),
nitric acid was used to leach the chalcopyrite and bornite. Linge (1976)
and others have noticed that leaching chalcopyrite with nitric acid does
not produce significant mineralogical phase changes as found when
leaching chalcocite. With this in mind, 0.2 g of copper sulfide was
placed (pure chalcopyrite or bornite) in 10 mL of heated 8N nitric acid
overnight. After leaching, the remaining solid materials were dried,
weighed, and dissolved in 5 mL Teflon beakers. XRD analyses of
powdered samples were used to identify mineralogical changes.
2.4. Copper Isotopic Composition of Minerals From Porphyry
Copper Deposits
The copper isotopic composition of hypogene chalcopyrites from
veins that contained high-temperature alteration silicate minerals (such
as biotite and potassium feldspar) from porphyry copper deposits
around the world (Table 2) were analyzed. In addition, supergene
chalcocites taken from enrichment blankets from Morenci and Silver
Bell porphyry copper deposits in Arizona were analyzed. For each
5236 R. Mathur et al.

Table 2. ␦65Cu‰ from high-temperature copper sulfides. pellets. IEC is necessary because the concentration of other ions in the
pellet is high and could cause matrix effects when analyzing for copper.
Sample name/Location Phase ␦ Cu‰*
65
Session** For example, test solutions were analyzed that contained proportions of
the NIST copper isotope standard mixed with ICPMS standard solu-
Tt-1, El Teniente bornite 0.37 1 tions of Si and Zn at various molar ratios. Si and Zn were investigated
Tt-1, El Teniente bornite 0.37 1 because Si impurities may have resulted due to the quartz in the
Tt-1, El Teniente bornite 0.41 2 chalcocite sample, whereas Zn is added to all analyses for mass bias
Tt-2, El Teniente chalcopyrite 0.01 1 correction. The standard solutions analyzed contained 0.008 mM Cu
Tt-2, El Teniente chalcopyrite ⫺0.07 2 (500ppb in 10 mL aliquot), and the other elements were added to the
Tt-3, El Teniente chalcopyrite 0.31 1 solutions to attain different proportions of Cu:element to test for a
Tt-4, El Teniente chalcopyrite 0.21 1 matrix effect. Figure 1 demonstrates that matrix effects are not common
Tt-5, El Teniente chalcopyrite ⫺0.70 1 when Si or Zn are present at twice the molar concentration of the
Tt-6, El Teniente chalcopyrite ⫺0.15 1 copper because the error associated with the standard analysis does not
Tt-7, El Teniente chalcopyrite ⫺0.51 1 change significantly. However, when the proportions of Cu:element are
Tt-8, El Teniente chalcopyrite 0.58 1 greater than a factor of two, it is observed that the analyzed value for
El-1, El Salvador chalcopyrite 0.81 1 the test solution lay outside the 2␴ range for the standard.
Esc-1, Escondida chalcopyrite 0.42 1 To avoid these and other matrix effects, IEC was conducted on the
C-1, Chuquicamata chalcopyrite ⫺0.09 1 solutions of dissolved bacteria pellets. Similar types and concentrations
C-1, Chuquicamata chalcopyrite 0.17 2 of acids for column elution (7N HCl ⫹ 0.001%H2O2) and resin (AG
C-2, Chuquicamata chalcopyrite ⫺0.11 1 MP-1 anion exchange resin) were used as in Maréchal et al. (1999).
C-2, Chuquicamata chalcopyrite ⫺0.03 2 The resins were rinsed and decanted five times in 18 ⍀ water to remove
Coya-1, Collahuasi chalcopyrite 0.38 1 particle fines, then settled in 18 ⍀ water. The volume of resin used was
Coya-2, Collahuasi bornite 0.64 1 1.6 mL, and we followed the purification protocol for copper outlined
M-1, Mocha chalcopyrite ⫺0.06 1 by Maréchal et al. (1999). The IEC procedure was tested with a mixed
T-1, Toquepala chalcopyrite ⫺0.44 1 solution prepared to contain 0.87 mM iron and 1.9 mM copper. One
Ers-1, Ertsberg chalcopyrite 0.17 1 hundred ⫾ 11% recovery of copper and no iron were found in the IEC
Gras-1, Grasberg chalcopyrite ⫺0.18 1 separation of this solution. In these experiments, the copper started
Pan-1, Panguna chalcopyrite ⫺0.39 1 coming off the resin at the 8th mL rather than the 10th mL; therefore,
copper elutions were collected between the 8th and 18th mL of elution
Notice that all values are within the reported 2␴ ⫽ 0.16‰. from the column (as noted in Table 2).
* ␦65Cu reported in per mill with respect to the 976NIST standard, as It has been well-documented that the ion-exchange resin can con-
demonstrated in Eqn. 1, with 2␴ ⫽ 0.16‰. tribute to fractionation of transition metal isotopes because of fraction-
** Indicates within-run and between-session variability of the Cu ation between adsorbed metal on the ion exchange sites within the resin
ratio measured. and the aqueous ions in eluted fluids (Anbar et al., 2000; Maréchal and
Albaréde, 2002). To be certain that the resin used for separation did not
cause fractionation of copper in the T. ferrooxidans pellet samples, we
analyzed the Cu isotopic composition of a high-concentration copper
analysis, ⬃0.2 g of each copper mineral were completely dissolved in
fluid we prepared and that was processed through the IEC. Without
heated 8N nitric acid overnight and analyzed isotopically (next sec-
using the IEC chemistry to separate copper (in other words, simply
tion).
diluting the high-concentration copper sample), the high-concentration
copper fluid was measured to have a ␦65Cu ⫽ 5.3 ⫾ 0.16‰ (abiotic day
3. ANALYTICAL METHODS 7; Table 1). Consistent with this, the same undiluted sample after
3.1. Concentrations of Cu and Fe treatment by IEC yielded a value for ␦65Cu within error of the diluted
sample (abiotic day 7; Table 3). Thus, at the percent recovery level of
Concentrations of Cu and Fe were measured by high resolution
inductively-coupled-plasma mass spectrometry (HR-ICP-MS; Finnigan
MAT Element I). All solution samples were acidified and diluted in 2%
nitric acid for chemical analysis. Fe and Cu concentrations were de- 0.5
termined by standard calibrations with the instrument in medium res- 500ppb NIST 976 Cu std + 500ppb Zn std
olution and indium was used as an internal standard. 0.4 500ppb NIST 976 Cu std + 1ppm Zn std
Cell pellets from the 30-day biotic experiment were rinsed four times 500ppb NIST 976 Cu std + 2 ppm Si std
0.3 500ppb NIST 976 Cu std + 4 ppm Si std
with 18 ⍀ water to remove the copper-rich medium and then dried
overnight. The dried pellet was weighed and dissolved in 2 mL of 0.2
d Cu

