APPLIED MICROBIOLOGY
Vol. 12, No. 2, p. 122-126 March, 1964
Copyright © 1964 American Society for Microbiology
Printed in U.S.A.
Leaching of Chalcopyrite with Thiobacillus ferrooxidans: Effect
of Surfactants and Shaking
D. W. DUNCAN, P. C. TRUSSELL, AND C. C. WALDEN
British Columbia Research Council, University of British Columbia, Vancouver, British Columbia, Canada
Received for publication 29 October 1963
ABSTRACT
DUNCAN, D. W. (British Columbia Research Council,
University of British Columbia, Vancouver, B.C., Canada),
P. C. TRUSSELL, AND C. C. WALDEN. Leaching of chalcopyrite
with Thiobacillus ferrooxidans: effect of surfactants and shaking.
Appl. Microbiol. 12: 122-126. 1964.-The rate of leaching of
chalcopyrite by Thiobacillus ferrooxidans has been greatly
accelerated by using shaken flasks in place of stationary bottles
or percolators. A further increase in rate and extent of leaching
was obtained by the use of Tween 20, 40, 60, and 80, Triton
X-100, Quaker TT 5386, and Hyamine 2389. Tween 20 was the
most effective surfactant. No individual component of the
Tween molecule was responsible for the improved leaching.
The Tween-to-chalcopyrite ratio is more important than the
Tween-to-medium ratio. The effect of the surfactants is probably
due to increased contact between the mineral surface and the
organism, and shaking provides the necessary oxygen. Rates and
yields obtained by use of surfactants and shaking as aids to
microbiological leaching approach those obtained with acidified
erric sulfate leaching.
The microbiological release of copper and other metals
from their sulfides is relatively slow, compared with the
rate obtained with acidified ferric sulfate solutions. Using
acidified ferric sulfate in a batch system, Colombo and
Frommer (1962) released 58 % of the copper from a
Michigan copper ore in 4 hr and over 90 % in 2 to 3 weeks.
Using percolators and microorganisms, Bryner et al.
(1954) released only 2.8 % of the copper from one chalcopy-
rite sample in 70 days and 6.6 % from another sample in
56 days. Also using percolators, Malouf and Prater (1961)
leached 50 % of the copper present in their sample of
chalcopyrite in about 170 days and a total of 60 % in 470
days. Razzell (1962) released 25 % of the copper from
chalcopyrite in 60 days with percolators and 35 % in 100
days in stationary flint-glass bottles. After a 7-month
leach, using stationary bottles, Razzell claimed that "80%
of the copper in chalcopyrite is converted to acid-soluble
copper but only 45 % of this is soluble at pH 2.5." Later,
Razzell and Trussell (1963a) improved this yield to 40 %
of the copper in 55 days. These excessively long leach
times and usually poor yields limit the practical applica-
tion of microbiological leaching.
Factors demonstrated to affect the rate and extent of
biological leaching are particle size, tenmperature, pH,
aeration, inoculum size, and exposure to ultraviolet light
122
(Bryner et al., 1954; Bryner and Anderson, 1957; Silver-
man and Lundgren, 1959b; Malouf and Prater, 1961;
Silverman, Rogoff, and Wender, 1961; Razzell and
Trussell, 1963a, b; Sutton and Corrick, 1963). Optimal
conditions include a particle size of -325-mesh, a
temperature near 35 C, a pH between 2 and 3, rapid
aeration, a large inoculum, and protection from sunlight.
MIicrobiological leaching of sulfide ores is most
commonly studied with air-lift percolators (Bryner et al.,
1954), Warburg respirometers (Beck, 1960), and stationary
leach bottles (Razzell and Trussell, 1963b). Percolators
and stationary leach bottles normally meet all the require-
ments for optimal leaching, with the possible exception of
aeration. Anaerobic conditions do not occur in the Warburg
apparatus, but its size and principle of operation render it
unsuitable for evaluating many practical leaching
variables.
A practical method which produces rapid aeration and
permits evaluation of the experimental variables is the
use of Erlenmeyer flasks on a shaking apparatus. All the
conditions for optimal leaching can be encompassed with
such an arrangement, except possibly the necessity for a
finite contact time between the ore particle and the
bacterium.
If Thiobacillus ferr ooxidans attacks the crystal lattice of
sulfide minerals directly, then the rate of leaching miiay be
increased by providing for intimate contact between the
organisms and the surface of the mineral. Starkey, Jones,
and Frederick (1956) showed that the rate of oxidation of
sulfur by T. thiooxidans in a liquid medium was increased
by shaking with surface-active agents. More recently,
Schaeffer and Umbreit (1963) showed that T. thiooxidans
produces a surface-active agent that wets the sulfur to be
oxidized. The present paper presents the effect of surface-
active agents and shaking on the biological leaching of
chalcopyrite by T. ferrooxidans.
