FEMSMicrobiolosyLetters 2 (1977) 163-165
0 CopyrightFederationof EuropeanMicrobiologicalSocieties
Publishedby Elmvier/North-HoIlandBiomedicalPress
T H E O C C U R R E N C E OF T H E R M O P H I L I C I R O N - O X I D I Z I N G B A C T E R I A IN A COPPER
LEACHING SYSTEM
J.A. BRIERLEY and S.J. IDCKWOOD
Departsqent of Biology. New Mexico Institute of Mining and Technology, Socorro. New Mexico 87801, U.S.A.
Received 18 July 1977
18H20, 4.0; MgSO4 • 7H20, 3.0; Ca(NO3)2 • 4H20,
0.1 ; MnSO4 • H20, 0.05; Na2SO4,0.05; KCI, 0.05;
the pH was adjusted between 2.6-3.0 with 1-12SO4.
The medium was sterilized, 121°C for 15 rain, in
50 ml quantities in alundnium fog.covered conical
flasks. 2 ml of sterile 25% (w/v) FeSO4 - 7H20 were
added to each flask. Where :indicated, yeast extract
(Difco, Detroit, Michigan)was added to the medium
in a final concentration of 0.02% (w/v).
Cultures were incubated at 50°, 55° or 60°C in a
water bath without shaking.
1. Introduction
The leaching of metals, specifically copper, from
metallic.sulfide rrdne waste material in large dumps
involves the activity of iron-oxidizing thiobacilli.
Localized areas within leach dumps have been observed where temperatures have approached 80°C
[1 ]. Thus, there has been speculation that thermophilic bacteria may be present in or near the hot
zones of leach dumps. However, no thermophilic,
iron-oxidizingbacteria have been found in copper
leaching dumps of the western United States [2].
A recent study [3] was conducted with copper
mine waste material in a large insulated cylinder
(10.7 m height, 3 m diemeter) containing 1.63
• l0 s kg of mine waste. Various parameters were
monitored including temperatures within the column
and numbers of Thiobacillus ferrooxidans present. In
the bottom portion of the tank the temperature
increased to a maximum of 60°C following 300 days
leaching. T. ferrooxidans declined from 106 cells/g
when the temperature reached 45°C to less than 10
cells/g near 60°C.
Following the increase in temperature, samples
were collected for determination of the possible presenc~ of thermophilic, iron.oxidizing bacteria• Our
research has confirmed the presence of this type of
bacterium in a large scale leach test facUity.
2.2. Ferrous iron analysis
The colorimetdc method of Grat-Cabanac [5] was
used to determine the concentration of ~ u b ] e ferrous iron.
2.3. Viable cell count
A most-probable-number method [6] was used to
determine the number of thermophilic, iron-ox/dizin~
bacteria present within the mine-waste material. The
tubes were incubated at 50°C, and yeast extract
(0.02% w/v) was used with the medium. The results
were considered either positive or negative for lpro~ch
after 21 days incubation.
2• Materiab and Metho~h
3. Reml,'s
2.1. Odmve cond~tions
The presence of thermophilic, iron-oxidizingbacteria was determined by inoculat/r~ the medium with
1 g of ndne-waste sample. The leach tank was sampled at 3.6 m (level l), ?.0 m (level 2), 8.5 m (level 3)
The mediw~'t used [4] consisted of the following
(g/l): (NH4)~SO4, ! .0; K2HPO4, 0.1; ~J2(SO4)3
163
164
large n u m b e r Of Gram,negative rods o f Various sizes:
Using t h e culture grown on yeast extract as inocu.
lure, three sets o f flasks were run: (1) medium plus
iron;(2) medium plus yeast extract; (3) medium plus
iron plus y e a s t extract. The inoculated fl~sks a n d controis were incubated at 50°, 55 ° or 60°C.: Iron analy;
ses were done on the two sets containing iron, and
these results are summarized in Table 2.
Microbial iron-oxidation occurred at 50 ° and
55°C. All flasks were checked microscopically. No
bacteria were found in any flask incubated at 60°C.
At 50 ° and 55°C the flasks with yeast extract and
iron contained large bacterial populations resembling
those described earlier. The flasks with only iron
showed n o bacteria. A few small rods were visible in
the flasks at 50 ° and 55°C containing on W yeast
extract.
The number o f yeast extract-requirinil iron.
oxidizing microbes capable o f growth at 50°C was
determined using a most-probable-number method.
The results are presented in Table 3.
The cell concentration was greatest in the lower
areas o f the leach-tank which were higher in temperature. At the surface (9.8 m above the bottom), the
leach solution was applied at ambient temperature,
thereby cooling the ore bed and possibly accountint;
for the low numbers o f iron-oxidizing bacteria capable o f growth at 50°C at level 4.