7NHCl ⫹ 0.01% peroxide, and copper was separated from the pellet 0.1
65

through wet ion-exchange column chemistry (described in detail be-

low). 0
-0.1
3.2. Copper Isotope Analysis
-0.2
Sample preparation for Cu isotope analysis on the Micromass Iso- -0.3
probe (multicollector inductively-coupled-plasma mass spectrometer
[MC-ICPMS] at the University of Arizona) varied for the three types of -0.4
samples analyzed: fluid digestates from copper minerals containing -0.5
⬎10 mM Cu, leach fluids with aqueous copper ⬎0.15 mM Cu, and
fluids from bacteria that contained 30 mM copper. 2 4 6 8
Fluid digestates and leach fluids from experiments with ⬎10 mM Cu Day of Cu std analysis
did not require ion chromatography for chemical separation. All of the
copper analyses for these samples were centrifuged and diluted with Fig. 1. Measured ␦65Cu for a Cu standard spiked with Zn and Si,
2% nitric acid to 0.008 mM Cu for analysis. Because copper is the plotted as a function of the analytical sessions over 153 days. Observed
dominant element in solution, the dilution eliminated any possible 2␴ ⫽ 0.16‰ for n ⫽ 76. Matrix effects (measured values outside 2␴,
matrix effects from other ions in solution (Zhu et al., 2000). as indicated by open symbols) were observed for solutions containing
In contrast, a wet ion exchange chromatography (IEC) procedure ⬎2 ppm of Zn or Si in solution. ␦65Cu presented as per mill with
was used to separate Cu from the solutions derived from bacteria respect to the 976NIST copper standard.
 Cu isotopes in the supergene environment 5237

Table 3. ␦65Cu‰ for column elutions. 4. RESULTS

Elluant Volume Matrix analyzed* % Cu** ␦ Cu‰

65
4.1. Results of 30-Day Leach Experiments
Rinse 21mL 0.5 NHNO3 bd nm
XRD patterns do not reveal any phase changes for the
Rinse 6mL 7NHCl ⫹ 0.01% H2O2 bd nm
Sample 2mL 7NHCl ⫹ 0.01% H2O2 bd nm chalcopyrite 30-day batch experiments, whereas diffraction
Rinse 8mL 7NHCl ⫹ 0.01% H2O2 bd 11.65 patterns from the chalcocite leach experiments reveal that chal-
Rinse 2mL MO water bd nm cocite (Cu2S) changed to digenite (Cu9S5) in 7 days and to
Collect 3mL 7NHCl ⫹ 0.01% H2O2 21.0 5.42 covellite (CuS) after 14 days. Table 1 summarizes the phases
Collect 7mL 7NHCl ⫹ 0.01% H2O2 79.1 5.42
Rinse 2mL MO water bd nm observed from the XRD patterns. XRD does not identify amor-
Rinse 7mL 7NHCl ⫹ 0.01% H2O2 bd nm phous precipitates. For instance, although yellow-orange coat-
ings were observed on the residual minerals throughout the
bd ⫽ below detection limit; nm ⫽ not measured. leach period for all the 30-day batch experiments with copper
* Indicates the normality of acids used.
sulfide minerals, the XRD did not reveal the presence of sec-
** Indicates % of total Cu eluted from the column.
ondary precipitates of crystalline iron oxide.
The fraction of the total starting Cu present as aqueous Cu
our IEC treatment, the chromatography does not produce isotopic (Table 1) increased with time for all experiments, but was
fractionation in these samples. higher for chalcocite than chalcopyrite at every leach period. In
Once the high-copper samples were diluted and the copper in the chalcopyrite experiments, aqueous concentrations (Fig. 3) of
bacteria pellets was purified with IEC, the solutions were injected into
copper were lower in the biotic as opposed to abiotic experi-
the MC-ICPMS using a microconcentric nebulizer to increase sensi-
tivity. All solutions were diluted and prepared so that the concentration ments. In contrast, in the chalcocite experiments the concen-
of copper in the solutions was 500 ppb Cu. The nebulizer flow was trations of aqueous copper in the biotic experiments were
adjusted so that the intensity of the 63Cu beam remained constant at 2 greater than the abiotic experiments (Table 1; Fig. 3). The
V. Because the signals were so large, our “blank” or background peaks concentration of iron in leach solutions for the biotic and
of copper at 4 mV are insignificant when considering the overall error.
Two sets of 20 ratios were collected for every sample measured. All abiotic experiments with chalcopyrite and chalcocite generally
ratios are reported here according to the following equation: decreased with time (Table 1; Fig. 3).
After the 30-day batch experiments with chalcocite, minerals