MATERIALS AND MIETHODS
The culture of T. fenrooxidans used in this study was
isolated by Razzell and Trussell (1963a) from the mine-
water of the Britannia IMine of the Howe Sound i\lining
Co. The organism was grown at 35 C in medium 9K
(Silverman and Lundgren, 1959a) on a gyratory shaker.
The sample of museum-grade chalcopyrite was crushed
and ground to pass through a 325-mesh sieve. Chemical
VOL. 12) 1964 LEACHING OF CHALCOPYRITE 123
analysis showed the chalcopyrite (CuFeS2) to be 95.35 % indicate that shake flasks produce higher yields than
pure on the basis of copper and 96.64 % pure on the basis of stationary bottles, and thus are preferred for studying
iron. microbiological leaching. The control shake flask, contain-
The leaches were carried out in 250-nd Erlenmeyer ing T. ferrooxidans but no surfactant, leached 35 % more
flasks containing 1.0 g of -325-mesh chalcopyrite and 75 copper in 33 days than did the corresponding stationary
ml of medium 9K (pH 2.5) in which the iron solution was bottle. The results (Table 2) show also that Tween 20
replaced by distilled water. The required amount of was the only surfactant in the anionic nonionic group
surfactant was added on a per cent by volume basis. The which improved the biological leaching of copper.
uninoculated control flasks contained Hg++ as a growth The improvement was particularly pronounced in the
inhibitor. shake flasks, where the release of copper was doubled.
The flasks were inoculated with three drops of an 8- Triton X-100 gave a final yield similar to the inoculated
to 10-day-old culture of T. ferrooxidans which had been control, but initial leaching rates were greater.
harvested according to the procedure of Lazaroff (1963), Table 3 presents the results with cationic surfactants in
washed twice with pH 2 sulfuric acid, and resuspended in shake flasks. Armour 461, Nopco CVT, and Ultrawet 40
pH 2.5 sulfuric acid at 100 times the original concentration.
The flasks were incubated in the dark at 35 C on a gyratory TABLE 2. Effect of various anionic and nonionic surfactants on
the release of copper from museum-grade chalcopyrite by
shaker (New Brunswick Scientific Co., Inc., New Bruns- Thiobacillus ferrooxidans
wich, N.J.) at 180 rev/min.
Periodically, the flasks were analyzed for soluble copper Leach Copper concn (ppm)
Surfactant Concn method
by adding 4 ml of 1.9 N ammonium hydroxide to 1 ml of 0 days 28 days 33 days
sample, centrifuging at 500 X g for 10 min to pre-
cipitate the iron, and reading the optical density at
620 m,u (Mehlig, 1941). When necessary, the residue from Inoculated control 0 Stationary 204 760 800
0 Shake 204 1,100 1,085
the copper determinations was dissolved in 0.5 ml of Uninoculated control 0 Stationary 259 335 335
6 N HCl, and iron determinations were carried out with 0 Shake 259 425 400
thiocyanate (Sandell, 1959). Duponol 80 0.01 Stationary 197 830
The surface active agents used are listed in Table 1. 0.01 Shake 197 965
Their properties and manufacturers are listed in Igepal CO-630 0.01 Stationary 190 650
0.01 Shake 190 755
Detergents and Emulsifiers Annual (Anonymous, 1963). Petrowet R 0.005 Stationary 200 760
0.005 Shake 200 950
RESULTS Petrowet WN 0.01 Stationary 197 800
The surfactants listed in Table 1 were initially evaluated 0.01 Shake 197 805
Triton X-100 0.005 Stationary 194 710
at the 0.001, 0.002, 0.005, and 0.01 % levels for their 0.005 Shake 194 960
effect on the oxidation of the iron in medium 9K by T. Tween 20 0.01 Stationary 197 960
fenrooxidans. Geigy Amine T, Naccanol NR, and Triton 0.01 Shake 197 2,320
X-400 inhibited iron oxidation at all concentrations,
whereas the others permitted oxidation at some concentra-
TABLE 3. Effect of various cationic surfactants on the release of
tions. copper from museum-grade chalcopyrite by Thiobacillus
Certain of those anionic and nonionic surfactants which ferrooxidans in shake flasks
were noninhibitory to iron oxidation were evaluated
Copper concn (ppm)
at their maximal noninhibitory concentration for their Surfactant ConcnSuran Inocu-
Inocu-
0 days 19 days 26 days
effect on the biological leaching of chalcopyrite, in both
stationary bottles and shake flasks. The results (Table 2)
Inoculated control 0 + 181 660 805
Uninoculated control 0 - 181 290 335
TABLE 1. Surface-active agents Armour 461 0.005 + 188 400
Anionic Cationic Nonionic 0.001 + 188 700
0.005 - 188 290
Duponol 80 Armour 461 Igepal CO-630 Hyamine 2389 0.001 + 180 1,215
Petrowet R Hyamine 2389 Span 20 0.001 - 180 355
Petrowet WN Geigy Amine T Span 80 Nopco CVT - 0.001 + 184 775
Naccanol NR Triton X-100 0.001 - 184 335
Nopco CVT Tween 20 Quaker TT5386 0.005 + 177 1,205
Quaker TT 5386 Tween 40 0.01 - 177 300
Quaker TT 5518 Tween 60 Quaker TT5518 0.005 + 177 355
Triton X-400 Tween 80 0.01 - 177 310
Ultrawet 40 Tween 81 Ultrawet 40 0.005 + 183 725
Tween 85 0.005 - 183 325
124 12 DUNCAN, TRUSSELL, AND WALDEN APPL. AIICROBIOL.