"[ABLE i
Cor~--.,nt':,ztion of ferrous iron in flasks -'ontaining 1.0 g of ore
~mple as inoculum
Fe 2+ concentration (ppm)
Lacation
Temperature (~C)
Day 0
Day 7
Day 13
Level I
L~el 2
Level 3
L~:vel 4
50
50
50
50
1375
1375
1335
1420
290
105
400
Control t~
50
1450
340
1520
22
14
9
55
1260
Level I
Level 2
revel 3
Lcvel 4
(ontrol a
60
60
613
61)
60
1480
1425
1490
1465
1325
1350
1385
1300
1285
1235
il50
1090
1025
1110
1025
a The controls were sterilized ore samoles added to the
growth medium.
a~d 9.3 m (level 4) above ~he tank bottom. Table 1
shows that microbial catalyzed iron oxidation took
place at all levels at 50oC • only slight spontaneous
oxidation, due to the h~gh temperature, took place at
60°C. When subculturing e f the bacteria from 50°C
flasks w.~s attempted, growth was not initiated unless
0.02% ~east extract was added. Microscopic analysis
clone on the yeast extract-grown bacteria revealed a
TABLE 2
The effects of temperature and the presence or absence of y,~ast extract on bacterial iron-oxidation
Fe2+ concentration (ppm)
Media
Temperature eC}
Day 0
Day 1
Day 2
Day 3
Day 4
Day $
Day 6
Fe
Fe - c,mtrol
Fe + yeast extract
Fe + yeast extract - control
50
50
50
50
1490
1440
1470
1500
1550
1580
1310
1500
1560
1440
955
1490
1440
1400
770
1440
1405
1500
515
1440
1420
1460
240
1440
1375
1400
6
1400
I'e
Fe - c~,ltrol
Fe + yeast extract
I.e + yet,st extract - control
55
55
55
55
1625
1680
1560
1400
1300
1460
1075
1460
1140
1380
910
1440
I100
1400
605
1620
1040
1460
440
1300
1090
1460
170
1400
985
1380
3
1350
le
Fe - col~arol.
Fe + yeast extract
Fe + yeast extract - control
60
60
60
60 '
1675
1400
1560
1120
1440
1400
1440
1460
1375
1370
1440
1440
1360
1280
1325
1350
1335
1320
1320
1320
-
-
I65
TABLE 3
Concentration of iron-oxidizingmicrobes capable of growth
at 50°C
Sample location
Cells/gsample
Level 1
Level 2
Level 3
Level 4
1.1 104
3.5 • 103
1.4 • 104
4.0 • 10 ]
4. Discussion
The results of these experiments have demonstrated the pre~nce of thermophilic, iron-oxidizing
bacteria in a large scale leach system, it is believed
that the source of these microbes was either the
crushed mine waste and/or the leach solution (22.7
• 10 a 1) transported from an active leach dump to the
test facility,
No "pure culture" has yet been isolated from the
enrichment, which contains bacteria very similar to a
thermophilic Thiobacillus-type bacterium from Icelandic thermal areas [7]. This rod-shaped microbe has
been shown to oxidize ferrous iron at 50°C and also
has a yeast extract requirement for growth [8].
The actual or pot ential role for the rod-shaped
thermophilic, iron-oxidizing bacteria in leaching is
presently unknown. However, other iron-oxidizing
thermophiles belonging to the genus Sulfolobus have
been shown to catal,/ze the leaching of molybdenum
and copper from wastes and concentrates at 60°C
[9,10], Further study is in progress to determine the
ability of the thermophilic, iron-oxidizing bacterium
to leach metal sulfides.
it has been suggested [3,11 ] that temperature
increases in leach dumps may be associated with bacterial oxidation of iron and sulfides. However, neither
study conclusively established the cause-effect associ.
ation between heating and bacterial activity• Yet, if
such an association does exist, then the thermophilic,
iron-oxidizing bacteria could increase dump temperstufts beyond the range of the mesophilic T. ferrooxidans commonly found in leach dumps.
Further study is needed to establish the role of
thermophilic, iron-oxidizing bacteria in leaching, their
distribution in leach dump environments, possible
effect on leach dump temperatures and apparent nee~t
for yeast extract.
Acknowledgements
This research was supported in part by the Kennecott Copper Corporation and the National Science
Foundation (RANN) grant AER-76-O3758. The
authors thank Mr. William Herring~on for technical
assistance•
References
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[2l Brierley, C.L. (1976) Technical Report GO1441 lOrood. 1 pp. 22-23, U.S. Bureau of Mines, Department
of the Interior, Washington,D.C.
[31 Murr, L.E., Cathles, L.M., P.¢~eie,D.A., Iiiskey, J.B.,
Popp, C.J., Brierley,J.A., Bloss, D., Berry, V.K., Schl/tt.
WJ• and ilsu, PC. (1977) Proc. Am. Nuc. Sot., m
14] Bryner, L.C. and Anderson, L. (1957) Ind. Eng. Chem.
49,1721-1724.
151 Grat-Cabanac,M. (1951) Anal. Chem. Acta 5,116118.
161 Collins, C.H. and Taylor, C.E.D. (1967) M i c r o b k ~
Methods, pp. 152-159, Plenum Press, New York.
[7] LeRoux, N.W., Wakerley, D.S. and Hunt, S.D. (1977) J.
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[8] Brierley,J.A. and LeRoux, N.W. (1977)GBF Moao.
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[9] Brierley,C.L• (1~74) J. Less-CommonMetals 36,237 247.
[10] B~'~erley,C•I_. (1977) Dev. Ind. ;JicrobioL/n press.
[ 11] Lyal~ova, N.N. (1960) Microbiology29, 281-283.