冢冤 冥冣
65 Cu
sample
63 Cu
␦65Cu‰ ⫽ ⫺1 ⫻ 1000 (3)
65 Cu
std
63 Cu
where the standard (std) was the NIST 976 Cu standard. Results are
reported as an average for each run. The standard deviation for mea-
sured ␦65Cu within each run was less than 0.01‰.
However, the major concern surrounding isotope data obtained dur-
ing an analytical session is the measurement error associated with mass
fractionation within the instrument due to variations in operating con-
ditions (Maréchal et al., 1999). To constrain the errors associated with
copper isotope analyses on our instrument, we compared all of the
copper ratios to the NIST 976 copper standard (Eqn. 3) using standard-
sample-standard bracketing. The 2␴ error for the variation of the
standard for eight analytical sessions over 5 months was observed to be
⫾0.16‰ (Fig. 1). Calculated errors using the Zn doping (Maréchal et
al., 1999) technique to correct for mass bias produced similar errors.
To test long-term reproducibility further, both fluid digestates from
copper minerals and leach fluids with aqueous copper were analyzed on
different run days. Results from these tests (Table 2) demonstrate that
the isotopic ratios of samples are reproducible within the reported error.
The errors for these measurements are larger than those presented in
Maréchal et al. (1999) and Zhu et al. (2000, 2002). These workers
report errors of ⫾0.1‰ and ⫾0.02‰, respectively, for long-term
reproducibility; however, our errors are small compared to the isotopic
shifts observed for experiments reported here.
To demonstrate the reliability of the measurements further, we
analyzed high-temperature, hypogene copper-rich minerals (chalcopy-
rite and bornite) collected from porphyry copper deposits around the
world (Table 2; Fig. 2). These samples were dissolved in a similar
manner as the solid copper mineral precipitates. As demonstrated by
Maréchal et al. (1999), Zhu et al. (2000, 2002), Larson et al. (2003),
and Graham et al. (2004), copper minerals that form in high-tempera-
ture environments do not demonstrate significant copper isotopic frac-
tionation when compared to the NIST 976 Cu standard; deviation from
the NIST 976 standard of these copper-rich minerals is approximately
⫾1‰. Figure 2 demonstrates that the hypogene minerals analyzed in
this contribution are in accordance with previous copper isotope mea-
surements of similar materials.
remaining in both the abiotic and biotic experiments show
␦65Cu values less than the starting materials. In contrast, the
␦65Cu values of the solid material from the experiments with
chalcopyrite remained within 2␴ of the starting material in both
the abiotic and biotic leach experiments.
Copper isotopic values of the aqueous Cu released during
abiotic leaching of both chalcopyrite and chalcocite are heavier
than the starting material and decrease with time (Fig. 4). In
contrast, the fluids from biotic leach experiments with chal-
copyrite and chalcocite remain at relatively constant ␦65Cu
values throughout the leach cycle. These constant values are, in
general, isotopically indistinguishable from the values in start-
ing materials.
4.2. Bacterial Uptake Experiment
In the 30-day biotic uptake experiments, the dissolved cop-
per in the Cu-rich medium has a measured ␦65Cu of 2.55 ⫾
0.16‰ at the start of the experiment and 2.50 ⫾ 0.16‰ at the
end of the experiment. The bacteria pellets centrifuged from
these solutions have significantly heavier isotope values than
the original copper medium in which they grew (5.59 ⫾ 0.16‰
average value for flasks 1 and 2; Table 4). The dried pellet
combined from two flasks by centrifugation for this experiment
weighed 0.24 g. The calculated concentration of Cu in the
pellet is 19 g Cu/kg dry weight bacteria.
TEM images and EDS spectra of bacteria collected from this
experiment also reveal that T. ferrooxidans cells contain ele-
vated concentrations of copper (Fig. 5). In contrast, EDS ele-
ment maps of the mineral precipitates and polymeric material
that formed in the medium during the experiment, when ob-
served under the SEM and TEM, did not reveal copper-rich
areas. No diffraction patterns indicating crystallinity were de-
5238 R. Mathur et al.
Fig. 2. Plot of ␦65Cu‰ from high-temperature copper sulfide min-
eralization from various porphyry copper deposits. ¦ ⫽ chalcopyrite
samples and ⽧ ⫽ bornite samples, and the open square is average
␦65Cu‰ ⫽ 0.09 (solid blank line ⫾ 2␴ ⫽ 0.8, dashed lines) of all
copper sulfides. ␦65Cu presented as per mill with respect to the
976NIST copper standard.
tected in the particulates associated with cells despite several
attempts to collect them.
4.3. Partial Dissolution Experiments
During the 24-h partial dissolution experiments, ⬃30% of
each sample dissolved (measured residue minerals weighed
0.15 g). The mineral composition as identified by XRD did not
change after partial acid digestion for both experiments. Partial