had no effect on leaching, whereas Quaker TT 5518 was
inhibitory. The only systems to release more copper thani
the controls were those containing Hyamine 2389 and
Quaker TT 5386. The uninoculated surfactant flasks in
Table 3 showed that the surfactants themselves had no
ability to leach chalcopyrite.
Of the four surfactants that accelerated the leaching of
chalcopyrite, Tween 20 was the most effective and Triton
X-100 was considered to have potential value because of
the rapid initial rate of leaching it produced. A more
detailed study of the optimal concentration of these two
surfactants was made (Table 4). The results indicate that
in 18 days Tween 20 released more copper than did
Triton X-100 and that the optimal concentrations were
0.002 to 0.003 %c for Triton X-100 and 0.003 to 0.004'S,
for Tween 20. After 18 days of leaching, 0.002CJ Triton
X-100 released twice as much copper as the inoculated
control, and 0.003 %. Tween 20 released three times as
much.
In working with stationary bottles, Razzell and Trussell
(1963a) postulated that an insoluble copper-iron complex
interfered with the estimation of copper released from
chalcopyrite. Before the addition of 2 volumles of 6 x
HCI, they recovered only 65 % of the released copper.
Upon termination of the leach reported in Table 2, certain
flasks were examiinled for their soluble copper content.
Results given in Table 5, for analyses made before and
after the solutions are adjusted to a concentration of 10 %
HCl, show that at least 95 %O of the copper was in solution
before the HCl was added.
The increase in soluble copper in 10 % HCl solutionis
(Table 5) is an anialytical artifact. Determiiinationi of
known amounts of copper in 10 % HCl gave recoveries of
105 "/'. When the Tween 20 sample was miiade to 10 /c
HCl, stirred, filtered, and the pH adjusted to 2, the
increase in determiined copper was negligible (Table 5).
Therefore, no insoluble copper-iron complex formls under
the conditions employed here, and the addition of HCl
in the final determination of copper was henceforth
omitted.
TABLE 4. Effect of concentration of T'riton X-100 and 7'ween 20 on the mictobiological release of copper from
museum-grade chalcopyrite
Surfactant Concn Inoculated
0 days
Since Tween 20 (polyoxyethylene sorbitan moniolaurate)
was the most effective surfactant investigated, other
related compounds were examiiined. Laurie acid, stearic
acid, oleic acid, and sorbitol at the 0.001 %G level had no
effect on the rate or extent of leaching. Palimiitic acid
inhibited the release of copper completely for 27 days anid
then slow leaching began. At the 0.003 %/ level, Span 20,
Span 80, Tween 81, and Tween 85 did not imlprove
leaching, although Span 80 caused leaching to start at
3 days instead of 6. Inoculated flasks containing Tween
40, 60, and 80 all started to leach rapidly and gave inm-
proved yields over control flasks.
These results indicate that Ino inidividual coimlponieint of
the Tweens is responsible for the improved leaching. The
poor results with the Spans indicate that the polyoxy-
ethylene side chain is necessary, and that it canniot be
shortened was indicated by the lack of effect of Tween 81.
Three fatty acid groups, as in the trioleate, Tween 85,
were as undesirable as the lack of the polyoxyethylene
side chain in Span 80. Improved leaching is associated
only with the molecular constituents as comiibined in
Tween 20, 40, 60, and 80.