dissolution of the copper minerals (Table 5) in nitric acid did
not produce copper isotopic shifts in the leach fluid in compar-
ison to complete dissolution.
5. DISCUSSION
5.1. Mass Balance
To highlight mechanisms, a mass balance for each of the
reactions is calculated using
␦65Cumin
o
⫽ ␦65Cuaqfaq ⫹ ␦65Cuminfmin ⫹ ␦65Cubfb (4)
Here, ␦65Cuomin describes the isotopic composition of the start-
ing mineral, ␦65Cumin describe isotopic composition of residual
minerals, the subscript aq refers to Cu in aqueous leach fluid,
and subscript b refers to Cu in the bacteria pellet. In addition,
fi ⫽ ratio of the mass of copper in phase i divided by the total
mass of copper in the starting material (0.53 g Cu for chal-
copyrite and 1.09 g Cu for chalcocite).
Equation 4 balances the measured values of faq, fmin,
␦65Cumin, and ␦65Cuaq within error for the abiotic leach exper-
iments for both chalcocite and chalcopyrite (Table 6). This
abiotic mass balance suggests sources and sinks for heavy and
light reservoirs where isotopically heavy aqueous Cu is in
solution, and isotopically light Cu is precipitated out of solu-
tion.
Equation 4 does not balance the measured values of faq, fmin,
␦65Cumin, and ␦65Cuaq for the biotic leach experiments with
chalcocite (Table 6). To understand how the bacterial interac-
tion in the experiment may cause the imbalance, we isolated the
bacteria in separate experiments (termed the 30-day bacterial
uptake experiment) because we could not separate cells from
residual mineral powders. This comparison between the uptake
experiment and the leach experiment is an imperfect one,
because the final concentration of aqueous Cu in the cellular
uptake experiment (50 mM) differed from the final Cu concen-
tration in the biotic chalcocite (114 mM) and chalcopyrite (14
mM) experiments. Nonetheless, the bacteria uptake experiment
most likely best models the biotic chalcocite experiment. The
bacterial uptake experiments indicated that the copper concen-
tration is 19g Cu/kg bacteria pellet when T. ferrooxidans is
grown for 30 days in copper-rich growth medium, and the
pellet is isotopically heavy (␦65Cub ⫽ 5.59 ⫾ 0.16‰). If
hypothesized that this mass of copper (0.005 g) and the isotopic
composition of Cu is also entrained in the bacteria in the 30-day
biotic chalcocite experiments (and an average of the three
biotic chalcocite experiments fb ⫽ 0.01) we calculate that
␦65Cubfb values that are within mass balance for the reported
values except for the last chalcocite biotic experiment (Table
6). This deviation can be explained if more cellular mass can be
assumed in the chalcocite biotic 30-day experiment than mea-
sured in the uptake experiments. Therefore, an assumed larger
mass of bacteria could account for the missing heavy copper.
Thus, the results are well explained by a scenario in which
bacterial uptake of Cu in the 30-day mineral leaching experi-
ments were similar to the mineral-absent bacterial uptake ex-
periment in amount and isotopic composition (␦65Cub ⫽ 5.59
⫾ 0.16‰).
For Eqn. 2 of the biotic chalcopyrite experiment, we infer
that less Cu was taken up into T. ferrooxidans grown with
chalcopyrite. This inference is consistent with the lower extent
of oxidation in the chalcopyrite compared to chalcocite exper-
iments and the significantly lower values of dissolved Cu in the
presence of the chalcopyrite at 30 days (Fig. 3). However,
Figure 4 clearly demonstrates that the isotopic value of aqueous
Cu released during abiotic chalcopyrite oxidation differs from
the biotic release. We can clarify this observation by the fol-
lowing model calculations: If we assume that biotic uptake in
the chalcopyrite experiment can explain why the concentration
of Cu in solution at the end of the biotic experiment is lower
than Cu concentration at the end of the abiotic experiment, we
calculate that 0.005 g of Cu (average of the three biotic chal-
copyrite experiments fb ⫽ 0.06) was taken up by Thiobacillus
cells. Assuming further that the isotopic composition of the
cellularly incorporated Cu was ␦65Cub ⫽ 5.59 (as in the cellular
uptake experiment, Table 5), the value of ␦65Cub fb for the
biotic chalcopyrite experiment is calculated to be 0.3, which
balances Eqn. 4 within error. This calculation demonstrates
why the abiotic release of Cu from chalcopyrite results in
isotopically heavy Cu in solution, whereas the biotic release of
Cu results in isotopically indistinguishable Cu from the starting
chalcopyrite in solution.
5.2. Mechanisms for Copper Isotope Fractionation
In this discussion we argue for the simplest mechanism for
the cause of isotope fractionation that is consistent with our
results, although clearly other mechanisms may be possible.
Fractionation of isotopes such as those presented (Table 1)
could occur during one or more fractionation steps, followed by
separation of the fractionated Cu reservoirs. Because of the
 Cu isotopes in the supergene environment 5239

Fig. 3. Plot of the concentration of iron and copper in the (a) abiotic 30-day batch experiments with chalcocite, (b) biotic
30-day batch experiments with chalcocite, (c) abiotic 30-day batch experiments with chalcopyrite, and (d) biotic 30-day
batch experiments with chalcopyrite.

complexity of our experiments, we consider the following
possible mechanisms as both fractionating and separating steps:
(1) release of copper from sulfide minerals, (2) adsorption or
precipitation of copper on or with iron oxides, (3) precipitation
of copper as a secondary sulfide, and (4) adsorption or uptake
of copper on, or by the cells.
Mechanism 1, fractionation during dissolution, is not the
most likely mechanism for fractionation of copper because we
observed no copper isotope fractionation during the 24-h partial
dissolution experiments.
Mechanism 2 relies upon incorporation or adsorption of
copper into or onto the iron oxides that precipitate during the
experiment. From a crystallographic standpoint, copper does
not easily substitute for Fe in the iron oxide crystal structure
(Waychunas, 1991). However, adsorption of Cu to the surface
of iron oxides is a possibility. Many studies have examined
adsorption of metals onto iron oxides (e.g., Parkman et al.,
1999; Jang et al., 2003 and references therein). For example,
Christl and Kretzschmar (1999) examined the role of copper
adsorption at the hematite-water interface. They found that at
pH ⬍4, copper does not adsorb onto the surface of hematite
regardless of the concentration of copper in the solution (20 –
100 mM). Given that our experiments were run at pH 2 to 3,
their results may suggest little to no copper adsorption onto iron
oxides. Consistent with this, data shown in Figure 3 document
that Cu and Fe behave differently in the experiments, and we
observed that suspended mineral precipitates observed under
EDS did not contain significant Cu. Therefore, adsorption of
Cu onto iron oxide surfaces most likely does not account for the
fractionation of copper isotopes in these experiments.
Mechanism 3, the precipitation of new copper minerals,
could explain the variations seen in both the abiotic 30-day
batch experiments with chalcopyrite and chalcocite. The mass
balance documented between the fluid and the residue in the
abiotic experiments ((Eqn. 4); Table 6) is consistent with this
idea. One possible explanation for the observed fractionation is
that the average copper coordination for Cu in the chalcocite-
to-covellite series of copper sulfide transformations correlates
5240 R. Mathur et al.

Fig. 4. Plot of ␦65Cu‰ in residual minerals and copper leach fluid vs. time for (a) abiotic 30-day batch experiments with
chalcocite, (b) biotic 30-day batch experiments with chalcocite, (c) abiotic 30-day batch experiments with chalcopyrite, and
(d) biotic 30-day batch experiments with chalcopyrite. Thick black line indicates the copper isotopic signature of the starting
sulfide material. Symbols with fill indicate fluids and symbols without fill indicate residual materials. Dashed lines indicate
2␴ error (0.16‰) of the starting material. ␦65Cu presented as per mill with respect to the 976NIST copper standard.