Results obtained in evaluating the effect of Tween 20,
40, 60, and 80 at four different concentrations are given
in Table 6. 1\Iaxinmal effect is displayed by Tween 20 at
the 0.001 %O level, which gave 2.25 timies as iiiuch copper
as the inoculated conitrol. In uninoculated leaches, 0.0005 %,
of the Tween surfactants leached no miiore copper than the
control, and about one-tenth as miiuch as the inioculated
control.
Wheni the rate of leachinig declined in the Tween 60
flasks, the 9K imiediumn and Tween 60 were replaced in
the 0.003 and 0.005 71 flasks. Instead of restoring the
initial leach rate, leachinig stopped coiimpletely (Table 6).
Leaching rates for all concentrations of Tweeni 20
listed in Table 6 are shown graphically in Fig 1. For
0.003 and 0.001 % Tween 20, rates and final yields are
alnmost identical. The rate of leaching with the 0.00057%
Tween 20 flask was appreciably slower than that with
the higher concentrations of Tween 20.
Concn of copper (ppm)
Yield of copper
4 days 7 days days 14 days 18 days

7, (7,
Inoculated control 0 <50 130 210 960 1,215 1,265
Uninoculated control 0 <50 90 145 175 265 375
Triton X-100 0.005 <50 1,575 2,090 2,485 2,330 2,510 55
0.004 <50 1,685 2,030 2,410 2,330 2,610 55
0.003 <50 1,645 1,925 2,290 2,450 2,650 58
0.002 <50 1,625 1,925 2,335 2,480 2,680 63
0.005 <50 <50 75 100 120 185 8
Tween 20 0.010 <50 1,765 2,310 2,840 2,720 2,950 63
0.005 <50 1,765 2,410 2,940 3,040 3,200
0.004 <50 1,980 2,535 3,090 3,330 3,670 75
0.003 <50 1,965 2, 625 3,250 3,350 3,670 79
VOL. 12) 1964 LEACHING OF CHALCOPYRITE 125

The previous experiments had shown that 0.001 to The overall low yields with 4 g of chalcopyrite were
0.003 % Tween 20 gave optimal leaching of chalcopyrite caused probably by a suboptimal chalcopyrite-to-medium
under the conditions employed. Table 7 presents the re- ratio, or possibly by an inhibitory effect of copper con-
sults of an experiment set up to determine whether this centrations in excess of 10,000 ppm. A high chalcopyrite-
optimum involved the ratio of Tween to medium or to-medium ratio may reduce access of bacteria to ore sur-
Tween to chalcopyrite. With 0.003 % Tween 20, the per- faces.
centage release of copper from 1 and 2 g of chalcopyrite Data in Tables 4 and 6 indicate that with 1 g of chalco-
was the same and from 4 g only slightly less. At a Tween pyrite in 75 ml of medium concentrations of Tween 20
20 concentration of 0.006 %, the yield of copper from 2 g most effective in releasing copper were 0.003 % or less.
of chalcopyrite was the same as for 0.003 % Tween 20. Above this concentration, copper release was inhibited.
With 4 g of chalcopyrite, the yields were decreased slightly, On the other hand, in Table 7, 0.006 % Tween 20 does not
but the percentage of copper released was slightly greater depress copper release from 2- and 4-g samples of chalco-
with 0.006 % Tween 20 than with 0.003 or 0.012 %o. pyrite. These results indicate that the Tween-to-chalco-
pyrite ratio is more significant than the Tween-to-medium
TABLE 5. Soluble copper before and after making the ratio.
solution up to 10% HCI
DIsCUSSION
Flask in 10%copper
Soluble copper Soluble HCI Soluble
before HCI Beck (1960) showed that, during the oxidation of iron by
mg mg % resting-cell suspensions of T. ferrooxidans, the rate of
Duponol 80. ............ 65.6 69.2 95 oxygen consumption is high. Thus, during active metabo-
Petrowet R ............ 69.3 70.6 98 lism the oxygen in solution may be depleted faster than it
Petrowet WN .......... 55.5 58.5 95 is replaced. This danger is particularly great with station-
Tween 20 .............. 259.9 260.7* 100 ary leach flasks where the only mechanism for oxygen
*
The pH of the filtrate was adjusted to 2 before copper deter- transfer is diffusion.
mination. In the time intervals involved in these experiments, the
stationary flasks consistently gave lower yields than did
TABLE 6. Percentage yield of copper in 24 days from -325-mesh
museum-grade chalcopyrite with various Tweens and the shake flasks. Slow diffusion of oxygen into the solution
Thiobacillus ferrooxidans probably was responsible for depressing the leaching rates.