with the copper isotope variation of acid-sulfate leach solutions
in abiotic leach experiments of chalcocite. They suggest that
the change in copper bonding in the Cu mineral during acid-
sulfate leaching causes an equilibrium copper isotope effect.
Because similar phase changes occurred during our batch leach
experiments, crystallographic changes in mineral residues and
Cu isotopic differences in these minerals could explain our
observations. Rouxel et al. (2004) also demonstrated that oxi-
dized aqueous copper acquires a 3‰ heavier copper isotopic
value during remobilization of copper in hydrothermal fields in
the Mid-Atlantic Ridge, and they attributed the fractionation to
crystallographic changes in the copper sulfide mineralogy.
Assuming mechanism 3 is correct, the definition of a frac-
tionation factor, ␣aq-min
␣aq⫺min ⫽ (␦65Cuaq ⫹ 1000) ⁄ (␦65Cumin ⫹ 1000)
The fractionation factor is determined at isotopic equilibrium.
In our experiments, we did not prove isotopic equilibrium.
However, to understand our system, we have calculated values
for these fractionation factors for every time point in our
experiment (Table 1); these values may or may not represent
equilibrium values of ␣aq-min and are similar to values pre-
sented by Ehrlich et al. (2004) and Zhu et al. (2002). Zhu et al.
(2002) conducted an abiotic reduction experiment in dilute
nitric acid where Cu mineral was precipitated from HNO3-KI
solution containing 0.05 M Cu (NO3)2. The experiment pro-
duced isotopically light precipitates containing Cu⫹ (average
␦65Cumin ⫽ ⫺3.4 ⫾ 0.01‰) and slightly isotopically heavier
fluids (average ␦65Cu of 0.53 ⫾ 0.01‰). They calculated
values of ␣aq-min of ⬃1.004 for their experiments, and they
argued their values represented equilibrium.
For the chalcopyrite experiments, XRD did not reveal the
(5)
presence of newly formed sulfide minerals after 30-day, and the
␦65Cu value of the residual material does not differ from the
 Cu isotopes in the supergene environment
Table 4. Results from 30-day batch experiments with copper-rich
medium.
Sample number Description ␦65Cu‰*
1 flask 1 ⫹ 2** 5.54
5.65
2 flask 3*** 4.16
4.31
3 starting medium 2.71
2.72
4 Cu nugget 2.55
* ␦65Cu reported in per mill with respect to the 976NIST standard, as
demonstrated in Eqn. 1, with 2␴ ⫽ 0.16‰, during one session.
** Bacteria pellet from combination of two flasks to increase con-
centration of copper in the pellet.
*** Bacteria pellet from one flask.
starting material. However, in these experiments, aqueous cop-
per is isotopically heavier than the starting material. We infer
that other copper phases are present in trace quantities in these
experiments, consistent with the observation that powder dif-
fraction cannot detect phases ⬍5%, or that there are amorphous
phases not detected by XRD. For example, Hackel et al. (1995)
identified, through the use of Auger electron spectroscopy and
X-ray photoelectron spectroscopy, the presence of metal-rich
sulfide minerals on the surface of sulfuric-acid leached chal-
copyrite. Todd et al. (2003) further examined chalcopyrite
surfaces with Cu and Fe edge spectroscopy and found that in
low pH solutions, the chalcopyrite surface is characterized by a
layer that is metal-deficient compared to the bulk stoichiome-
try. Given that these experiments are relatively similar in de-
sign, small quantities of metal-bearing and isotopically distinct
sulfide minerals may have formed on the surface of the chal-
copyrite. Therefore, the copper isotope fractionation recorded
in the leach fluids from the chalcopyrite could be evidence that
copper-rich sulfide minerals precipitated on the surface of the
chalcopyrite.
The fourth mechanism, cellular uptake or adsorption, is not
needed to explain mass balance within error as exemplified in
Eqn. 4 for the biotic chalcopyrite experiment. However, the
difference between the isotopic values of aqueous Cu released
during biotic and abiotic oxidation of chalcopyrite is best
explained by cellular uptake of ⬃0.0045 g of Cu with ␦Cu ⫽
5.59 ⫾ 0.16‰, as discussed earlier. Furthermore, this mecha-
nism explains the results for both the biotic 30-day batch
experiments with chalcocite and the bacterial uptake experi-
ment. If the bacteria are not considered in the mass balance
expression 4 for the biotic 30-day batch experiments with
chalcocite, mass balance cannot be achieved. The bacteria are
therefore inferred to be a sink for the heavy copper. Specifi-
cally, in the final collection of the biotic batch experiments with
chalcocite, there must be at least 0.2 g of bacteria that contains
19g Cu/kg bacteria pellet present for mass balance. Our exper-
iments are consistent with 0.24 g dry weight for the bacteria in
the chalcocite experiment (Table 5). As shown in Figure 5,
each bacterium is observed to be coated by Cu and Fe-contain-
ing particles, presumed to be oxides. Interestingly, these Cu
oxides must be isotopically heavy (Table 5), in contrast to the
newly precipitated Cu-sulfide residual minerals in abiotic ex-
periments (Table 1), which are isotopically light.
5241
Further clarification as to how the bacteria interact with
copper to produce these shifts involves understanding how the
organism uses copper. At least four biologic pathways can be
envisioned in which the copper isotopes could be fractionated
by bacteria. First, Cu uptake into a biofilm could cause the
relative enrichment of one isotope. Kinzler et al. (2003) de-
scribes the complex processes that occur within biofilms and
points out that trace metals are often complexed to polymers in
biofilms. Secondly, the uptake of Cu through the cell mem-
brane could selectively favor one isotope either due to transport
down concentration gradients or via carriers. Third, the copper
fractionation could be related to active sites on enzymes in the
bacteria where different coordination sites within these organic
molecules favor one isotope or another. It has recently been
demonstrated, for example, that different oxidation states of Cu
are favored by differently structured copper-bearing proteins
(Peariso et al., 2003). Conceivably, the selection of Cu⫹ or
Cu2⫹ by different enzymes could produce the isotopic shifts.
Finally, cells can cause precipitation of metals at their outer
membrane surface.
Given the high concentration of copper measured in the T.
ferrooxidans, fractionation by cellular intake is not reasonable
because the observed concentration of copper is so high as to
presumably be toxic. No metal precipitates were observed
within polymeric material associated with cells. A more likely
explanation for Cu uptake by cells is precipitation of Cu min-
erals on external membranes as suggested by Figure 5. Perhaps
copper precipitation on membranes may represent a process by
which the organism protects itself against high concentrations
of potentially toxic metals. The inference that mineral precip-
itates around cell membranes are oxides rather than sulfides
may further suggest that Cu entrained in secondary oxide
minerals is isotopically heavy as compared to Cu in secondary
sulfide minerals. In these experiments, precipitation of isotopi-
cally heavy Cu-Fe amorphous nanoparticulates are an impor-
tant vital effect that contributes to the isotopic value of Cu
released to solution during sulfide oxidation.
5.3. Supergene Environments
During supergene leaching, Cu is leached from chalcopyrite
in the oxidizing vadose zone and becomes concentrated as
precipitated chalcocite and covellite in a reducing environment
below the water table. Cu from the chalcocite/covellite can in
turn be leached (as uplift occurs and exposes the inner portions
of the system) and precipitated again as a new, lower, enrich-
ment blanket as chalcocite and covellite. These experiments are
thought to roughly mimic conditions found in natural super-
gene environments. As demonstrated by Bladh (1982), fluids
existing in ambient atmospheric conditions provide enough O2
(fO2 ⫽ 10-2 atm.) to generate dilute sulfuric acid of low enough
pH to dissolve and mobilize metals in sulfide minerals.
As originally suggested by Shields et al. (1965) and con-
firmed by the results presented here, the precipitated copper
minerals in the enrichment blanket should acquire a heavy
copper isotopic value during multiple leach events with or
without bacteria (Fig. 4). In each leaching event, chalcocite will
dissolve oxidatively to release isotopically heavy copper from
the solid reservoir into solution. If bacteria are present, uptake
of Cu as nano-oxides will drive aqueous Cu back toward
5242 R. Mathur et al.

Fig. 5. TEM image and EDS spectra of transition metals imaged in cell pellets of T. ferrooxidans. Note the thin black
rims on the outer cell walls of the bacteria. These black rims most likely indicate amorphous copper-iron oxide minerals.

starting material values. However, with or without bacteria, the
chalcocite transforms into isotopically lighter covellite during
leaching. The aqueous copper is either lost from the system or
precipitates in the enrichment blanket as copper supergene
minerals. As multiple supergene leaching events transpire, su-
pergene minerals become progressively enriched in the heavier
isotope as compared to the originally emplaced hypogene cop-
per (␦65Cu ⫽ 0‰, Fig. 2).
A cross-sectional model of the copper isotope composition
of supergene minerals within an enrichment blanket is not
 Cu isotopes in the supergene environment 5243

Table 5. Results from 24-hour partial dissolution experiments with The following equations describe the change in isotopic
copper sulfide minerals. value of Cu in the mineral and aqueous species during leaching
of chalcopyrite as a function of extent of reaction (Faure and
Sample name Phase Amount dissolved ␦65Cu‰*
Mensing, 2005). The chalcopyrite is assumed to be of known
Ertzberg Chalcopyrite complete dissolution 0.17 isotopic value at time zero, ␦65Cuomin:
Etzberg Leach Chalcopyrite partial dissolution 0.21
␣⫺1
Collahuasi Bornite complete dissolution 0.64 ␦65Cumin ⫽ (␦65Cumin
o
⫹ 103)fmin ⫺ 103 (6)
Collahuasi Leach Bornite partial dissolution 0.62
␣-1
␦ Cuaq ⫽ (␦ Cu
65 65 o
min ⫹ 10 )␣f
3
min ⫺ 10
3
(7)
* ␦65Cu reported in per mil with respect to the 976NIST standard, as
demonstrated in Eqn. 1, with 2␴ ⫽ 0.16‰. The physical model is based on average ␣ values derived from
the abiotic and biotic leach experiments (Table 1) and the
following assumptions:
possible to construct given the limited nature of these experi-
ments. The copper isotopic composition of copper minerals 1. The hypogene chalcopyrite is emplaced with a value of
throughout the enrichment blanket is most likely variable given ␦65Cuomin ⫽ 0‰.
the differences seen in the digenite and covellite residues from 2. The only source of copper is chalcopyrite. No further copper
the leach experiments. However, the copper isotopic composi- is added to the system, which is open only to oxygen, carbon
tion of a processed copper nugget from Morenci (␦65Cu ⫽ 2.5) dioxide, and water.
has the same copper isotope value as the chalcocite sampled 3. The fractionation factor for leaching chalcopyrite,
from the enrichment blanket. This most likely indicates the ␣aq-chalcopyrite, is 1.0012 for the abiotic model and 1.0004 for
average copper isotope signature from all of the mined super- the biotic model. These values represent values averaged for
gene ore at Morenci is relatively similar to the copper isotope all measurements for each chalcopyrite experiment (Table
signature of chalcocite sample used for the leach experiments. 1). Whereas in the biotic case the value is determined from
Therefore, single analyses of chalcocite may provide some differences well within the margin of error of measurement,
insight into the overall leaching process that has occurred in the we include that calculation here simply to exemplify our
geological past. best estimate of how such fractionation factors would allow
To quantify this relationship, we constructed a Rayleigh both aqueous and mineral Cu to evolve over time for the
distillation model using the calculated fractionation factors biologic system.
from the abiotic and biotic leach experiments to predict how the 4. During the first leaching event, 65% of the copper is leached
copper isotopic composition of supergene minerals would and 35% remains in minerals in the leach cap. These ap-
change during sequential leaching and reworking of copper in proximate proportions were determined by examining how
the Morenci and Silver Bell supergene environments (Fig. 6). copper is currently distributed in Morenci. At Morenci,

Table 6. Mass balance for components involved in leach cycle*.