Concn Inoculated Tween 20 Tween 40 Tween 60 Tween 80 Control
In view of this, shake flasks have replaced the use of
stationary bottles for studying microbiological leaching in
this laboratory.
0 -3 The possibility of oxygen deficiency also exists with
0 + 38 percolators. From the descriptions and illustrations of
78*
0.0005 - 4 4 4 4 percolators given in the literature (Bryner et al., 1954;
0.0005 + 62 50 54 45 Bryner and Anderson, 1957; Malouf and Prater, 1961;
0.001 + 86 74 74 Sutton and Corrick, 1963), the columns are flooded with
0.003 + 80 77 57t 75 nutrient solution to a height of 3 to 5 cm above the surface
0.005 + 76 64 58t 66 of the sand and ore. The liquid that is entering the top of
* Maximum after 109 days. this column of ore may be saturated with oxygen, but
t Media replacement experiments. with the rapid rate at which oxygen is consumed, at least
during iron oxidation, anaerobic conditions probably occur
TABLE 7. Effect of variation in the ratio of the concentration of
Tween 20 to amount of -325-mesh museum-grade chalcopyrite
on the release of copper by Thiobacillus ferrooxidans
2
0.
(L
iL-l Concn of Tween 20
2
z
0
.4
Amt of 0 0.003%* 0.006%* 0.012%*
cr
I.-
chalcopyrite
z
w
u
z
2 Amt of Yield Amt of Yield Amt of Yield Amt of Yield
0
u
Cu Cu Cu Cu
x
w
a.
a.
g mg % mg % mg % mg %
0
u
1.0 126.0 38 18.2t 5t
224.4 68
2.0 455.1 69 442.1 67
4.0 771.1 59 815.3 62 749.9 57
TIME IN DAYS

FIG. 1. Effect of various concentrations of Tween 20 on the release * On basis of 75 ml of liquid volume.
of copper from chalcopyrite. t IlJninoculated control
126 DUNCAN, TRUSSELL, AND WALDEN
in the lower reaches of the columiin. The improved release
of copper from chalcopyrite by T. ferrooxidans by use of
shake flasks in the absence of a surfactant (Table 6) sup-
ports this hypothesis.
These conclusions are in agreement with the results ob-
tained by Audsley and Daborn (1962) during the leaching
of uranium ores in percolators. They found that, in a deep
column of uranium ore plus pyrite, over 83 % of the ura-
nium had been released from the top two-fifths and only
32 % from the bottom one-fifth. When the frequency at
which the mineral surfaces of another deep column were
wetted was decreased from daily to weekly intervals, an
increased rate of leaching was obtained. They concluded
that this was due to a greater volume of air in the voids.
The addition of Hyamine 2389, Quaker TT 5386, Triton
X-100, and Tween 20, 40, 60, and 80 to the leach solutions
aided the microbiological leaching of chalcopyrite. The
surfactants probably wetted the chalcopyrite surface, per-
mitting more rapid contact and more intimate association
by the bacteria. The rapid aeration of the systenm provided
the necessary oxygen.
Shaking might be expected to mrinimize residence time
of the organism on the mineral surface. Starkey et al.
(1956) obtained poorer oxidation of sulfur on a reciprocal
shaker than on a rotary shaker, which they attributed to
sulfur accumulating on the necks of the flasks above the
liquid surface. Another possibility may be that the snap-
like motion of a reciprocating shaker limits the contact
time of the bacteria on the sulfur particles. In the studies
reported here, a rotary shaker was used, and the motion it
imparts to the system may not hinder bacterial contact
with the mineral surfaces.
The increased rate and extent of biological leaching of
chalcopyrite, by use of surfactants and shaking, increases
the commercial attractiveness of this process. Yields of
copper from chalcopyrite as high as 85 % in 24 days have
been obtained with T. ferrooxidans, Tween 20, and aera-
tion. Initial rates are much higher than these figures indi-
cate; release of copper over the first 5 to 7 days has been
10 % per day. These rates and yields are much higher than
the previously reported maximums of 60 % copper in 470
days by 1\Ialouf and Prater (1961) and 40 % copper in 60
days by Razzell and Trussell (1963b). They approach the
results obtained by Colombo and Frommer (1962) during
chemical leaching of chalcocite.
The shake-leach process as applied to other minerals
and ores was presented elsewhere (Duncan and Trussell,
in press). These authors showed that vastly improved
leach rates and yields result with improved aeration but
that only museum-grade chalcopyrite, museum-grade
APPL. MICROBIOL.
millerite, and two copper ores responded to the presence of
Tween 20.
ACKNOWLEDGMENT
The authors wish to acknowledge the technical assist-
ance of H. Kurtz.
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