Sample Leach day ␦65Cuaqfaq ␦65Cuminfmin ␦65Cubfb ␦65Cumino

Chalcocite

Abiotic

7 1.29 1.44 0 2.74

14 1.65 1.28 0 2.93
30 1.65 1.06 0 2.72

Biotic

7 0.64 1.84 0.2 2.67

14 1.42 1.27 0.2 2.88
30 1.59 0.30 0.2 2.09

Chalcopyrite
Abiotic

7 0.22 0.33 0 0.54

14 0.30 0.23 0 0.53
30 0.36 0.45 0 0.81

Biotic

7 0.08 0.62 0.2 0.70

14 0.13 0.29 0.2 0.61
30 0.14 0.24 0.2 0.58

* ␦65Cu reported in per mill with respect to the 976 NIST standard.
5244 R. Mathur et al.
Fig. 6. Predicted leach events using Rayleigh distillation models.
Solid lines indicate copper isotope composition of the chalcocites from
Morenci and Silver Bell porphyry copper deposits in Arizona. Each
filled symbol on the graph represents the predicted ␦65Cu value of
chalcocite after one leaching/precipitation event as supergene enrich-
ment progresses (see text). Circles indicate abiotic processes and
squares indicate biotic processes. ␦65Cu presented as per mill with
respect to the 976NIST copper standard.
several enrichment blankets are mined; the average enrich-
ment blanket is 130 m thick and contains 0.47 wt% Cu. The
leach cap is 82 m thick and contains 0.1 wt% Cu (Enders,
2000). We assume that, after the first leach event, ⬃65% of
the total copper within the deposit is now present in the
enrichment blanket. Of course, as pointed out by Enders
(2000), lateral transport of copper into exotic periphery
deposits may also have occurred; therefore, the system may
not have been closed with regard to total movement of
copper. Nonetheless, our model assumes a closed system.
5. Of the 65% of the total copper that is removed from the
leach cap, we assume all of the copper precipitates as
chalcocite in the enrichment blanket. The assumed value of
␣aq-chalcocite is 1.003 for the abiotic model and 1.0009 for the
biotic model (Table 1).
6. During each of the subsequent leach events, all of the copper
is derived from chalcocite in the last-formed blanket. For
each leach event, 65% of the copper in the blanket is
removed and is precipitated quantitatively in the next
blanket.
To test these models, we compared the predicted copper
isotopic composition of chalcocite derived from the model to
the copper isotopic composition of one sample of chalcocite
taken from one enrichment blanket from the Morenci and
Silver Bell porphyry copper deposits, respectively. Results of
field observations and study of enrichment blankets of deposits
in the American Southwest reveal a complex process of uplift
history and related lowering of the water table (Titley, 1983).
These coupled events have resulted in the formation, destruc-
tion, and reprecipitation of chalcocite enrichment blankets over
time.
The copper isotopic compositions of the two chalcocites are
plotted on Figure 6 along with the predicted changes of the
copper isotopic ratios from the Rayleigh fractionation models.
The overlap of the modeled copper isotopic composition of
chalcocite with the copper isotopic composition of the chalcoc-
ite from Morenci suggests that multiple leaching events were
needed to explain the isotopic systematics. If the system was
abiotic, 10 events are consistent with our data, whereas 27
leaching events would have been needed if Thiobacillus had
been important in the supergene system. Strong geologic evi-
dence based on mineral textures and radiometric dating are
consistent with multiple supergene leaching events at this site
(Titley and Marozas, 1995; Enders, 2000); however, these
authors could only suggest that the number of leach periods
was greater than two or three.
The heavy copper isotopic composition found in both en-
richment blanket chalcocites and these calculated high number
of leach events could be consistent with enrichment occurring
incrementally rather than as discrete enrichment/leaching
events separated in time. In other words, the water table may
have slowly dropped and the copper isotope composition of the
chalcocites may now reflect the slow evolution of the migration
of enrichment to its current level. There is no geologic evidence
to support this idea, in fact Cook (1994) dated alunites from
supergene blankets across the southwestern United States and
interpreted the scatter of alunite ages to represent several epi-
sodes of enrichment throughout the geologic past. This evi-
dence indicates that there are distinct intervals of enrichment
and that the enrichment process does not appear to be a con-
tinual process once erosion is initiated.
Field studies of oxidized sulfide ores and outcrop features at
Silver Bell (Lopez and Titley, 1995) have revealed the presence
of at least two former dated enrichment blankets above the
present enriched Silver Bell ores. Those superjacent blankets
have lost most of their copper through oxidation, leaching, and
movement to the new level in the North Silver Bell deposit. The
sample of chalcocite from Silver Bell (␦65Cu ⫽ 6.54‰) con-
tains copper derived by oxidation and leaching of Cu from the
multiple enrichment blankets above it. Thus, although our
model is too simplistic, it provides a basis for future modeling,
and it emphasizes that the isotopic values of supergene miner-
als are consistent with many stages of leaching and enrichment.
An essential prediction from this model is that the “lighter”
copper isotope values must exist somewhere in the copper
deposit. There are two possible reservoirs for this light copper:
(1) The light copper could be in the leach cap. Currently the
leach cap is ⬃82 m thick and contains ⬃0.1% copper (Enders,
2000) and could be used to make the mass balance. However,
there has been significant erosion in the deposit (upwards of 1
Km; Enders, 2000), and it would difficult to place an exact
constraint on the amount of missing copper. (2) The copper
could have moved laterally and essentially “leaked” out of the
system and formed what is termed “exotic” copper deposits.
One or a combination of both these sources could provide an
explanation for where copper minerals with light copper iso-
tope values exist.
Future models must incorporate more complexities. For ex-
ample, during the sequential leaching events, copper from
primary chalcopyrite in the ore deposit is probably added to the
system. In contrast, in our simplified model, 35% of the original
chalcopyrite was left behind, unleached. If additional hypogene
chalcopyrite were considered in the model, this source would
significantly change the overall copper isotopic composition of
the fluid by lowering its copper isotopic composition. There-
fore, the values modeled here represent minimum copper iso-
tope values per leach cycle.
In addition, copper-rich phases other than chalcopyrite exist
 Cu isotopes in the supergene environment
in hypogene ores. It is possible that minerals such as bornite,
primary chalcocite, and others could yield different fraction-
ation factors. Furthermore, copper not only precipitates as
copper sulfide phases in the supergene system, but also precip-
itates as sulfates and oxides (Titley, 1978). Copper leached
from primary chalcopyrite is also observed to precipitate as
chalcocite and other phases, even in the leach cap. These
copper precipitates are not accounted for in our model but
could be incorporated in future models.
Despite the simplistic nature of our model, our calculations
confirm that multiple leaching and precipitation events must
have occurred at both Morenci and Silver Bell deposits. If
Thiobacillus was involved in the leaching, more leach events
are warranted to explain the data. Our experiments further
document the possibility of a vital effect during oxidative
dissolution of Cu sulfides; Thiobacillus promotes the formation
of isotopically heavy Cu nano-oxide particles that may accu-
mulate in the leach caps. If this is true, it may be possible to
find such particulate material (e.g., Sillitoe et al., 1996) and
isotopic values of biotic and abiotic reactions during leaching.
Further research into the systematics of Cu isotopes should
refine such models further.
6. CONCLUSIONS
The experiments conducted in this study demonstrate that
during the oxidation of copper via abiotic leaching, Cu released
to solution is isotopically heavy because Cu mineral precipi-
tates entrain isotopically light Cu. Interestingly, Thiobacillus
cells promote the formation of isotopically heavy Cu oxide
nanoparticles during such leaching. This is the first study to
document the complexity of copper isotopic shifts during abi-
otic and biotic supergene processes. The results suggest that
supergene minerals should possess heavier copper isotope val-
ues than their parent material. Further experiments, mineralog-
ical observations, and modeling using isotopes of Cu and other
elements will undoubtedly provide insights into the supergene
process.
Acknowledgments—We would like to thank Art Rose, Steve Young,
Bryn Kimball, Henry Ehrlich, and Richard Thompson for their insight-
ful comments and discussion concerning this project. We would also
like to thank Maria Orosz and Lawrence Mutti for their help with the
diffraction of minerals and Ed Ripley and two anonymous reviewers
for their helpful comments. We would finally like to thank Phelps
Dodge for providing the chalcocite samples and John Uhrie for assis-
tance in completing this project.
Associate editor: E. M. Ripley
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